Application de l'immunolocalisation à la recherche de la cellule souche endothéliale cornéenne humaine

Le contrôle de la transparence de la cornée dépend de l'intégrité de l'endothélium cornéen mono-stratifié qui est classiquement considéré dès la naissance, dépourvu de capacité de régénération chez l homme. Dans des conditions pathologiques conduisant à la cécité par œdème cornéen, les pertes significatives en cellules endothéliales (CE) ne sont pas remplacée efficacement, ce qui signifie que ni de nouvelles CE provenant de cellules souches (CS), ni la division des cellules voisines des lésions ne peuvent contribuer à la régénération endothéliale. Toutefois, plusieurs travaux ont prouvé depuis 25 ans que les CE possédaient une capacité proliférative résiduelle ex vivo et deux équipes ont suggéré l existence de CS ou de progéniteurs à la périphérie de l endothélium cornéen. Dans notre travail de thèse, nous avons tout d'abord optimisé, en la systématisant, une technique d immunomarquage spécialement adaptée à l'endothélium cornéen intact de cornées montées à plat. A l issue de ces développements, nous disposons de protocoles simples de fixation à température optimale et de démasquages antigéniques susceptibles de permettre la révélation de nombreuses protéines. A partir d une importante série de cornées humaines non conservées et d autres conservées en organoculture, et grâce à cet outil désormais efficace, nous avons étudié le cycle cellulaire des CE et la localisation de potentielles CS sur l endothélium cornéen humaine. Nos résultats démontrent que dans ces conditions, les CE expriment de façon homogène des régulateurs positifs (PCNA, MCM2, cycline D1, cycline E et cycline A) et des régulateurs négatifs du cycle cellulaire (P16, P27); certaines particularités ont par ailleurs pu être décrites de façon innovante, comme la localisation cytoplasmique diffuse de MCM2, paranucléaire de la cycline D1, l absence de P21. L ensemble des marquages pourrait suggérer que les CE sont arrêtées en fin de G1, après le point de restriction et que de nombreux mécanismes de réparation de l ADN sont mis en jeux dans les CE exposées à un stress oxydant important tout au long de l existence. Nous avons identifié une nouvelle organisation de la micro-anatomie de la périphérie et de l'extrême périphérie de l endothélium où des cellules regroupées en multiples clusters pluristratifiés semblent alimenter des colonnes de CE radiaires longues d'un millimètre. Ces éléments, associés à l observation d'une moindre différenciation et d une compétence proliférative plus élevés en périphérie suggèrent un nouveau modèle d homéostasie endothéliale humaine in vivo: toute la vie, des CS périphériques alimentent de façon très lente la périphérie cornéenne en CE qui migrent de façon centripète pour assurer la stabilité du centre cornéen dont les propriétés optiques primordiales sont sous-tendues par un endothélium qui ne perd que 0,6% de CE par an. A la différence de l épithélium cornéen, ce système ne peut être accéléré lors de circonstances pathologiques. Les perspectives de nos travaux sont désormais d essayer d isoler de l extrême périphérie les CS endothéliales ou les progéniteurs et de les cultiver en recréant un microenvironnement favorable. La possibilité de produire un grand nombre de CE in vitre non pas à partir de CE sénescentes prélevées sur la totalité de l endothélium comme cela a été tenté par la passé, mais cette fois à partir de CS ou des progéniteurs ouvriraient la voie d'une véritable thérapie cellulaire endothéliale. L'enrichissement des greffons pendant la durée de leur conservation à la banque de cornée pourrait constituer une première étape majeure avant d envisager créer de novo un endothélium sur un support greffable pour une greffe endothéliale du 3e type qui deviendrait ainsi enfin indépendante des aléas de la découpe du greffon. Enfin, l ïdentification de la CS endothéliale et de son microenvironnement permettra aussi d'envisager une thérapie cellulaire in vivo pour traiter les stades précoces des pathologies endothéliales cornéennesThe control of corneal transparency depends on the integrity of the mono-stratified corneal endothelium, which is considered devoid of regenerative capacity after birth in humans. In pathological conditions leading to blindness by irreversible corneal edema, the significant losses of endothelial cells (ECs) are not replaced efficiently, indicating that neither new ECs derived from stem cells (SC) nor the division of ECs neighboring the lesions can contribute to a form of regeneration. However, several works of the last 25 years demonstrated that ECs possess residual capacity of proliferation ex vivo, and more recently, two teams suggested the existence of SC or endothelial progenitors located in the corneal periphery. In this thesis work, we have firstly optimized an immunostaining technique specially adapted to intact endothelium of flat mounted whole corneas. Consequently, we now have simple protocols of fixation at the right temperature, and of antigen retrieval that allow detecting multiple proteins with a clear subcellular localization. Using important series of non-stored and of organ cultured corneas, and thanks to this technique, we investigated the cell cycle status of ECs and the location of potential SC in human corneal endothelium. Our results indicate that ECs homogeneously express positive regulators (PCNA, MCM2, cyclin D1, cyclin E and cyclin A) as well as negative regulators of the cell cycle (P16, P27); several original descriptions have been made: diffuse cytoplasmic location of MCM2, paranuclear location of cyclin D1, absence of P21. The expression patterns suggest that ECS could be arrested after the restriction point of the G1 phase and that numerous mechanism of DNA repair are stimulated in ECs exposed to an important oxidative stress throughout live. We identified a novel organization of the micro-anatomy of the endothelial periphery and extreme-periphery, where cells accumulate in multiple small multilayered clusters connected radial centripetal cell rows of nearly 1 mm of length. Associated with the observation of a lesser differentiation and an increased proliferation capacity in this area, these elements suggest a novel model of endothelial homeostasis in humans: during life, SC continuously and extremely slowly sustain the endothelial periphery with new ECs that migrate toward centre forming cells rows. These cells ensure the stability of the center, which optical fundamental properties require a perfect stability, as indicated by an annual cell loss of only 0.6%. Contrary to the corneal epithelium, this system is incapable of accelerations in case of important cell loss. Further studies are necessary to understand this limitation. Our works offer several perspectives: the next step is to isolate the SC or the progenitors from the extreme periphery and to cultivate them in an adapted microenvironment. The possibility to cultivate endothelial cells directly from SC or progenitors and not, as previously tried in the past, from senescent EC from the whole endothelium open the way of a true endothelial cell therapy. The increase of endothelial cell density during corneal storage by eye banks could be a first step before developing bioengineered endothelium on a specific carrier that could be implanted in the recipient eye. Finally, the identification of SC and of its microenvironment would allow developing the basis of an in vivo cell therapy able to treat early stages of endothelial dysfunctionsST ETIENNE-Bib. électronique (422189901) / SudocSudocFranceF

