D Servant

E Drumez

S Raynal

A L Demarty

A Salembier

M H Deschepper

M A Bizet

A Pisanu-Zimny

J B Culem

J Labreuche

A Duhamel

G Vaiva

Élaboration et qualités psychométriques d'une échelle de bien-être au travail. Étude SERENAT auprès de salariés vus en médecine du travail

Keywords: Mental health, Questionnaire, Depression, Anxiety, Psychometrics, Occupational stress, Happiness

soutien social et le contrôle au travail protègent la santé mentale alors que les exigences élevées et le déséquilibre effort-récompense sont des facteurs de risque d'anxiété et de dépression. Il n'existe actuellement aucun indicateur global permettant de mesurer à la fois l'état de santé mentale et les conditions de travail. L'objectif de cette étude est de construire puis d'étudier les qualités psychométriques d'une échelle de bien-être au travail l'échelle Serenat, dans le but de sa validation.

Méthodes

L'échelle Serenat est un auto-questionnaire composé de 20 items évalués selon une échelle de Likert de 0 à 3 pour un score total allant de 0 à 60. Les items ont été construits à partir des données de la littérature et d'une évaluation qualitative préalable auprès de salariés. De janvier 2014 à mai 2017, 193 sujets vus en médecine du travail ont été inclus dans cette étude transversale. La validation comprend un diagnostic de qualité des items et de structure des données, la consistance interne, la reproductibilité intraobservateur et la consistance externe.

Résultats

L'échelle Serenat a montré une très bonne qualité des items, avec un taux de non-réponse maximal de 0,01 % par item, et aucun effet plateau mis en évidence.

L'analyse factorielle permet de conclure à l'unidimensionnalité de l'échelle. Le coefficient α de Cronbach mesurant la consistance interne était de 0,89. Le coefficient de corrélation intra-classe pour la reproductibilité intra-observateur était de 0,89. L'échelle Serenat était corrélée aux scores HADS (r=-0,54; p<0,001), STAI-Y (r=-0,78; p<0,001) et BDI-13 (r=-0,57; p<0,001).

Conclusion L'échelle de bien-être au travail Serenat montre de bonnes propriétés psychométriques en vue de sa validation définitive. Elle pourrait être utile aux médecins du travail dans le dépistage individuel et collectif.

Mots clés

Santé mentale ; Questionnaire ; Dépression ; Anxiété ; Psychométrie ;

Stress professionnel ; Bonheur Enregistrement de l'essai : ClinicalTrials.gov ID: NCT02905071.

Introduction

La santé psychologique au travail est un sujet majeur de santé publique. Elle a été principalement étudiée à partir du concept de stress au travail. Plus d'un tiers des européens ayant une activité professionnelle perçoivent un niveau de stress au travail élevé [START_REF]OSH in figures : stress at work -facts and figures[END_REF]. Il désigne à la fois la perception des conditions de travail et les symptômes physiques et psychologiques de la réponse au stress. Le stress au travail est responsable d'un coût élevé lié aux conséquences médicales et psychologiques, à une perte de productivité et à un absentéisme [START_REF] Hassard | Calculating the Cost of Work-Related Stress and Psychosocial Risks[END_REF]. Le stress perçu est corrélé positivement avec le niveau d'anxiété, les ruminations, les perturbations émotionnelles et l'humeur dépressive [START_REF] Lee | Perceived stress and self-esteem mediate the effects of workrelated stress on depression[END_REF] La notion de bien être psychologique a été proposée dans différents domaines comme la santé mentale, la psychologie positive et les sciences de gestion. Elle se définit non seulement par l'absence de symptômes sur un plan de la santé de l'Organisation mondiale de la santé (OMS) mais par une perception subjective psychologique de satisfaction et de bien-être [START_REF] Diener | Subjective well-being[END_REF]. Deux approches du bien être psychologique ont été proposées en se référant au concept de bonheur des philosophes grecs, l'une dite hédonique vue sous l'angle des émotions positives et de la satisfaction dans la vie [START_REF] Keyes | Optimizing well-being: the empirical encounter of two traditions[END_REF] et l'autre dite eudémonique dans le sens de la réalisation personnelle et de l'accomplissement [START_REF] Ryff | Happiness is everything, or is it? Explorations on the meaning of psychological well-being[END_REF][START_REF] Waterman | The implications of two conceptions of happiness (hedonic enjoyment and eudaimonia) for the understanding of intrinsic motivation[END_REF]. Le bien être psychologique pourrait être appréhendé selon ces deux dimensions qui lorsqu'elles sont prises en compte simultanément apparaissent très fortement corrélées et la question a été posée de leur distinction. D'une façon générale, le bien-être est aujourd'hui défini comme un état psychologique positif qui écarte les aspects négatifs de la santé mentale [START_REF] Diener | Assessing subjective well-being: Progress and opportunities[END_REF]. Une revue recense 99 instruments proposés pour évaluer le bien être dans différents contextes (population générale, milieux universitaires, soins…) et montre qu'ils abordent de très nombreuses dimensions dont la plus fréquente est la dépression suivie de l'anxiété [START_REF] Linton | Review of 99 self-report measures for assessing well-being in adults: exploring dimensions of well-being and developments over time[END_REF].

La L'objectif de cette étude est d'explorer les qualités psychométriques de cette échelle de bien être au travail, que nous avons appelé Serenat, en consultation de médecine du travail.

Sujets et méthodes

Construction de l'échelle

A partir des données de la littérature et des données recueillies auprès de consultations dans le service de santé au travail, une liste de (alpha=2,5 %, bêta=20 %), en considérant un ICC attendu de 0,80, 50 sujets sont requis.

Un autre groupe de 50 sujets a été tiré au sort pour évaluer la liaison entre l'échelle développée et les trois autres échelles (validité externe).

Qualité des items

Des analyses descriptives ont été réalisées pour chaque item. Le nombre de données manquantes par question a été relevé. En cas de taux de données manquantes importantes (>20 %), l'item doit être supprimé de l'échelle. La distribution des items a été examinée pour détecter si des questions avaient des modalités trop fréquentes (>50 %).

Analyse factorielle

Une analyse en composantes principales (ACP) avec rotation varimax a été utilisée pour vérifier la structure de l'échelle. Le nombre de dimensions a été évalué selon le critère du pourcentage de variance expliquée, à l'aide de la procédure décrite par Horn (32) et à l'aide de la corrélation entre chaque item et les composantes principales. Une corrélation supérieure à 0,40 était considérée comme suffisante.

Consistance interne

La consistance interne reflète l'homogénéité des items qui constituent chaque dimension et a été mesurée à l'aide du coefficient α de Cronbach. La consistance interne d'une dimension a été considérée comme bonne lorsque ce coefficient est supérieur à 0,70 (33).

Test-restest

La reproductibilité intra-observateur a été évaluée à l'aide du coefficient de corrélation intraclasse (ICC) entre les deux passages de l'échelle de bien-être à une semaine d'intervalle.

La reproductibilité a été considérée comme excellente si la valeur de ce coefficient est supérieure ou égale à 0,75 [START_REF] Hernaez | Reliability and agreement studies: a guide for clinical investigators[END_REF]. La méthode de Bland et Altman (34) a également été employée pour évaluer le test-retest.

Consistance externe

La consistance externe permet de s'assurer que la nouvelle échelle est bien corrélée avec d'autres échelles validées mesurant un phénomène proche. Ces corrélations ont été évaluées par des coefficients de corrélation de Spearman. Chaque coefficient doit être supérieur à 0,40.

Influence des caractéristiques démographiques

Analyse factorielle

Une analyse en composantes principale (ACP) avec rotation varimax a été réalisée sur les 20 items de l'échelle. La première valeur propre expliquait 33,3 % de la variance de l'échelle. Le graphique de la variance expliquée pour chaque composante principale ainsi que la méthode de simulation développée par Horn ont permis de conclure à l'unidimensionnalité de l'échelle Serenat (Fig. 1). Une seule question avait une faible corrélation avec la première composante principale (Tableau 2). Cet item a été conservé car jugé cliniquement important.

Conclusion

Le

Déclaration de liens d'intérêts

Les auteurs ne déclarent aucun conflit d'intérêt en lien avec cette étude.

  De nombreux questionnaires et échelles ont été utilisés dans le but d'évaluer le stress au travail dont on peut distinguer deux types. D'une part les questionnaires centrés sur la perception des conditions de travail qui appréhendent des dimensions comme les exigences liées au travail, la latitude décisionnelle, le soutien social (4) et l'effort et la reconnaissance (5). De nombreuses études ont établi une corrélation entre les conditions de travail perçues par le sujet comme stressantes et la survenue d'une symptomatologie dépressive (6) ou d'un épisode dépressif majeur (7). D'autres part, les échelles dimensionnelles évaluant des symptômes en relation avec le stress (échelle de stress perçu, échelles d'anxiété et de dépression…) qui ne sont pas spécifiques au travail et ont été utilisés dans les études sur des populations d'employés (8,9). Un rapport publié en 2008 à l'initiative du ministère du travail recommandait de construire un indicateur global évaluant simultanément les conditions sociales de travail et l'état psychologique du sujet (10). Malgré ces recommandations faites depuis dix ans aucun indicateur global évaluant à la fois les symptômes psychologiques en lien avec le travail et les conditions sociales du travail n'a été proposé et validé sur le plan psychométrique. Un point qui mérite d'être souligné est que si les questionnaires et échelles de stress au travail mesurent différentes dimensions, ils ont en commun d'apprécier le versant négatif du travail comme les contraintes, le mal être, la souffrance, les conséquences psychiques.

  dépressive trop proche selon les auteurs de la santé psychologique[START_REF] Bietry | Proposition d'une échelle de mesure positive du bien-être au travail (EPBET)[END_REF].Sur la base d'interview ils retrouvent huit principales dimensions du bien-être au travail : « sentiments », « relations aux collègues », « environnement physique de travail », « management », « temps sociaux », « rémunération », « valeurs », « organisation ». Puis en croisant avec les données de la littérature et des analyses factorielles ils finissent par retenir quatre dimensions pour bâtir un instrument : relation aux collègues, ambition d'épanouissement personnel, rapport au temps satisfaisant, environnement physique du travail. Les études évaluant une corrélation entre la dimension bien-être au travail et un faible niveau de symptômes psychologiques sont pratiquement inexistants. A notre connaissance une seule étude à partir d'un échantillon de 205 salariés d'un hôpital vu en médecine du travail montre une corrélation inverse significative entre le niveau de bien-être déterminé par une échelle visuelle analogique et les scores au questionnaire GHQ (coefficient de corrélation =0,898, p≪0,0001) (21). Dans une approche médicale du bien-être au travail, il apparaît nécessaire de prendre en compte à la fois l'absence de symptômes médicaux physiques et psychologiques au travail, les expériences de vie générales (p. ex. : satisfaction dans la vie) et les expériences au travail (p. ex. : satisfaction au travail, attachement au travail).En nous référant à toutes les données vues précédemment, nous avons construit une échelle de bien être au travail dans le but de proposer un indice. Il explore à la fois la perception positive des facteurs organisationnels, l'absence de symptômes psychologiques, la perception positive de l'état de sa santé en lien avec le travail (émotion positive et épanouissement personnel). Il s'agit d'un instrument de bien être spécifique au travail où chaque item se rapporte exclusivement au travail.

3 . 1 .

 31 Afin d'évaluer l'influence des caractéristiques démographiques sur le score total de l'échelle de bien-être, des analyses bivariées ont été employées. Pour les variables quantitatives, des tests de corrélations de Spearman ont été utilisés, pour les variables qualitatives binaires des tests de Mann-Whitney ont été utilisés, et pour les variables qualitatives à plus de deux modalités des tests de Kruskall-Wallis ont été utilisés. Des tests bilatéraux ont été réalisés avec un niveau de significativité de 5 %. Les analyses statistiques ont été effectuées à l'aide du logiciel SAS (SAS Institute version 9.4). Description de la population Un total de 193 patients a été inclus dans l'étude. Parmi les participants, 34,0 % étaient des hommes (n=65) et 66, 0 % des femmes (n=126) ; 81,7 % travaillaient à temps plein. Le score total moyen à l'échelle de bien-être au travail était de 43,1±8,4. Parmi les 193 patients, 38 patients ont participés au test-retest et 47 patients à la validation externe. Les caractéristiques démographiques de la population totale et des deux sous-populations (utilisées pour la validation externe et pour le test-retest) sont décrites dans le tableau 1. Tableau 1. Caractéristiques démographiques des différentes populations d'analyse3.2. Qualité des itemsLa proportion de réponses manquantes par item était faible, avec un maximum de 2 réponses manquantes, soit 0,01 % des réponses, permettant de conclure à une bonne qualité des données. Treize items parmi les 20 questions de l'échelle (65,0 %) n'avaient aucune réponse manquante ; 96,0 % (n=186) des patients avaient répondu à l'ensemble des questions de l'échelle, 6 patients n'avaient pas répondu à un item et un patient n'avait pas répondu à deux items. Aucun effet plateau n'a été observé pour les différents items (échelle de Likert à 4 points).

Fig. 1 .

 1 Fig. 1. Scree plot des valeurs propres de l'ACP avec les 20 items de l'échelle Serenat sur la population totale (n=193)

Fig. 2 .

 2 Fig. 2. Graphique de Bland et Altman représentant la reproductibilité intra-observateur (n=39)

Fig. 1 .Fig. 2 .

 12 Fig. 1. Scree plot des valeurs propres de l'ACP avec les 20 items de l'échelle Serenat sur la population totale (n=193)

  Le questionnaire a été proposé à 50 salariés lors de leur visite médicale périodique dans un service de médecine du travail autonome. Il leur a été demandé de choisir les items qui leur semblaient les plus représentatifs de leur bien-être au travail parmi la liste proposée.Au final, 20 items ont été sélectionnés comme les plus importants par les salariés. Dix items Trois auto-questionnaires d'évaluation des symptômes anxio-dépressifs ont été utilisés. L'échelle « Hospital Anxiety and Depression Scale » (HADS)[START_REF] Zigmond | The hospital anxiety and depression scale[END_REF] est un auto-questionnaire de 14 items, côtés par des échelles de Likert allant de 0 à 3, permettant de dépister des troubles anxieux et dépressifs. Le questionnaire est composé de deux parties permettant l'obtention de deux sous-scores : un sous-score anxiété (HADS-A) et un sous-score dépression (HADS-D). Plus le score est élevé, plus la symptomatologie est sévère. Le questionnaire HADS a été validé dans de nombreuses études en populations médicales non psychiatriques et chez des salariés ou chez des sujets soumis à un stress professionnel[START_REF] Bjelland | The validity of the Hospital Anxiety and Depression Scale: An updated literature review[END_REF]. Nous utilisons la version française du questionnaire qui a été validée à partir d'un échantillon de patients hospitalisés en médecine interne[START_REF] Lepine | Evaluation de l'anxiété et de la dépression chez des patients hospitalisés dans un service de médecine interne[END_REF]. En considérant 10 patients par variable, nous avons inclus dans cette étude 200 sujets. Parmi les 200 patients inclus, un groupe de 50 sujets a été constitué par tirage au sort pour évaluer la reproductibilité intra-observateur (test-retest). Le questionnaire a été administré la première fois lors de la visite programmée pour l'étude et les patients devaient le remplir une nouvelle fois une semaine plus tard. La

	concernaient l'état psychologique au travail : détente (item1), qualité de sommeil (item 2),
	concentration (item3), forme (item 4), sentiment de sécurité (item 5), satisfaction (item 6),
	qualité des relations et contacts au travail (item 7) tension agressivité (item 8), optimisme
	pour l'avenir professionnel (item 9), équilibre vie personnelle (item 10). Les dix autres items
	concernaient les conditions et l'organisation du travail : confort de l'environnement
	professionnel (item 11), contrainte temporelle (item 12), sentiment de compétence (item
	13), latitude décisionnelle (item 14), intérêt et variété du travail (items 15), reconnaissance L'Inventaire d'Anxiété Trait-État adapté du « State-Trait-Anxiety Inventory » STAI-Y
	reproductibilité a été estimée par le coefficient de corrélation intra-classe (ICC). Le nombre
	de sujets nécessaire a été déterminé de la manière suivante. Selon Hernaez (31), un ICC
	inférieur à 0,4 est considéré comme mauvais, entre 0,40 et 0,59 acceptable, entre 0,60 et
	0,75 bon et supérieur à 0,75 excellent. Pour montrer que l'ICC est supérieur à 0,60

40 items a été élaborée par le premier auteur (DS) à partir des termes les plus souvent cités. Ces termes concernaient l'état de santé et le bien-être en lien avec le travail (détente, sommeil, concentration, forme), le milieu professionnel dont notamment le contenu du travail, son organisation, son cadre et ses perspectives (variété des tâches, horaires, confort), la qualité des relations interpersonnelles (supérieur hiérarchique, collègues, clients) et la satisfaction, la confiance, la perspective, la capacité d'action et de décision.

(item 16), soutien professionnel (item 17), communication et transfert d'information (item 18), conflit humain (item 19), horaires de travail satisfaisant (item 20). Chaque item a été noté sur une échelle de Likert à 4 points allant de 0 à 3. Les réponses de chaque item ont été recodées afin qu'une valeur élevée indique une bonne condition de travail. Le score total correspond à la somme des réponses des questions répondues. Le score total varie de 0 à 60, où un score élevé correspond à un bien-être optimal au travail. La moyenne du temps de passation se situe autour de trois minutes.

2.2. Sujets

De janvier 2014 à mai 2017, l'étude a été proposée à tous les patients majeurs, assurés sociaux, en activité professionnelle depuis au moins trois mois, consultants dans des services de médecine du travail (autonome ou inter-entreprises) lors de leur visite périodique, lors d'une visite à la demande du salarié ou du médecin du travail. L'étude a reçu un avis favorable du Comité de protection des personnes (CPP) du CHRU de Lille n° CPP 13/50.

Les patients consentant à participer à l'étude ont tous répondu au questionnaire de bienêtre au travail au moment de l'inclusion dans l'étude. Deux sous-groupes de patients ont été constitués par tirage au sort : un sous-groupe répondant à trois auto-questionnaires d'évaluation des symptômes anxio-dépressifs (HADS, STAI-Y et BDI) au moment de l'inclusion et un sous-groupe répondant une nouvelle fois au questionnaire de bien-être au travail une semaine plus tard par courrier.

Les patients occupant un statut intérimaire ou en arrêt de travail, consultant lors d'une visite de pré-reprise ou de reprise, ne sachant pas lire, incapable de répondre à l'échelle, incapable de consentir et les femmes enceintes n'ont pas été inclus dans l'étude.

2.3. Evaluation des symptômes anxio-dépressifs

(Spielberger, 1993)

est un auto-questionnaire de 20 items, côtés de 1 à 4, évaluant les sentiments d'appréhension, la tension, la nervosité et l'inquiétude que le sujet ressent au moment de la consultation soit dans l'instant (anxiété État forme A) ou l'anxiété ressentie généralement (anxiété Trait forme B)

[START_REF] Spielberger | Manual for the State-Trait Anxiety Inventory (Form Y) Self-evaluation questionnaire[END_REF]

. Plus le score est élevé, plus l'anxiété ressentie au quotidien est forte. Le questionnaire a été traduit et validé en français et nous avons utilisé cette version de la STAI Y-B pour l'étude

[START_REF] Bruchon-Schweitzer | Inventaire d'anxiété état-trait forme Y (STAI-Y) C.D Spielberger[END_REF]

.

L'inventaire abrégé de Dépression de Beck BDI-13

[START_REF] Beck | Assessment of depression: the depression inventory[END_REF]

est une version abrégée de 13 items de l'auto-questionnaire de dépression de Beck

[START_REF] Beck | Psychometric properties of the Beck Depression Inventory: Twenty-five years of evaluation[END_REF]

, permettant une évaluation rapide de la symptomatologie dépressive. Plus le score est élevé, plus le niveau de dépression est haut. Nous avons utilisé la version française validée (29).

2.3. Analyses statistiques 2.3.1. Calcul du nombre de sujets

Selon les recommandations de De Vet

[START_REF] Vet | Measurement in medicine: a practical guide[END_REF]

, il faut disposer d'au moins 4 à 10 sujets par variable, avec un minimum de 100 sujets.

  bien-être au travail est aujourd'hui mieux défini et de nombreux rapports d'expertise Le bien-être au travail est aujourd'hui conceptualisé comme une entité associant des affects positifs au travail qui prévalent sur les affects négatifs et des expériences de développement des potentialités de l'individu et de son accomplissement[START_REF] Warr | How to Think About and Measure Psychological Well-being[END_REF].

	Nous avons choisi cette approche qui reflète selon nous les dimensions explorées par
	l'échelle : l'absence de symptômes physiques et psychiques liés au travail et une perception
	des conditions sociales, d'environnement et d'organisation du travail satisfaisantes pour la
	personne. Cet état de bien-être au travail correspondrait à un état de santé psychologique
	évalué par l'absence d'un niveau significatif des dimensions anxieuses et dépressives.
	L'échelle Serenat pourrait être utile comme instrument de dépistage global, court,
	synthétique, et de passation aisée, afin que son utilisation soit possible par le plus grand
	nombre.
	Remerciements	Les auteurs remercient les médecins de prévention qui ont réalisé les
	inclusions dans cette étude. Les Dr Claude Bailly, Marie-Aude Bizet, Juliette Caudrelier,
	Marie-Hélène Deschepper, Damien Duquesne, Sonia Dymny, Pascale Kopciuch,
	Dominique Fourmaintraux, Alexandra Salembier.
	recommandent le développement d'instruments validés. L'Organisation internationale du
	travail des Nations Unies (ILO) donne comme définition « Le bien-être au travail se rattache
	à tous les aspects de la vie au travail, de la qualité et la sécurité de l'environnement de
	travail, à comment les travailleurs se sentent à propos de leur travail, leur environnement
	de travail, le climat au travail et l'organisation du travail. »
	Un groupe d'experts réuni à l'initiative de l'OMS retient comme proposition que le bien être
	global doit être appréhendé de façon subjective et objective et encourage le développement
	d'instruments évaluant le bien-être dans le domaine de la santé (41).