Comparison of corneal endothelial mosaic according to the age: the corimmo 3D project

International audienceAim: The human corneal endothelium is a monolayer of flat hexagonal cells. It is a nearly regular hexagonal tessellation during the first years of life, but with age, becomes less regular in shape and size. The aim is to evaluate geometrically the age of an endothelial mosaic.Material and methods: Segmented endothelial mosaics of healthy subjects of different age groups are compared by morphological criteria. The mosaics are studied according to their age group (decades), their age and their location (center or mid-periphery of the cornea). The measures used are: the cell density, the Ripley's L function and the cell area and perimeter density.Results: These measures point out the endothelial cell density decrease, the cell area, perimeter and diameter increase, the cell heterogeneity increase, and the differences between central and mid-peripheral cells increases with age.Conclusion: These measures are able to characterize healthy mosaics

Innovative, non-contact wide field imaging of corneal endothelium

International audienceIn this paper, we investigated the possibility of getting wide-field images of corneal endothelium for patients. An optical set-up coupled to a numerical reconstruction based on Structured Illumination Mircoscopy (SIM) has been developped in order to isolate the tiny volume wich contains the endothelial mono-layer found at the inner surface of the cornea. At this moment, this imaging system seems compromised for patients and futhur refinement are investigated for stored humans corneas banks

3D Map of the Human Corneal Endothelial Cell

Corneal endothelial cells (CECs) are terminally differentiated cells, specialized in regulating corneal hydration and transparency. They are highly polarized flat cells that separate the cornea from the aqueous humor. Their apical surface, in contact with aqueous humor is hexagonal, whereas their basal surface is irregular. We characterized the structure of human CECs in 3D using confocal microscopy of immunostained whole corneas in which cells and their interrelationships remain intact. Hexagonality of the apical surface was maintained by the interaction between tight junctions and a submembraneous network of actomyosin, braced like a drum. Lateral membranes, which support enzymatic pumps, presented complex expansions resembling interdigitated foot processes at the basal surface. Using computer-aided design and drafting software, we obtained a first simplified 3D model of CECs. By comparing their expression with those in epithelial, stromal and trabecular corneal cells, we selected 9 structural or functional proteins for which 3D patterns were specific to CECs. This first 3D map aids our understanding of the morphologic and functional specificity of CECs and could be used as a reference for characterizing future cell therapy products destined to treat endothelial dysfunctions

A new HPLC-ESI-MS/MS method to characterize and quantify phosphatidyl-choline with VLC-PUFA: Application to human retina