Dans cette perspective le bien-être au travail intègre à la fois des aspects affectifs (émotion et humeur) et cognitif (perception d'accomplissement) auxquels on peut ajouter la perception des conditions de travail

[START_REF]Well-being at work: creating a positive work environment Literature Review Luxembourg: Publications Office of the European Union[END_REF]

.

  Les valeurs sont exprimées en effectif (pourcentage) et en moyenne±écart-type pour l'âge. Corrélation de chaque item de l'échelle Serenat avec la première composante principale issue de l'ACP avec les 20 items du questionnaire de bien-être au travail sur la population totale (n=193) Corrélation entre le score Serenat et les trois auto-questionnaires (n=47) Influence des caractéristiques démographiques sur le score Serenat (n=193)

	Tableau 1 Tableau 2 Tableau 3 Tableau 4
	Caractéristiques démographiques des différentes populations d'analyse
					Médiane
	Variables qualitatives HADS HADS-D Homme HADS-A Sexe	Population (intervalle pour le test-interquartiles) Coefficient de corrélation N Population -0,54 totale (n=38) -0,59 65 45 (38 à 51) (n=193) retest Première composante Population P-value P-value pour la validité <0,001 (n=47) principale <0,001 externe -0,40 0,005 0,12
	Hommes Q1 Détente et calme STAI-Y Femme		65 (34,0) -0,78 126	10 (26,3) 43 (37 à 48)	18 (38,3) 0,65 <0,001
	Age, années Q2 Souci professionnel et endormissement 40,2±10,1 BDI-13 -0,57 Catégories socio-professionnelles	41,3±10,3	40,5±9,4 0,56 <0,001 0,011
	Travaillant à temps plein Q3 Concentration sur le travail Cadres et professions supérieures	152 (81,7)	26 (72,2)	38 (82,6) 0,57
	Catégories socio-professionnelles Q4 Forme au travail Professions intermédiaires	63	44 (38 à 49)	0,73
	Cadres et professions supérieures Professions intermédiaires Employés Ouvriers Q5 Sentiment de sécurité au travail Employés Q6 Satisfaction du travail accompli Ouvriers Q7 Qualité des contacts et des relations au travail 63 (33,3) 52 (27,5) 69 (36,5) 5 (2,7) 52 69 5 Q9 Optimisme pour l'avenir professionnel Temps plein 152 Q8 Tension et agressivité à mon égard Temps de travail	13 (35,1) 10 (27,0) 14 (37,9) 0 42 (36 à 46) 46 (38 à 50) 47 (46 à 55) 44 (37 à 49)	0,65 15 (31,9) 0,71 15 (31,9) 0,53 16 (34,1) 0,52 1 (2,1) 0,64 0,77
	Q10 Contribution du travail à l'équilibre personnel Temps partiel 32	43 (38 à 48)	0,44
	Q11 Confort de l'environnement du travail Q12 Contraintes temporelles du travail Variables quantitatives	N	Coefficient de corrélation	0,66 0,53 P-value
	Q13 Sentiment de compétence pour le travail Age 189	-0,09	0,24	0,19
	Q14 Latitude décisionnelle Niveau d'étude		189	-0,07	0,56	0,35
	Q15 Intérêt pour le travail et tâches variées			0,51
	Q16 Reconnaissance professionnelle				0,65
	Q17 Soutien au sein du travail				0,51
	Q18 Communication et circulation des informations		0,68
	professionnelles
	Q19 Gestion des difficultés et des conflits professionnels	0,44
	Q20 Horaires permettant de concilier vie personnelle et
	professionnelle
	Pourcentage de variance expliquée				33,3 %

Position du problème Le bien-être au travail est aujourd'hui un enjeu majeur de


Timoh ; K Nyangoh

; Le Lous

; Lavoué

Dr Dion Ludivine
email: ludivine.dion@chu-rennes.fr

Première étape vers la transplantation utérine chez la femme : mise en place d'un modèle animal (avec vidéo) First step to human uterus transplantation : setting-up of an animal model (with video)

Première étape vers la transplantation utérine chez la femme : mise en place d'un modèle animal avec vidéo

First step to human uterus transplantation : setting up of an animal model with video

Introduction

La transplantation utérine (TU) connaît un essor depuis la première naissance après greffe d'utérus issue de l'essai suédois du Pr Brännström(1). On compte à ce jour plus de 50 transplantations utérines dans le monde et 13 naissances vivantes d'enfants en bonne santé(2). Une analyse macroscopique du greffon a été réalisé 30 minutes après la greffe. Un doppler des artères utérines a été réalisé en fin de procédure ainsi qu'un test au vert d'indocyanine.

Une IRM pelvienne avec injection de produit de contraste a été réalisé à J7. L'utérus a été explanté à J10.

Chirurgie : appel le schéma anatomique (Figure 1) et la vidéo.

Résultats

Les différents temps de la technique chirurgicale sont détaillés dans la vidéo.

Nous avons réalisés 6 auto transplantations utérines. Les 6 utérus ont blanchi après perfusion de sérum hépariné à 4°C. Trente minutes après greffe notre 1 er greffon n'était pas vascularisé, concernant les 5 autres greffons, une bonne perfusion a été constaté (recoloration, saignement à la piqûre). Deux truies sont décédées dans les premières 12h (hémopéritoine et détresse respiratoire aiguë). L'hémopéritoine serait dû à un fuite en regard d'une anastomose vasculaire qui se serait démasquée au réveil de l'animal (sutures vasculaires intactes à l'autopsie). Le devenir de chaque animal est détaillé dans le tableau 1.

Le test au vert d'indocyanine a permis d'objectiver la perfusion de notre greffon et de repérer d'éventuelle fuite au niveau des anastomoses. Les acquisitions IRM à J7 ont permis de montrer la possibilité de celle-ci de visualiser ou non la perfusion de l'utérus après injection de gadolinium. Lors de l'explantation à J10, 2 utérus étaient nécrosés et 2 utérus étaient viables. Les temps opératoires sont passés de 8h30 pour le 1 er animal à 6h pour le dernier.

Discussion/Conclusion

Nos résultats montrent que la TU est une procédure complexe même pour une équipe

  De nombreuses équipes s'intéressent au développement cette technique pour traiter l'infertilité utérine absolue. L'une des première étape est de mettre en place un modèle animal. Cette étape animale pour les équipe est nécessaire car elle permet d'augmenter le taux de succès de la transplantation utérine de 56% à 82%(3). De plus l'Agence National de la santé et du médicament (ANSM) indique comme un pré-requis cette étape pour éventuellement délivrer un agrément à une équipe pour la TU. Nous avons mis en place un modèle porcin d'auto-transplantation utérine. Le but de cet article est de montrer les étapes chirurgicales de la TU dans un modèle animal au travers d'une vidéo de technique chirurgicale. De plus nous détaillons les principaux résultats sur le devenir des animaux et les utérus auto-transplantés. Matériel et méthodes L'étude a été réalisée après accord du comité rennais d'éthique en matière d'expérimentation animale et validée par le ministère de l'éducation nationale, de l'enseignement supérieur et de la recherche (2017083120283994-V2-APAFiS #11100). Animaux : Les animaux étaient des truies Yucatan multipares provenant de l'élevage de l'INRA St Gilles (35). Plan expérimental : nous avons réalisés des auto-transplantations utérines chez la truie Yucatan multipares. L'hystérectomie a été réalisée, puis l'utérus a était placé dans un récipient contenant de la glace et perfusé par une solution héparinée à 4°C. Après 60 minutes d'ischémie froide, l'utérus a été transplanté en orthotopique, les anastomoses terminolatérales entre les veines utéro-ovariennes et les veines iliaques externes puis entre les artères utérines et les artères iliaques externes ont été réalisées. Les temps opératoires ont été noté.

  Figure 1

Figure 1 :

 1 Figure 1 : utérus de truie à gauche : schéma anatomique à droite : représentation des anastomoses vasculaires post-greffe UR : urétère, AU : artère utérine ; VU : veine utérine ; AIE : artère iliaque externe ; VIE : veine iliaque externe ; VO : veine ovarienne ; VUO : veine utéro-ovarienne ; VII : veine iliaque interne, AII : artère iliaque interne


Sven F Plappert

Falk W Liebner

Johannes Konnerth

Jean-Marie Nedelec
email: jean-marie.nedelec@sigma-clermont.fr

Anisotropic Nanocellulose Gel-Membranes for Drug Delivery: Tailoring Structure and Interface by Sequential Periodate-Chlorite Oxidation

Keywords: 2, 3-dicarboxyl cellulose, cellulose nanofibers, anisotropic gel membranes, transdermal drug delivery patches, non-steroidal anti-inflammatory drugs, piroxicam

This study investigates periodate-chlorite oxidation as a pretreatment to tailor the surface charge density of cellulose nanofibers employed in open-porous anisotropic hydrogel membranes for transdermal drug delivery. The obtained materials feature high specific surface (≤ 500 m 2 g -1 , BET), small average pore size (ca. 40 nm) and tunable surface charge, which are key properties for adsorption and slow release of charged drug molecules. Loading of the non-steroidal anti-inflammatory drug (NSAID) piroxicam (PRX) into the membranes confirmed that the extent of loading is governed by surface charge density and carboxylate group content, respectively, which can be controlled by the oxidation procedure within the range of 0.74 to 2.00 mmol g -1 . Prolonged release of PRX over several hours was observed upon exposure of the loaded membranes to simulated human skin fluid demonstrating the applicability as drug delivery patches.