Purpose: Mutations in the ELOVL4 gene have been found in Stargardt-like macular dystrophy or STD3. Previous studies have shown that ELOVL4 is involved in the biosynthesis of very long chain polyunsaturated fatty acids (VLC-PUFA). The aim of this work was to develop a HPLC-ESI-MS/MS method of characterization and quantification of dipolyunsaturated phosphatidyl-choline (PC) molecular species containing VLC-PUFA and to apply it on retinas from human donors. Methods: Eyeballs were collected from calf as well as from nine human donors (body donation to Science). The neural retina was dissected from the RPE/choroid. Following lipid extraction, phosphorus content of total phospholipids was determined.Using a triple quadrupole MS instrument, PC molecular species were structurally characterized by collision-induced dissociation in the negative mode with a method based on normal-HPLC-ESIMS/MS. PC molecular species were then quantified using precursor ion scanning of m/z 184amu in the positive mode. Results: The characterization of PC species was done on bovine retinas. Among them, 28 were dipolyunsaturated PC species containing one VLC-PUFA (C24 to C36) with three to six double bonds. VLC-PUFA were always in the sn-1 position whilst PUFA at the sn-2 position was exclusively docosahexaenoic acid (DHA, C22:6.n-3). Most of these VLC-PUFA-containing dipolyunsaturated PC were detected and quantified in human retinas. The main represented compounds were those having VLC-PUFA of 32 carbon atoms (C32:3, C32:4, C32:5 and C32:6) and 34 carbon atoms (C34:3, C34:4, C34:5 and C34:6). Dipolyunsaturated PC with 36:5 and 36:6 were detected in lower quantities. Conclusions: This new HPLC-ESI-MS/MS method is sensitive and specific enough to structurally characterize and quantify all molecular species of PC, including those esterified with VLC-PUFA. This technique is valuable for a precise characterization of PC containingVLC-PUFA in retina and may be useful for better understanding their implication in the pathogenesis of STD3

Handheld In Vivo Reflectance Confocal Microscopy for the Diagnosis of Eyelid Margin and Conjunctival Tumors

OBJECTIVE To assess the diagnostic accuracy of handheld in vivo reflectance confocal microscopy (IVCM) for the diagnosis of eyelid margin and conjunctival tumors. DESIGN A prospective observational study was conducted at University Hospital of Saint-Etienne from January 2, 2011, to December 31, 2016 (inclusion of patients until December 31, 2015, and follow-up until December 31, 2016). A total of 278 consecutive patients with eyelid margin or conjunctival lesions were included. Conjunctival lesions were diagnosed with a conventional clinical examination using a slitlamp and by handheld IVCM. Final diagnoses were established by histopathologic examination for 155 neoformations suspicious for being malignant through clinical and/or IVCM examination that were excised and on follow-up of 12 months or longer for the remaining 140 lesions. MAIN OUTCOMES AND MEASURES Sensitivity, specificity, and positive and negative predictive values for malignant tumors of the conjunctiva and eyelid margin were calculated using clinical examination with slitlamp and handheld IVCM. RESULTS In the 278 patients (136 [48.9%] females; mean [SD] age, 59 [21] years), a total of 166 eyelid margin and 129 conjunctival lesions were included in the analysis. Of the 155 excised neoformations with a histopathologic diagnosis, IVCM showed higher sensitivity compared with clinical examination conducted with the slitlamp for malignant tumors of the eyelid margin (98%vs 92%) and conjunctiva (100% vs 88%). The specificity for malignant eyelid margin tumors was higher for IVCMthan for slitlamp examination (74%vs 46%), but slightly less for malignant conjunctival tumors (78%vs 88%). Analysis of all neoformations (155 excised and 140 in follow-up) confirmed these differences in the diagnostic accuracy of the clinical examination and IVCM. The presence of hyperreflective Langerhans cells mimicking malignant melanocytes was the main cause for misdiagnosis of malignant conjunctival tumors with IVCM. CONCLUSIONS AND RELEVANCE Handheld IVCM could be a useful tool for the identification of malignant conjunctival tumors. Further studies are required to confirm the usefulness of this device and identify possible features that can differentiate Langerhans cells from malignant melanocytes to prevent the misdiagnosis of melanoma using IVCM

Lipid Composition of the Human Eye: Are Red Blood Cells a Good Mirror of Retinal and Optic Nerve Fatty Acids?