INTRODUCTION

Assembly of colloidal particles such as nanocellulose into porous bulk structures can afford materials of high specific interface which can be utilized for applications relying on interfacial chemical and physical processes, such as chemi-and physisorption. In case the interstitial liquid phase filling the interconnected pore system of the colloidal solid is water, respective

2 materials are classified as hydrogels. The latter have demonstrated great potential for biomedical applications including wound dressings [START_REF] Hakkarainen | Nanofibrillar cellulose wound dressing in skin graft donor site treatment[END_REF][START_REF] Leppiniemi | 3D-Printable Bioactivated Nanocellulose-Alginate Hydrogels[END_REF], cartilage implants [START_REF] Ahrem | Laserstructured bacterial nanocellulose hydrogels support ingrowth and differentiation of chondrocytes and show potential as cartilage implants[END_REF], scaffolds for tissue engineering [START_REF] Bonilla | Micromechanical model of biphasic biomaterials with internal adhesion: Application to nanocellulose hydrogel composites[END_REF][START_REF] Deng | Layer-by-layer structured polysaccharides film-coated cellulose nanofibrous mats for cell culture[END_REF][START_REF] Liu | High internal phase emulsions stabilised by supramolecular cellulose nanocrystals and their application as celladhesive macroporous hydrogel monoliths[END_REF][START_REF] Van Vlierberghe | Biopolymer-based hydrogels as scaffolds for tissue engineering applications: a review[END_REF][START_REF] Zaborowska | Microporous bacterial cellulose as a potential scaffold for bone regeneration[END_REF] and drug delivery [START_REF] Abeer | A review of bacterial cellulose-based drug delivery systems: their biochemistry, current approaches and future prospects[END_REF][START_REF] Hujaya | Polyion complex hydrogels from chemically modified cellulose nanofibrils: Structure-function relationship and potential for controlled and pH-responsive release of doxorubicin[END_REF][START_REF] Paukkonen | Nanofibrillar cellulose hydrogels and reconstructed hydrogels as matrices for controlled drug release[END_REF]. Next to structure derived properties like porosity, specific surface and permeability, biocompatibility is a key feature required for these applications. As biocompatibility is inherent to cellulose and its derivatives obtained by oxidative modification [START_REF] Hujaya | Polyion complex hydrogels from chemically modified cellulose nanofibrils: Structure-function relationship and potential for controlled and pH-responsive release of doxorubicin[END_REF][START_REF] Kumar | HNO3/H3PO4-NANO2 mediated oxidation of cellulose -preparation and characterization of bioabsorbable oxidized celluloses in high yields and with different levels of oxidation[END_REF][START_REF] Mou | 2,3-Dialdehyde nanofibrillated cellulose as a potential material for the treatment of MRSA infection[END_REF][START_REF] Syamala Devi | Biosoluble surgical material from 2,3dialdehyde cellulose[END_REF], nanocellulose has been gaining considerable attention as building block of novel functional materials for biomedical applications. In particular cellulose nanofibers (CNF) which are highly anisometric nanoparticles consisting of biosynthetically [START_REF] Fernandes | Nanostructure of cellulose microfibrils in spruce wood[END_REF][START_REF] Sethaphong | Tertiary model of a plant cellulose synthase[END_REF] aligned cellulose molecules are interesting building blocks in this respect. Next to nanocellulose hydrogels also dried materials such as foams [START_REF] Löbmann | Cellulose Nanopaper and Nanofoam for Patient-Tailored Drug Delivery[END_REF][START_REF] Svagan | Solid cellulose nanofiber based foams -Towards facile design of sustained drug delivery systems[END_REF] and aerogels [START_REF] Li | Nanoformulations of quercetin and cellulose nanofibers as healthcare supplements with sustained antioxidant activity[END_REF][START_REF] Zhao | Polyethylenimine-grafted cellulose nanofibril aerogels as versatile vehicles for drug delivery[END_REF] have been proposed for drug delivery. The latter however, require rewetting to trigger the drug release which, in turn, necessitate sufficient dimensional stability to resist capillary forces, but bear the advantage of an easier storage.

CNF can be obtained from virtually any natural cellulose source by mechanical delamination.

The required energy input for such processes can be significantly reduced by introduction of repulsive electrostatic forces which can be accomplished by (e.g. oxidative) chemical surface modifications. To this end sequential periodate-chlorite oxidation (H. [START_REF] Liimatainen | Enhancement of the nanofibrillation of wood cellulose through sequential periodate-chlorite oxidation[END_REF][START_REF] Tejado | Energy requirements for the disintegration of cellulose fibers into cellulose nanofibers[END_REF] has recently emerged as efficient means of introducing surface charges onto cellulose pulp in form of carboxylate moieties. While (meta)periodate selectively and simultaneously converts the two vicinal secondary hydroxyl groups of the anhydroglucose units (AGU) constituting cellulose into aldehydes, chlorite ions further oxidize the latter to carboxyl moieties. The utilized periodate can be easily regenerated by an facile ozone [START_REF] Koprivica | Regeneration of Aqueous Periodate Solutions by Ozone Treatment: A Sustainable Approach for Dialdehyde Cellulose Production[END_REF] or hypochlorite treatment (Henrikki Liimatainen, Sirviö, Pajari, Hormi, & Niinimäki, 2013). Mechanical delamination -aided by electrostatic forces of the oxidized pulp cellulose -can yield fully individualized nanofibers, the latter featuring a very low polydispersity with regard to diameter. These CNF are well dispersible in aqueous media and the obtained dispersions are highly stabilized by electrostatic forces arising from the abundant surface charges. Compared to the frequently applied (2,2,6,6-tetramethylpiperidin-1-yl)oxyl-mediated (TEMPO) oxidation (A. [START_REF] Isogai | TEMPO-oxidized cellulose nanofibers[END_REF] which also promotes mechanical delamination of cellulose, sequential periodate-chlorite oxidation introduces two carboxyl moieties per oxidized AGU (C2, C3 position) instead of one in C6 position, enabling the introduction of higher surface charge densities.

Self-assembly of individualized cellulose nanofibers into gel and membrane structures can engender highly interfacial anisotropic materials featuring unique properties. Respective CNF membranes have been thus recently tested as templates for transparent, flexible devices (Matti S. [START_REF] Toivonen | Ambient-Dried Cellulose Nanofibril Aerogel Membranes with High Tensile Strength and Their Use for Aerosol Collection and Templates for Transparent, Flexible Devices[END_REF], light diffuser coatings (M. S. [START_REF] Toivonen | Anomalous-Diffusion-Assisted Brightness in White Cellulose Nanofibril Membranes[END_REF] and filter devices for aqueous ultra-(M. [START_REF] Visanko | Porous thin film barrier layers from 2,3-dicarboxylic acid cellulose nanofibrils for membrane structures[END_REF]Miikka Visanko, Liimatainen, Sirviö, & Hormi, 2015) and gaseous aerosol- [START_REF] Nemoto | Simple Freeze-Drying Procedure for Producing Nanocellulose Aerogel-Containing, High-Performance Air Filters[END_REF]Matti S. Toivonen et al., 2015) filtration. In case the cellulose nanofiber gel-membranes -obtained by slow vacuum filtration assembly -are oven dried to afford dense layers they are referred to as nanopapers [START_REF] Benítez | Cellulose nanofibril nanopapers and bioinspired nanocomposites: a review to understand the mechanical property space[END_REF].

In this study we investigate the assembly of nanofibrillated 2,3-dicarboxyl cellulose (nf-DCC) or simply "CNF" into anisotropic layered membranes for transdermal drug delivery systems utilizing their high internal surface and tunable content of surface charges for drug loading. In particular loading and release of the potent nonsteroidal anti-inflammatory drug (NSAID) piroxicam (PRX) was investigated. PRX has both analgesic and antipyretic activity and is frequently applied for treatment of acute and chronic rheumatoid arthritis or osteoarthritis [START_REF] Rafiee-Tehrani | In vitro release studies of piroxicam from oil-in-water creams and hydroalcoholic gel topical formulations[END_REF]. However as gastrointestinal disorders [START_REF] Laporte | Upper Gastrointestinal Bleeding Associated with the Use of NSAIDs[END_REF]) may occur upon oral administration, transdermal administration [START_REF] Rafiee-Tehrani | In vitro release studies of piroxicam from oil-in-water creams and hydroalcoholic gel topical formulations[END_REF] is commonly recommended. PRX exhibits poor solubility in many solvents including water potentially constituting an obstacle for its applications.

Adsorptive loading of considerable quantities of the drug onto CNF membranes of high PRX affinity and large specific surface is demonstrated to render respective materials attractive as a transdermal drug delivery patch since it overcomes the above obstacle.

MATERIALS AND METHODS

Materials

Dissolving-grade hardwood pulp (TCF bleached, CCOA [START_REF] Potthast | A novel method for the determination of carbonyl groups in cellulosics by fluorescence labeling. 3. Monitoring oxidative processes[END_REF] 24.3 µmol g -1 C=O, FDAM [START_REF] Bohrn | The FDAM method: determination of carboxyl profiles in cellulosic materials by combining group-selective fluorescence labeling with GPC[END_REF] 13.9 µmol g -1 COOH, Mw 303.7 kg mol -1 , 50 %w) obtained from an industrial magnesium bisulfite process was utilized as starting material. Sodium (meta)periodate, acetic acid, sodium chlorite and sodium hydroxide were purchased (Sigma-Aldrich, Vienna, Austria) at the highest purity grade available and used without further purification. Also, piroxicam (PRX) with a purity of ≥98 % (TLC) was purchased from Sigma-Aldrich and used without further purification. 0.1 M sodium acetate buffer was prepared by adding 0.1 M acetic acid to a 0.1 M sodium acetate solution until a pH of 5 was reached. Hydrophilic polyvinylidene difluoride (PVDF) membranes (Durapore®, DVPP, Ø 47 mm, H 125 μm, 650 nm pore size) were purchased from Merck KGaA.

Sequential regioselective oxidations

Various oxidative pretreatments based on sequential periodate and chlorite oxidations were investigated as means of pulp modification prior to mechanical delamination of pulp fiber cellulose. The regioselective sequential oxidation (H. [START_REF] Liimatainen | Enhancement of the nanofibrillation of wood cellulose through sequential periodate-chlorite oxidation[END_REF][START_REF] Tejado | Energy requirements for the disintegration of cellulose fibers into cellulose nanofibers[END_REF] is a facile tool for introducing charged moieties (specifically carboxylates) into the pulp materials facilitating delamination and liberation of cellulose nanofibrils.

In brief never-dried dissolving-grade pulp was disintegrated in deionized water using a blender and NaIO4 was added to the pulp suspension to start the first oxidative modification.

1.2 L of 1 wt% pulp suspension was oxidized under constant stirring (600 rpm) using 46 mmol of NaIO4 at 50° C. While the reagent concentration was the same for all variants, reaction time was varied between 2, 3.75 and 4.5 hours to obtain three different degrees of oxidative modification. The reactions were stopped by filtration and washing with deionized water.

The obtained 2,3-dialdehyde cellulose (DAC) samples with different degrees of oxidation were immediately oxidized in a second step by NaClO2 to yield 2,3-dicarboxyl cellulose. For the second oxidative step the entire obtained amount of DAC was suspended in 1 M acetic acid and 12 g of NaClO2 was added and left to react for 40 h under constant agitation. The reaction was stopped again by filtration and extensive washing with deionized water until the electric conductivity dropped below 25 μS cm -1 . Finally, the concentration of the suspensions was adjusted to 5 g L -1 and pH was set to 8 by addition of 0.1 M NaOH to ensure a sufficient amount of Na + counter ions to saturate the introduced carboxylate moieties.