International audienceBACKGROUND: The assessment of blood lipids is very frequent in clinical research as it is assumed to reflect the lipid composition of peripheral tissues. Even well accepted such relationships have never been clearly established. This is particularly true in ophthalmology where the use of blood lipids has become very common following recent data linking lipid intake to ocular health and disease. In the present study, we wanted to determine in humans whether a lipidomic approach based on red blood cells could reveal associations between circulating and tissue lipid profiles. To check if the analytical sensitivity may be of importance in such analyses, we have used a double approach for lipidomics. METHODOLOGY AND PRINCIPAL FINDINGS: Red blood cells, retinas and optic nerves were collected from 9 human donors. The lipidomic analyses on tissues consisted in gas chromatography and liquid chromatography coupled to an electrospray ionization source-mass spectrometer (LC-ESI-MS). Gas chromatography did not reveal any relevant association between circulating and ocular fatty acids except for arachidonic acid whose circulating amounts were positively associated with its levels in the retina and in the optic nerve. In contrast, several significant associations emerged from LC-ESI-MS analyses. Particularly, lipid entities in red blood cells were positively or negatively associated with representative pools of retinal docosahexaenoic acid (DHA), retinal very-long chain polyunsaturated fatty acids (VLC-PUFA) or optic nerve plasmalogens. CONCLUSIONS AND SIGNIFICANCE: LC-ESI-MS is more appropriate than gas chromatography for lipidomics on red blood cells, and further extrapolation to ocular lipids. The several individual lipid species we have identified are good candidates to represent circulating biomarkers of ocular lipids. However, further investigation is needed before considering them as indexes of disease risk and before using them in clinical studies on optic nerve neuropathies or retinal diseases displaying photoreceptors degeneration

Ambiguïtés et leçon d'une liturgie laïque : la commémoration républicaine de la mort de Louis XVI dans quelques villes et villages du Nord de l'an V à l'an VII

Philippe Gain, Ambiguities and Lessons of a Lay Liturgy : the Republican Commemoration of the Death of Louis XVI in some Towns and Villages of the Nord from Year V to Year VII. The first French Constitution of 1791 holds among its provisions relative to the Executive that the person of the King is inviolable. Yet, a few months later, the sovereign, thrown down his throne, is executed. The First Republic sets itself, from then on, in the Nord department as everywere else in the country, to commemorate this founding act. The new ordo imposes itself to everybody with the force of the law from year V to year VII, as can be witnessed through the numerous mail exchanged between the representatives of the central power and the local administration. A lay liturgy, centered around the major theme of the « commemoration of the just execution of Louis Capet » under the tutelage of the Supreme Being, substituted itself then to the antique ritual of the Christian tradition...La première constitution française de 1791 porte dans ses dispositions relatives à l'Exécutif que la personne du roi est inviolable. Cependant, quelques mois plus tard, le souverain jeté bas du trône est exécuté. La Première République s'attache dès lors, dans le département du Nord comme dans l'ensemble du territoire national, à commémorer cet acte fondateur. Le nouvel ordo s'impose à tous avec la force de la loi, de l'an v à l'an vu, comme en témoigne l'importante correspondance échangée entre les représentants du pouvoir central et les administrations locales. Une liturgie laïque axée autour du thème principal de « la commémoration de la juste exécution de Louis Capet », sous le regard tutélaire de l'Etre Suprême, se substituait ainsi à l'antique rituel de la tradition chrétienne...Philippe Gain, Dubbelzinnigheid van een les in lekenliturgie : de republikeinse her- denking van de dood van Lodewijk XVI in enkele steden en dorpen in het noorden van het jaar V tot VII. De eerste franse grondwet van 1791 stelt inzake de uitvoerende macht dat de per- soon van de koning onaantastbaar is. Een paar maand later wordt de van zijn troon beroofde koning geëxecuteerd. De Eerste Republiek begint dan, in het noorden zoals in heel het land, die grensverleggende daad te herdenken. De nieuwe ordo wordt met de kracht van wet toegepast, van het jaar V tot VII, zoals blijkt uit de belangrijke briefwisseling tussen de vertegenwoordigers van het centrale gezag en de locale bes- tuurders. Een lekenliturgie omtrent de « herdenking van de rechtvaardige terechtstel- ling van Louis Capet » neemt, onder het toezicht van het Hoogste Wezen, de plaats in van het antieke christelijke ritueel.Gain Philippe. Ambiguïtés et leçon d'une liturgie laïque : la commémoration républicaine de la mort de Louis XVI dans quelques villes et villages du Nord de l'an V à l'an VII. In: Revue du Nord, tome 80, n°326-327, Juillet-décembre 1998. pp. 681-696

Propagande et contrebande royaliste dans le Nord et en Belgique (XIXe-XXe siècles)

Gain Philippe. Propagande et contrebande royaliste dans le Nord et en Belgique (XIXe-XXe siècles). In: Frontières. Actes du 125e Congrès national des sociétés historiques et scientifiques, « L’Europe », Lille, 2000. Paris : Editions du CTHS, 2002. pp. 261-266. (Actes du Congrès national des sociétés savantes, 125