Mechanical delamination

Suspensions of the oxidatively modified pulp were subjected to repeated highpressure homogenization until dispersions of individualized cellulose nanoparticles were obtained. In brief, the pulp suspensions (5 g L -1 , pH 8) were passed two consecutive times through the homogenizer (APV 1000, SPX Flow Technology, USA) at 60 MPa. To ensure complete separation of nanofibers the dispersions were then diluted to 2.5 g L -1 and homogenized again by two passes at 80 MPa. Finally, the obtained dispersions were filtered through a POR 3 glass frit to remove any macroscopic residues. Rotoevaporation was employed to concentrate the dispersion to the desired concentration of 5 g L -1 .

Atomic force microscopy (AFM)

Obtained dispersions were diluted to 5 mg L -1 and a drop was dried onto mica discs at 105 °C. AFM scans were taken in tapping mode on a Dimension Icon Scanning Probe Microscope (Bruker AXS, France; formerly Veeco) equipped with OTESPA cantilever and Nano-Scope V control station. Average dimensions (diameter, length) of CNF were calculated from the size of 30 randomly selected particles per sample.

Determination of carboxyl content

Automated conductometric titration (800 Dosino dosing device connected to an 856 conductivity module both from Metrohm, Switzerland) of the nanofibers was applied to determine the amount of carboxyl moieties on the nanofibers. Nanofiber dispersions (~ 2.5 g L - 1

) were freeze dried at -80° C and 100 mg of dry nanofibers were redispersed in 50 mL deionized water. The dispersions were acidified prior to titration by addition of 2.75 mL of 0.1 M hydrochloric acid. After acid-induced protonation, conductometric titration was accomplished by addition of 25 µL increments of 0.1 M NaOH every 30 seconds. After a total volume of 6 mL had been added, back-titration was conducted by step-wise addition of the same amount of 0.1M HCl (25 µL every 30 seconds). The obtained conductometric titration curves were evaluated by linear fitting and the carboxyl content was calculated as average from both the NaOH and HCl titration curves.

Electrophoretic mobility measurements

The electrophoretic mobility of nanofibers was determined at 25° C by an electrophoretic light scattering instrument (Zetasizer ZS, Malvern Instruments Ltd., U.K.). Dilute nanofiber dispersions were dialyzed against deionized water over two weeks with regular exchange of water to remove excess of sodium ions and other low molecular products potentially present in the dispersions after mechanical delamination. Dialyzed nanofiber dispersions diluted to 500 mg L -1 with various NaCl salt concentrations in the range of 2.5 to 100 mM were prepared and measured. The pH was determined with an InLab ® Expert Pro pH electrode connected to a seveneasy pH meter (both Mettler Toledo, USA) and adjusted to 6.25 by addition of small quantities of 0.1 M NaOH. All measurements were conducted within hours after the samples had been prepared. The zeta potential was calculated from the electrophoretic mobility according to the Smoluchowski and Ohshima-Henry [START_REF] Ohshima | Surface Charge Density/Surface Potential Relationship for a Cylindrical Particle in an Electrolyte Solution[END_REF][START_REF] Sato | Charging and Aggregation Behavior of Cellulose Nanofibers in Aqueous Solution[END_REF] equations.

Dynamic Light Scattering (DLS)

The size of nanofibers was also evaluated by DLS using a Zetasizer ZS (Malvern Instruments Ltd., Worcestershire, U.K.). Since an accurate measurement of nanofiber length by DLS is difficult, only a comparative estimation was conducted. Dialyzed nanofiber dispersion (500 mg L -1 ) set to pH 6.25 and 25 mM NaCl were measured as triplets with ten acquisitions each. Additionally, DLS was used to monitor potential aggregation of nanofibers in dispersion state.

Membrane preparation

Membranes were formed by vacuum-assisted sedimentation of concentrated aqueous nanofiber dispersions (5 g L -1 , not dialyzed) using vacuum filtration equipment and hydrophilic PVDF 650 nm filter discs supported on a Pore 3 glass frit. A TRIVAC-E2 two stage oil sealed rotary vane pump was employed to generate reduced pressure of about 10 mbar.

After one hour of filtration, excess nanofiber dispersion was poured off and the formed nanofiber membranes together with the PVDF filter were submerged in ethanol for another hour to allow homogeneous mixing with entrapped water. Then the formed nanofiber membrane was carefully peeled off from the PVDF filter support. Sodium counter ions were replaced by H + ions by placing the membranes for 1 hour into a mixture of 25 %v aqueous 1M HCl and 75 %v ethanol. Thereafter four solvent exchanges steps were conducted aiming to replace the remaining acidic water quantitatively by ethanol using a total ethanol volume equaling 100 times the volume of the membranes.

Field emission electron microscopy of membranes (FEG-SEM)

The morphology of the layered nanofiber membranes was studied by FEG-SEM. Dry membrane samples were prepared by extracting the obtained alcogels submerged in ethanol with supercritical CO2 aiming to avoid any capillary forces that may arise during drying and to preserve the porous membrane structure to full extent. Extraction was performed at 40° C and 9.5 MPa, maintaining a flow rate of 40 g min -1 CO2 for 4 hours. The dry samples were gold sputtered with an Edwards S150B and imaged on a Zeiss Supra 55VP (Oberkochen,

Germany) using a 0.5-2 kV beam.

Nitrogen sorption of membranes

Pore size distribution and specific surface area (SSA) of the membranes were assessed by nitrogen sorption experiments at 77K using supercritically dried (cf. above) samples. Prior to nitrogen sorption all samples were degassed at 50 °C for 20 hours (VacPrep 061, Micromeritics). Nitrogen adsorption and desorption isotherms of the degassed samples were recorded at 77 K on a TriStar II PLUS (Micromeritics, Merignac, France). The BET specific surface area was calculated from data points in the range of p/p 0 = 0.05-0.2 (ISO, 2010) of the adsorption branch using the Brunauer-Emmett-Teller (BET) equation [START_REF] Brunauer | Adsorption of Gases in Multimolecular Layers[END_REF]. The pore size distributions of the studied membranes were derived using the Barrett-Joyner-Halenda (BJH) method [START_REF] Barrett | The Determination of Pore Volume and Area Distributions in Porous Substances. I. Computations from Nitrogen Isotherms[END_REF] and applying the Broekhoff-de Boer model [START_REF] Broekhoff | Studies on pore systems in catalysts XIII. Pore distributions from the desorption branch of a nitrogen sorption isotherm in the case of cylindrical pores B. Applications[END_REF]) on the desorption branch.

Piroxicam loading and release from membranes

Aqueous loading and release of the nonsteroidal anti-inflammatory drug PRX from the various membranes was investigated by UV-Vis spectroscopy using a UV-2600 instrument (Shimadzu, Noisiel, France) quantifying the decrease / increase of PRX in respective aqueous media (see below). Linear PRX calibration curves in deionized water and 0.1 M acetate buffer (pH 5) were recorded covering the concentration range of 0.2 to 2.5 mg L -1 and using the respective absorption maxima at 353 nm and 360 nm.

Prior to drug loading the membranes were placed for 24 hours in excess of deionized water corresponding to 100 times the volume of that of the membranes. The latter were then transferred to a loading bath (400 mL, 25° C) containing 20 mg L -1 PRX. Drug loading was monitored throughout the entire residence time of 24 hours by quantifying the decrease of PRX content in the loading bath as effectuated by drug uptake and adsorption onto the nanofiber membranes.

The release of PRX from the nanofiber membranes was accomplished by placing the membranes into 400 mL sodium acetate buffer (0.1 M, pH 5) and quantifying the increase of PRX content in the aqueous phase.

RESULTS AND DISCUSSION

Sequential oxidative modification and mechanical delamination of the used dissolving pulp afforded entirely individualized cellulose nanofibers (cf. Figure 1) which can be referred to as nanofibrillated 2,3-dicarboxyl cellulose (nf-DCC) or simply "CNF" to emphasize their similarity to (charged) cellulose nanofibrils obtained by other approaches. Aiming to investigate membrane formation and PRX loading / release for samples of different charge density and nanomorphology, periodate was conducted for different time periods, i.e. 2, 3.75 and 4.5 hours (samples nf-DCC 1, nf-DCC 2 and nf-DCC 3). Conductometric titration confirmed that the duration of periodate oxidation is positively correlated with the amount of introduced carboxyl groups. Correspondingly, the degree of oxidation related to modified anhydroglucose units (AGU) increased in the order nf-DCC 1 (6.4%) < nf-DCC 2 (14.1%) < nf-DCC 3 (16.2%).

The introduced surface charges not only stabilize the nanofiber dispersion, by means of electrostatic repulsion [START_REF] Fall | Colloidal stability of aqueous nanofibrillated cellulose dispersions[END_REF], but also facilitate mechanical delamination of the oxidized pulp. This reduces the energy input required to separate and individualize the cellulose nanofibrils considerably (Akira Isogai & Bergström, 2018; A. [START_REF] Isogai | TEMPO-oxidized cellulose nanofibers[END_REF][START_REF] Tejado | Energy requirements for the disintegration of cellulose fibers into cellulose nanofibers[END_REF]. Both length and aspect ratio (a) of the nanofibers decreased with the degree of oxidation while the nanofiber diameter remained largely constant (cf. Figure 2). The particle size obtained by dynamic light scattering experiments, confirm the decreases of nanofiber length with increasing degree of oxidation (cf. Table 1). Yet the difference is less pronounced as suggested by AFM data. Here η denotes the viscosity while εr and ε0 correlate to the relative permittivity and permittivity of free space respectively. Although the Smoluchowski equation is also frequently applied for nanocellulose characterization [START_REF] Foster | Current characterization methods for cellulose nanomaterials[END_REF][START_REF] Safari | Electroacoustic characterization of conventional and electrosterically stabilized nanocrystalline celluloses[END_REF],

it can underestimate the zeta potential of cellulose nanofibers at lower salt concentrations [START_REF] Sato | Charging and Aggregation Behavior of Cellulose Nanofibers in Aqueous Solution[END_REF]. The small diameters of individualized and well-dispersed cellulose nano-fibers including the presented nf-DCC is in the range of their surrounding electric double layer at lower salt concentration which requires to account for the curvature of the nanofiber.

While the Smoluchowski equation is applicable for nanofibers oriented parallel to the electric field, nanofibers in perpendicular orientation is only accurate under consideration of their surface curvature. The electrophoretic mobility of cylinders oriented randomly within an electric field, as we assume for our dilute dispersions, is (De Keizer, [START_REF] De Keizer | Electrophoresis of randomly oriented cylindrical particles[END_REF]:

‖ 2 3
With μ‖ and μ⊥ representing the electrophoretic mobility parallel and orthogonal to the applied electric field respectively. Under consideration of the curvature of cylindrical particles oriented randomly within the electric field the zeta potential can be derived by the Ohshima-Henry [START_REF] Ohshima | Surface Charge Density/Surface Potential Relationship for a Cylindrical Particle in an Electrolyte Solution[END_REF][START_REF] Sato | Charging and Aggregation Behavior of Cellulose Nanofibers in Aqueous Solution[END_REF] equation:

3 2 1 . ! "# $ ! %
When keeping the pH constant (at pH 6.25) the zeta potential decreases with increasing electrolyte concentration (cf. Figure 2 d, e and f). This is in accordance with previous studies on carboxylated CNF [START_REF] Fall | Colloidal stability of aqueous nanofibrillated cellulose dispersions[END_REF][START_REF] Sato | Charging and Aggregation Behavior of Cellulose Nanofibers in Aqueous Solution[END_REF][START_REF] Wagberg | The build-up of polyelectrolyte multilayers of microfibrillated cellulose and cationic polyelectrolytes[END_REF] and cellulose nanocrystals carrying sulfate ester moieties [START_REF] Prathapan | Modulating the zeta potential of cellulose nanocrystals using salts and surfactants[END_REF].

While ζ S and ζ OH are in relative good agreement for higher salt concentrations (>50 mM), ζ OH is considerably higher for lower electrolyte concentrations (<50 mM). While the higher charged nanofiber samples nf-DCC 2 and 3 retain their colloidal stability in the studied range up to a 100 mM NaCl concentration, the less charged sample nf-DCC 1 reaches the limit of its stability at this salt concentration.

Considering their aspect ratio, the highly charged shorter nanofibers (nf-DCC 2 and 3) could also be classified as cellulose nanocrystals obtained by an oxidative mechanical approach rather than conventional acid hydrolysis. [START_REF] Zhou | Acid-Free Preparation of Cellulose Nanocrystals by TEMPO Oxidation and Subsequent Cavitation[END_REF] However the high amount of surface charges clearly differentiates them from cellulose nanocrystals [START_REF] Habibi | Cellulose nanocrystals: chemistry, self-assembly, and applications[END_REF] obtained by acid hydrolysis with zeta potentials of about -30 mV. The amount of surface carboxyl groups on the highly oxidized nanofibrils of the nf-DCC 3 sample also exceeds that of previously reported nf-DCC [START_REF] Hujaya | Polyion complex hydrogels from chemically modified cellulose nanofibrils: Structure-function relationship and potential for controlled and pH-responsive release of doxorubicin[END_REF][START_REF] Liimatainen | Enhancement of the nanofibrillation of wood cellulose through sequential periodate-chlorite oxidation[END_REF][START_REF] Plappert | Strain Hardening and Pore Size Harmonization by Uniaxial Densification: A Facile Approach toward Superinsulating Aerogels from Nematic Nanofibrillated 2,3-Dicarboxyl Cellulose[END_REF][START_REF] Visanko | Porous thin film barrier layers from 2,3-dicarboxylic acid cellulose nanofibrils for membrane structures[END_REF]. Compared to TEMPO-mediated oxidation (A. [START_REF] Isogai | TEMPO-oxidized cellulose nanofibers[END_REF] which is frequently applied for the preparation of CNF, sequential periodate-chlorite oxidation can be regarded superior for the introduction of higher amounts of surface charges. TEMPO oxidation relying on the electron transfer capabilities of a stable nitroxyl radical selectively converts the primary hydroxyl groups in C6 position of the AGU into carboxyl(ate) moieties (A. [START_REF] Isogai | TEMPO-oxidized cellulose nanofibers[END_REF].

Provided quantitative turnover, TEMPO-mediated oxidation maximally yields one carboxyl moiety per oxidized AGU. Sequential periodate-chlorite oxidation on the other hand simultaneously introduces two carboxyl moieties per oxidized AGU. On crystalline cellulose I surfaces TEMPO-mediated oxidation can only oxidize every second AGU due to the 180° rotation of every second AGU within the cellulose chain [START_REF] Montanari | Topochemistry of Carboxylated Cellulose Nanocrystals Resulting from TEMPO-Mediated Oxidation[END_REF][START_REF] Shinoda | Relationship between length and degree of polymerization of TEMPO-oxidized cellulose nanofibrils[END_REF]. For these limitations, zeta potential does not exceed -75 mV, hence limiting colloidal stability for CNF obtained by TEMPO oxidation regardless of the type of cellulose I substrate used [START_REF] Okita | Entire surface oxidation of various cellulose microfibrils by TEMPO-mediated oxidation[END_REF].

Highly anisometric colloids such as the well-dispersed CNF of this study can exhibit decreased degrees of (translational and rotational) freedom in dispersions of low solid content and volume fraction, respectively [START_REF] Mendoza | Gelation mechanism of cellulose nanofibre gels: A colloids and interfacial perspective[END_REF][START_REF] Nordenstrom | Formation of Colloidal Nanocellulose Glasses and Gels[END_REF][START_REF] Solomon | Microstructural regimes of colloidal rod suspensions, gels, and glasses[END_REF]. The solid content of the 0.5 wt% dispersions utilized for membrane formation corresponds to a 0.3 % volume fraction (φ). At this semi-dilute state (a -2 < φ < a -1 ) the nanofiber's translational freedom is already restricted whereas rotation around and vibration along the axis is not affected. Therefore, respective dispersions flow easily even though the nanofibers are entangled to some extent. Filtration of semi-dilute nanofiber dispersions therefore yields entangled layered gel structures. Submerging the formed anisotropic gels in the cellulose non-solvent ethanol leads to a strengthening of the structure due to the removal of tightly bound surface water as well-known from cellulose hornification. Moreover protonation of carboxyl moieties on the nanofiber surface as accomplished by acidification diminishes electrostatic repulsion [START_REF] Fall | Colloidal stability of aqueous nanofibrillated cellulose dispersions[END_REF][START_REF] Mendoza | Gelation mechanism of cellulose nanofibre gels: A colloids and interfacial perspective[END_REF] and enables further inter-particulate hydrogen bonding and van der waals interactions. Exchange of sodium ions by protons (H + ions) is also paramount for electrostatic adsorption of charged drug molecules.

The final membranes had a thickness of about 1 mm and a largely consistent nf-DCC content of about 22 mg cm -3 corresponding to a volume fraction of about 1.4 % compared while the employed CNF dispersion had a volume fraction of 0.3 %. Figure 3b) and reveal their interconnected nanoporosity constituted of voids not larger than 100 nm (cf. Figure 3 c,d). Nitrogen sorption isotherms of scCO2-dried nf-DCC membranes as recorded at 77 K correspond in shape the IUPAC type IV characteristic for mesoporous materials [START_REF] Thommes | Physisorption of gases, with special reference to the evaluation of surface area and pore size distribution (IUPAC Technical Report)[END_REF]. The pore size distribution calculated according to the BJH method was for all membranes centered around a maximum at about 40 nm. However, membranes composed of the longer nf-DCC 1 fibrils feature a considerably larger amount of mesopores compared to the other two membranes which are composed of shorter nanofibers (nf-DCC 2 and 3). On the other hand, the latter had also a considerable amount of small mesopores (< 10 nm). As suggested by BET calculations, all samples featured high specific surface areas ranging from 350 to 450 m 2 g -1 for the membranes composed of shorter nanofibers (nf-DCC 2 and 3) up to 500 m 2 g -1 for the membranes composed of the longer nanofibers (nf-DCC 1). The specific surface of the prepared membranes is in good agreement with that of previously reported aerogels [START_REF] Plappert | Strain Hardening and Pore Size Harmonization by Uniaxial Densification: A Facile Approach toward Superinsulating Aerogels from Nematic Nanofibrillated 2,3-Dicarboxyl Cellulose[END_REF] which were obtained by acid-induced gelation of nematically structured nf-DCC dispersions followed by scCO2 drying. The BET constant CBET [START_REF] Sing | The use of nitrogen adsorption for the characterisation of porous materials[END_REF] indicating the affinity of N2 for the surface decreased with increasing amount of surface carboxyl moieties. This could be an indication for adsorbed water [START_REF] Orsolini | Characterization of Pores in Dense Nanopapers and Nanofibrillated Cellulose Membranes: A Critical Assessment of Established Methods[END_REF] which remains on the membrane surface as hydrophilicity increases with the amount of introduced carboxyl moieties and could interfere with nitrogen adsorption. Upon placing the membranes in water prior to drug loading the membranes soften, become more elastic and swell again. When the membrane is immersed in the loading bath containing piroxicam (PRX), the zwitterionic drug infuses the highly porous nanofiber scaffold and adsorbs onto its large internal surface. Depending on the solvent environment PRX can form various tautomers including a zwitterionic structure in aqueous environments [START_REF] Gil | Femtosecond dynamics of piroxicam structures in solutions[END_REF][START_REF] Ivanova | Tautomeric transformations of piroxicam in solution: a combined experimental and theoretical study[END_REF]. Due to the comparatively low pKa value of its pyridyl nitrogen moiety, a cationic PRX tautomer is prevalent in acidic aqueous environments [START_REF] Gil | Femtosecond dynamics of piroxicam structures in solutions[END_REF]. This has been spectroscopically verified by the slight red shift (353 → 360 nm) of its adsorption maximum [START_REF] Gil | Femtosecond dynamics of piroxicam structures in solutions[END_REF] and was confirmed for a PRX solution in 0.1 M acetic acid buffer at pH 5 (cf. supporting information S2).

The high specific surface of CNF based materials has great potential with regard to adsorption of poorly water-soluble drugs [START_REF] Lobmann | Cellulose nanofibers as excipient for the delivery of poorly soluble drugs[END_REF]. This has been previously demonstrated for the sulfonamide derivative furosemide, a drug that is frequently used for treatment of hypertension and edema. It has been shown that adsorption is here governed by specific surface rather than by surface charge which has been explained by the specific mode of adsorption relating to deposition of the planar aryl moieties onto hydrophobic domains of cellulose and the resulting gain in entropy driven by release of surface bound water [START_REF] Lombardo | Toward Improved Understanding of the Interactions between Poorly Soluble Drugs and Cellulose Nanofibers[END_REF]. Different from furosemide, a positive correlation between surface charge density and amount of adsorbed drug was found for piroxicam (cf. Figure 5) when loaded in zwitterionic form at near neutral pH. This suggests electrostatic adsorption of the drug molecule at the nf-DCC interfaces which still requires a high specific surface area in addition to an adequate surface charge density. Considering a zwitterionic structure prevailing for PRX at loading conditions and the pKa range of the carboxyl groups residing at the interface of the nf-DCC membranes (pKa 3.66-4.76) [START_REF] Kim | Ion-exchange chromatography by dicarboxyl cellulose gel[END_REF], it is safe to assume that PRX will adsorb as cation and establish an electric double layer around the nf-DCC surface. PRX loading curves (Figure 5a-c) reveal that the equilibrium adsorption state had been reached for all membrane samples after 24 hours of residence time in the loading bath. The adsorbed amounts of PRX ranged from 30 to 60 mg per g of nf-DCC, depending on surface charge of the respective nf-DCC interfaces. Considering the nf-DCC density of the prepared membranes (22 mg cm -3 ), a maximum loading of 1.32 mg cm -3 could be achieved which exceeds the loading bath concentration (0.02 mg cm -3 ) by a factor of 66.

The release of PRX from the nf-DCC membranes was investigated using 0.1 M acetate buffer (pH 5) as these conditions resemble the slightly acidic nature and electrolyte content of human skin [START_REF] Draize | The Determination of the pH of the Skin of Man and Common Laboratory Animals[END_REF]. Release of the drug is triggered by the electrolyte infusing the membranes and gradually weakening electrostatic forces between the cationic drug molecules and the anionic membrane surfaces mainly by electrostatic shielding and cation exchange. The PRX release could be triggered by the induced pH change as well, however, this was not further investigated since it was not considered relevant for transdermal drug delivery but could be meaningful for oral drug administration.

All tested membranes released piroxicam at somewhat slower rate compared to its loading from near-neutral aqueous phase. Both uptake and release are characterized by high initial adsorption and desorption rates that increasingly abate with time. Compared to previously reported CNF nanopapers [START_REF] Löbmann | Cellulose Nanopaper and Nanofoam for Patient-Tailored Drug Delivery[END_REF] the release is the drug molecules is significantly increased. During preparation of such Nanopapers or films the porous structure and surface of the CNF is typically lost. On the other hand, the here described wet drug loading and release of CNF membranes utilizes the anisotropic porous gel structure feature high specific surface areas (cf. BET) for drug molecule adsorption and release. This enables to exploit the full potential of CNF as high surface area nanoparticles and demonstrates their applicability for aqueous sorption processes. Therefore the comparatively long equilibration times for both the loading and unloading process can be attributed at least in part to the high surface area mesoporous structure of the membranes which impedes diffusion of the drug molecules due to long, windy and tortuous narrow pathways through the nf-DCC scaffolds.

Due to the layered alignment of the nanofibers, diffusion orthogonal to the membrane planewhich corresponds to the mode of drug delivery for an application as transdermal patchesis stronger retarded than in plane. Compared to the aerogels materials [START_REF] Ulker | An emerging platform for drug delivery: aerogel based systems[END_REF][START_REF] Zhao | Polyethylenimine-grafted cellulose nanofibril aerogels as versatile vehicles for drug delivery[END_REF] the need for a employing supercritical drying of freeze drying technology is completely avoided by utilizing the gel membranes in the wet state while similar release times and profiles were observed.

CONCLUSION

Sequential periodate-chlorite oxidation of pulp cellulose prior to mechanical delaminationemployed to obtain well isolated cellulose nanofibrils -has been demonstrated to be an efficient measure to tune the surface charge of the nanoparticles in terms of carboxylate group content within the range of 0.74 to 2.00 mmol g -1 . While the completely individualized nanofibers all share similar diameters of about 2.5 nm, both length and aspect ratio decreased with increasing degree of oxidation. For the longest oxidation periods tested, the average length of the nanofibers dropped from about 375 to 95 nm (AFM) compared to short-term oxidation. This trend was confirmed by DLS.

The variation in DO allows for tuning the range of surface charge density as evident from electrophoretic mobility of respective dispersions that was determined for various electrolyte concentrations (2.5 to 100 mM NaCl). The Zeta potential of 0.05% (w/v) nf-DCC dispersions in aqueous 2.5 mM sodium chloride as calculated using the Smoluchowski equation varied from -44.5 to -87.2 mV and confirms that.increasing electrolyte concentrations entails a respective decrease of zeta potential. FEG-SEM images of the anisotropic membranes and nitrogen sorption experiments revealed a relatively narrow pore size distribution peaking at about 40 nm. Specific surface as determined by nitrogen sorption at 77K using the BET approach ranged from 350 to 500 m 2 g -1 with membranes composed of longer nanofibers featuring higher SSA values and higher nanopore volumes than membranes made from shorter nanofibers. Loading of wet membranes with the nonsteroidal anti-inflammatory drug piroxicam suggests that adsorption of the drug at near neutral pH is driven by electrostatic attraction between the cationic drug and the anionic internal surface of the nf-DCC membranes. This is evident from the extent of drug loading (30-60 mg g -1 ) which increased with surface charge density. Release of the drug into acetate buffer resembling human skin conditions was found to require longer time periods compared to the loading process. This confirms the applicability of the nanofiber membranes as patches for transdermal drug delivery. Furthermore, the anisotropic layered nanoporous structure of the membranes holds great potential for prolonged drug release orthogonal to the plane of the membrane.

Figure 1 :

 1 Figure 1: AFM images of nf-DCC samples differing by carboxyl group content (0.78 mmol g -1 COOH [a], 1.74 mmol g -1 COOH [b], 2.00 mmol g -1 COOH [c]).Increasing contents of surface carboxyl(ate) moieties were shown to enhance also the electrophoretic mobility, since the nanofiber diameter and hence surface to volume ratio remains largely constant. Zeta potential (ζ) is the electrical potential occurring at the phase boundaries of moving nanoparticles in dispersion state capable of exerting a force on other charged particles. It is commonly calculated from the electrophoretic mobility (μ) is the Smoluchowski equation:

Figure 2 :

 2 Figure 2: Conductive titration curves (a, b and c) of nf-DCC (100 mg, protonated form) dispersions and electrophoretic mobility (d, e and f) of corresponding dialyzed nf-DCC samples at different salt (NaCl) concentrations. Zeta potentials were calculated using the Smoluchowski and Ohshima-Henry equations (Ohshima, 1998; Sato et al., 2017), respectively.

Figure 3 :

 3 Figure 3: Picture of the nf-DCC 2 membrane [a] and corresponding FEG-SEM images showing the horizontally layered structure [b] and the nanoporous surface [c] and [d] at different magnifications. Supercritical fluid extraction allows for drying the nf-DCC gel (Plappert et al., 2017) membrane without causing structural damage to the nanofiber structure which would arise from capillary forces during conventional drying. This allowed for a detailed characterization of the anisotropic membranes by field emission gun scanning electron microscope (FEG-SEM) and

Figure 4 :

 4 Figure 4: Nitrogen adsorption (blue) and desorption (orange) isotherms [a, b and c]. Pore size distribution obtained via the BJH methods on the desorption branches of the isotherms [d, e and f] for respective nf-DCC membrane samples.

Figure 5 :

 5 Figure 5: Piroxicam loading in zwitter-ionic form (a-c, pH ≈ 6.8) and release as cation (c-e, acetate buffer pH 5) for membranes composed of nf-DCC 1 (a, d), 2 (b, e) and 3 (c, f) with the standard deviations displayed as shaded areas.

  nf-DCC nanofibers were assembled into anisotropic layered gel membranes by vacuum filtration. Treatment of the membranes with aqueous hydrochloric acid transforms surface carboxylate ions into carboxyl groups and triggers gel formation by interparticulate hydrogen bonding. Supercritical CO2 extraction of the gel membranes after replacing the interstitial water by ethanol -a solvent fully miscible with CO2 -allowed the nanomorphological characterization of the scaffold structures without causing any damage by the drying procedure.

Table 1 : Chemical properties and morphological characteristics of the prepared nf-DCC samples.

 1

	nf-DCC	COOH	DO	Length AFM	Diameter AFM Aspect ratio	Size DLS
		[mmol/g]	[%AGU]	[nm]	[nm]	[-]	[nm]
	1	0.78±0.03	6.36±0.30	375.1±203.7	2.47±0.67	152	365.2±68.6
	2	1.74±0.03	14.10±0.48	127.3±46.4	2.68±0.69	48	206.9±52.3
	3	2.00±0.11	16.16±0.89	95.8±53.9	2.72±0.95	35	182.2±10.1

Table 2 : Bulk density of scCO2 dried nf-DCC membranes and internal surface characteristics as deter- mined by nitrogen sorption experiments at 77K.

 2

	nf-DCC	Membrane density	Specific surface area	C-value (BET)
		[mg cm -3 ]	[m 2 g -1 ]	[-]
	1	21	502	107
	2	22	452	87
	3	22	349	68

ACKNOWLEDGEMENTS

Financial support by the French Agence Nationale de la Recherche (ANR-15-CE18-0031, OA-ANGIO-ANALGESIA) and the Austrian BMLFUW Ministry (WoodWisdom-Net+ project AeroWood) is gratefully acknowledged.


Guido Kroemer Kroemer

Laurence Zitvogel

Leptin-producing oncolytic virus makes tumorinfiltrating T cells fit, not fat

Keywords: adipokine, leptin, mitochondria, cancer, cytotoxic T cells, exhaustion, memory, oncolytic viruses immune checkpoint inhibitors, ACT: adoptive cell transfer, TIL: tumor infiltrating lymphocytes, TME: tumor microenvironment, TAA: tumor associated antigens, I-O: immuno-oncology: OV: oncolytic virus

Effective anticancer immunosurveillance after oncolytic viral infection is often hindered by the defective metabolic function of tumor infiltrating lymphocytes (TILs). A recent paper by

Rivadeneira et al. demonstrates that intratumoral delivery of leptin by a recombinant oncolytic vaccinia virus can metabolically enhance TIL effector and memory functions through improved mitochondrial oxidative phosphorylation, thereby enhancing therapeutic efficacy.

Main text

Immuno-oncology (I-O) therapies such as immune checkpoint inhibitors (ICIs) or adoptive T cell therapies (ACT) are improving cancer treatments, leading to regulatory approvals in a broad range of indications. However, only a minority of patients, especially those with immunologically "hot" tumors, i.e. cancers full of tumor-infiltrating lymphocytes (TILs), exhibit objective responses and survival benefits. Moreover, I-O therapies can be harmful at two levels. On the one hand, they can induce debilitating autoimmunity affecting any organ system. On the other hand, the excessive price tag attached to I-O treatments drains resources that might be invested more effectively in cancer prevention or low-cost therapies. In this issue of Immunity, Rivadeneira et al. (Rivadeneira et al., 2019) demonstrate that local delivery of the adipokine leptin by oncolytic viruses can metabolically reprogram newly generated antitumor T lymphocytes, protecting the host against aggressive melanoma and pancreatic cancers.

Intratumoral immunotherapy is a therapeutic strategy that aims at converting the tumor into its own vaccine, offering several advantages over systemic delivery. First, a high concentration of immuno-stimulatory products can be achieved in situ, while using small amounts of drugs. Second, local delivery of immunotherapies allows multiple combination therapies, while preventing significant systemic exposure and off-target toxicities. Third, local I-O may elicit a broad spectrum of T cell receptor (TCR) specificities across neo-antigens or tumor-associated antigens (TAAs) in a personalized manner, while being agnostic with regard to the relevant epitopes (Kepp et al. 2019).

One local I-O approach consists in the intratumoral injection of oncolytic viruses (OVs).

OVs have a tropism for malignant cells, thereby causing lysis of malignant cells with consequent local inflammation of the tumor microenvironment (TME). OV-mediated tumor cell killing may result in "immunogenic cell death", associated with effective cross-presentation of tumor antigens by dendritic cells to T lymphocytes, thus igniting a systemic "abscopal" immune response at distant sites [START_REF] Fend | Immune Checkpoint Blockade, Immunogenic Chemotherapy or IFN-α Blockade Boost the Local and Abscopal Effects of Oncolytic Virotherapy[END_REF]. Preclinical studies have confirmed that OVs can cause systemic reactivation and/or proliferation of adoptively transferred T cells and mediate synergistic antitumor effects with other I-O modalities, especially when OVs have been engineered to express immunostimulatory genes. Thus far, the sole FDA-approved oncolytic virus, T-VEC (official name: Talimogene Laherparepvec), a genetically modified herpes simplex-1 (HSV) virus expressing granulocyte macrophage colony-stimulating factor (GM-CSF), is used for the local treatment of inoperable melanoma [START_REF] Andtbacka | Talimogene Laherparepvec Improves Durable Response Rate in Patients With Advanced Melanoma[END_REF]. Clinical trials revealed that T-VEC can induce "abscopal" effects upon local injection into melanomas, thus reducing the volume of non-injected lesions, especially in the context of immune checkpoint blockade [START_REF] Ribas | Oncolytic Virotherapy Promotes Intratumoral T Cell Infiltration and Improves Anti-PD-1 Immunotherapy[END_REF]. Nonetheless, durable remission rates remain exceptional with this new class of I-O therapy [START_REF] Andtbacka | Talimogene Laherparepvec Improves Durable Response Rate in Patients With Advanced Melanoma[END_REF], highlighting the need to further elucidate the mechanisms of action of OVs. In fact, the immune cell subtypes contributing to OV-induced immune response have not been fully defined, and the functional status of newly infiltrating lymphocytes has been poorly investigated.

Cancer cells and TILs, including effector CD8 + T cells, have similar metabolic requirements and compete for nutrient resources, including glucose and amino acids, within the TME. Furthermore, tumors create a challenging environment characterized by hypoxia, Braf V600E Tyr2 Cre.ER mice using Western Reserve laboratory strain Vaccinia OV, the authors obtained substantial tumor growth retardation but failed to observe complete tumor eradication.

Single-cell sequencing of the OV-induced TILs led to the identification of a subset of CD8 + T cells harboring hallmarks of activation and exhaustion, such as high PD1 and TIM3 expression, coupled with reduced mitochondrial biogenesis.

Although leptin was first discovered as the satiety hormone, meaning that its (rare) genetic defect causes hyperphagy and obesity, this hormone produced by adipocytes, also known as an adipokine, is chronically elevated in the vast majority of obese patients. The rational to use leptin to metabolically reprogram the TME came from pioneering work highlighting that this adipokine could reverse the immunosuppressive effects of acute starvation in mice. Administration of leptin reinstated T helper 1 (Th1) immune responses, acting on activated T cells to stimulate glucose uptake and metabolism [START_REF] Lord | Leptin modulates the T-cell immune response and reverses starvation-induced immunosuppression[END_REF]). Rivadeneira et al. found higher expression levels of leptin receptors on PD1 + Tim3 + CD8 + T cells residing in tumor beds compared with secondary lymphoid organs or compared with PD1 - T cells and then observed that, in activated T cells in vitro, leptin boosted the mitochondrial mass and induced the phosphorylation of p38 MAPK as well as the transcription factor ATF2.

Encouraged by these findings, the authors then conducted several experiments to confirm the beneficial effects of leptin on TIL cell fitness in the hostile TME. First, genetic engineering of CL24 melanomas to enforce expression of leptin (CL24 leptin ) led to CD8 + T cell-dependent tumor growth retardation associated with the influx of cytotoxic T lymphocytes (CTLs) and NK cells into the tumor bed. Importantly, despite high PD1 and Tim3 expression levels, CD8 + T cells homing into CL24 leptin tumor beds were specific for cancer antigens, remained proliferative and polyfunctional, exhibited phosphorylation of PKB, p38 MAPK and STAT3, and showed increased mitochondrial biogenesis. Moreover, the anticancer effects of leptin release into the TME were lost when recombinant CL24 leptin tumor cells were inoculated into hosts harboring a T cell-restricted heterozygous deletion of the leptin receptor (Rivadeneira et al. Immunity in press).

Next, Rivadeneira et al. equipped OVs with a gene encoding leptin under the control of the Vaccinia p7.5 promoter. Local delivery of leptin by OV leptin into 5mm CL24 wt tumors recapitulated the effects observed with CL24 leptin cells, leading to tumor infiltration by proliferative polyfunctional CD8 + T lymphocytes with amplified mitochondrial mass, as well as to complete tumor eradication in 27% of the cases. These beneficial effects of OV leptin were corroborated in a second tumor model resistant to PD1 blockade, the mouse Panc02 pancreatic cancer model. Single-cell RNA sequencing of TILs in OV leptin tumor beds revealed a change in the effector memory phenotype of CD8 + cells, towards a more polyclonal and precursor memory CD127 + Tcf7 + profiling (Figure 1) as well as a type 1 IFN signature. Animals that received intratumoral OV leptin developed not only a memory T cell response against viral antigens, but more importantly, a protective immunity against tumor antigens (Rivadeneira et al. Immunity in press).

This study employed leptin therapeutically as a metabolic modulator of the immune response against cancer using oncolytic viruses that were poorly efficient in these tumor models resistant to anti-PD1 antibodies. This strategy may invigorate pre-existing immunity by stimulating PPARγ coactivator 1α dependent-mitochondrial oxidative phosphorylation, reinstating T cell fitness, similar to prior findings with agonistic CD137 antibodies [START_REF] Menk | 4-1BB costimulation induces T cell mitochondrial function and biogenesis enabling cancer immunotherapeutic responses[END_REF]).

However, it is intriguing to note that some of the findings apparently conflict with clinical data reporting superior clinical benefit of anti-PD1 and/or anti-PDL-1 Abs in obese melanoma patients [START_REF] Mcquade | Association of body-mass index and outcomes in patients with metastatic melanoma treated with targeted therapy, immunotherapy, or chemotherapy: a retrospective, multicohort analysis[END_REF]. Obesity is associated with inflammaging, hyperleptinemia and deregulated immune functions. An unfavorable role of leptin signaling in increasing PD-1 and other hallmarks of T cell exhaustion (Tim3, Lag3, Eomes and fatty acid oxidation) and culminating in accelerated tumor growth has been recently reported in obese tumor-bearing mice (Wang Z, et al. 2019). These discrepancies might stem from the differential effects of leptin on naive versus memory T cells, on the bioactivity of leptin on distinct cellular compartments of the TME (such as myeloid derived-suppressor cells or macrophages), or from the confounding factor of sex in these effects, PD1 blockade being more effective in obese males than obese females. It is of note that Rivadeneira et al. failed to obtain beneficial effects of systemic administration of recombinant leptin, suggesting that local delivery of this adipokine is key for the anticancer effects (Rivadeneira et al. Immunity in press). In addition, Rivadeneira et al. obtained most of the findings in lean but not obese tumor bearers, highlighting the yet-to-be-explored possibility of an acquired T cell resistance to leptin in organisms chronically exposed to obesity-related systemic hyperleptinemia.

Regardless of these open issues, the possibility to modulate immunometabolism by OV represents a promising strategy for stimulating anticancer immunity in a way that synergies with current systemic I-O strategies. Following local delivery leptin by OV-infected target cells, TILs become metabolically empowered, recover a polyfunctional phenotype (Ki67, IFNγ, TNFα) and acquire hallmarks of "memory resource" T cells (Tcf7, CD127, type 1 IFN signature). This metabolic switch is associated with complete tumor eradication, antigen spreading and long term protective immune responses against various tumor -associated antigens.

  acidosis and high concentrations of immunosuppressive metabolites. Chronic protein kinase B signaling in T lymphocytes induces a defect in transcription factor PPARγ coactivator 1α-(PGC-1α)-driven mitochondrial biogenesis and oxidative phosphorylation (Scharping et al. 2016) associated with T cell dysfunction. Thus, new approaches that combine existing I-O with strategies that overcome the metabolically challenging TME and enhance infiltration by, and longevity of, antitumor effector memory responses are actively investigated. Rivadeneira et al. sought to determine the immune cell subtypes contributing to OVinduced immune responses and to determine why OV responses are typically incomplete. When attempting to treat an oncogene-driven established melanoma line (CL24) derived from Pten f/f

  obesity on T cell function during tumor progression and PD-1 checkpoint blockade. Nat Med. 2019 Jan;25(1):141-151.

Figure 1 .

 1 Figure1. Metabolic reprograming of TIL by oncolytic viruses engineered to express leptin. Following oncolytic virus (OV) infection, melanoma cells undergo immunogenic cell death. T cell incomers that are newly recruited into the inflamed tumor bed (along with NK cells and monocytes) exhibit inhibitory (PD1, Tim3) and functional leptin receptors. However, such tumor infiltrating lymphocytes (TILs) are poorly proliferative, contain low mitochondrial mass, and are defective in T helper 1 (Th1) cytokine secretion and memory responses. Following local delivery leptin by OV-infected target cells, TILs become metabolically empowered, recover a polyfunctional phenotype (Ki67, IFNγ, TNFα) and acquire hallmarks of "memory resource" T cells (Tcf7, CD127, type 1 IFN signature). This metabolic switch is associated with complete tumor eradication, antigen spreading and long term protective immune responses against various tumor -associated antigens.

Acknowledgments: GK and LZ are the Ligue contre le Cancer (équipe labellisée); Agence National de la Recherche (ANR) -Projets blancs; ANR under the frame of E-Rare-2, the ERA-Net for Research on Rare Diseases; Association pour la recherche sur le cancer (ARC); Cancéropôle Ile-de-France; Chancelerie des universités de Paris (Legs Poix), Fondation pour la Recherche Médicale (FRM); a donation by Elior; European Research Area Network on Cardiovascular Diseases (ERA-CVD, MINOTAUR); Gustave Roussy Odyssea, the European Union Horizon 2020 Project Oncobiome; Fondation Carrefour; High-end Foreign Expert Program in China (GDW20171100085), Institut National du Cancer (INCa); Inserm (HTE); Institut Universitaire de France; LeDucq Foundation; the LabEx Immuno-Oncology (ANR-18-IDEX-0001); the RHU Torino Lumière; the Seerave Foundation; the SIRIC Stratified Oncology Cell DNA Repair and Tumor Immune Elimination (SOCRATE); and the SIRIC Cancer Research and Personalized Medicine (CARPEM).

Conflicts of interest:

GK is a scientific co-founder of Samsara Therapeutics. GK and LZ are co-founders of everImmune. LZ is member of scientific advisory board of Transgene and co-inventor of a patent dealing with "Combination of oncolytic virus with immune checkpoint modulators".

WT LEPTIN

Tumor regression