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Resume La dermatite atopique (DA), est une dermatose prurigineuse inflammatoire, frequente et chronique. Elle touche aussi bien les enfants que les adultes. Son etiologie est multifactorielle et implique des interactions complexes entre une predisposition genetique, une dysregulation du systeme immunitaire, une alteration de la barriere cutanee et l’environnement. Les interleukines (IL)-4 et 13 jouent un role cle. Le dupilumab, anticorps monoclonal dirige contre la sous-unite alpha commune aux recepteurs de l’IL-4 et de l’IL-13, est la premiere biomolecule approuvee pour le traitement de la DA moderee a severe. Il s’est revele tres efficace aussi bien dans les premiers essais cliniques de phases II et III que dans la vraie vie, avec une bonne tolerance generale. Cependant, une frequence assez elevee d’evenements indesirables oculaires (de 10 a 40 %), de type conjonctivite et syndrome sec, a ete rapportee. Leur prise en charge peut dans certains cas echouer et leur gravite imposer l’arret du traitement. De plus et de facon surprenante, ces complications oculaires n’ont pas ete decrites chez les patients traites par dupilumab pour d’autres pathologies telles que l’asthme. La comprehension des mecanismes moleculaires responsables est importante pour identifier les patients a risque et adapter leur prise en charge preventive et curative a la fois dermatologique et ophtalmologique.

PURPOSE: To evaluate the efficacy and safety of transepithelial corneal cross-linking (CXL) with supplemental oxygen. METHODS: This was a prospective, non-comparative, pilot cohort study conducted at the National Reference Center for Keratoconus (Toulouse, France) on patients with progressive keratoconus. Transepithelial, pulsed, accelerated CXL was performed in an oxygen-rich atmosphere. Oxygen goggles were applied to the eyes to maintain a high level of oxygen during treatment. The main efficacy outcome was the mean change from baseline in maximum keratometry (Kmax) and the secondary outcomes were the mean changes in flat keratometry (K1), steep keratometry (K2), mean keratometry (Km), corrected distance visual acuity (CDVA), uncorrected distance visual acuity (UDVA), and demarcation line depth. The safety outcomes were the incidence of adverse events, the mean change in pachymetry, and endothelial cell count. RESULTS: Thirty-four eyes of 34 patients were included. At 12 months postoperatively, the Kmax decreased by 1.56 ± 1.71 diopters (D) ( P < .0001) and CDVA improved by 0.093 ± 0.193 logMAR ( P < .02). The K2 and Km decreased by 0.51 ± 1.03 D ( P < .02) and 0.40 ± 0.78 D ( P < .01), respectively. There was no change in K1 and UDVA. The most frequent adverse event was corneal haze (64.78%). There were neither cases of infectious keratitis or loss of more than two lines in CDVA nor changes in pachymetry or endothelial cell count. CONCLUSIONS: Transepithelial CXL performed in an oxygen-rich atmosphere results in improved Kmax and CDVA with good safety. These promising findings suggest that this procedure could be safe and capable of halting the progression of keratoconus. [ J Refract Surg . 2021;37(1):42–48.]

OBJECTIVES To evaluate the success rate, efficacy, and safety of the ICD 16.5 mini-scleral gas permeable (GP) contact lens. METHODS This prospective study included referred consecutive patients with irregular corneas and severe ocular surface disease (OSD) in treatment failure. All patients were fitted with the ICD 16.5 mini-scleral GP lens. Even though we had some limited experience with scleral lenses, it was our first experience with the ICD 16.5 mini-scleral GP lens. Efficacy was assessed by comparing best-corrected visual acuity (BCVA) with the mini-scleral lens to baseline BCVA. A subjective visual functioning questionnaire (comfort score, visual quality score, handling rating, and wearing time) was administered in a face-to-face structured interview. RESULTS Thirty-nine eyes of 23 patients with a mean age of 43±16 years were included. Fitting indications were keratoconus (46%), post-penetrating keratoplasty (21%), other irregular astigmatism (15%), and severe OSD (18%). Twenty-five eyes (64%) were successfully fitted with an 18-month follow-up. The mini-scleral GP lens BCVA was 0.16 logarithm of the minimum angle of resolution (logMAR; 20/25) versus a baseline BCVA of 0.44 logMAR (20/63; P

Purpose To compare the outcomes of transepithelial photorefractive intrastromal corneal crosslinking (CXL) and photorefractive keratectomy (PRK) in eyes with low myopia. Setting Purpan Hospital, Toulouse, France. Design Prospective case series. Methods Myopic patients with a manifest refraction spherical equivalent (MRSE) of −1.00 to −2.50 diopters (D) and a cylindrical component of plano to −0.75 D were included. The dominant eye had PRK (PRK eyes). The nondominant eye had transepithelial photorefractive intrastromal CXL with riboflavin (ParaCel Part 1 and 2), 30 mW/cm2 pulsed ultraviolet-A irradiation centered on the pupil (Mosaic System) for 16 minutes and 40 seconds, and a supplemental oxygen delivery mask (CXL eyes). The primary outcome measure was the change in the MRSE. Other outcome measures were the uncorrected (UDVA) and corrected (CDVA) distance visual acuities, mean keratometry, and endothelial cell count (ECC) over a 6-month follow-up. Adverse events were assessed. Results Nineteen patients were included. By 6 months, the mean MRSE had decreased by 0.72 D ± 0.42 (SD) in CXL eyes and by 1.35 ± 0.46 D in PRK eyes (P Conclusions Photorefractive keratectomy provided better visual and refractive outcomes than transepithelial photorefractive intrastromal CXL. Transepithelial photorefractive intrastromal CXL, however, showed the potential refractive effect of CXL but with a limited magnitude of myopic correction.

ALK is a receptor tyrosine kinase, associated with many tumor types as diverse as anaplastic large cell lymphomas, inflammatory myofibroblastic tumors, breast and renal cell carcinomas, non-small cell lung cancer, neuroblastomas, and more. This makes ALK an attractive target for cancer therapy. Since ALK–driven tumors are dependent for their proliferation on the constitutively activated ALK kinase, a number of tyrosine kinase inhibitors have been developed to block tumor growth. While some inhibitors are under investigation in clinical trials, others are now approved for treatment, notably in ALK-positive lung cancer. Their efficacy is remarkable, however limited in time, as the tumors escape and become resistant to the treatment through different mechanisms. Hence, there is a pressing need to target ALK-dependent tumors by other therapeutic strategies, and possibly use them in combination with kinase inhibitors. In this review we will focus on the therapeutic potential of proapoptotic ALK-derived peptides based on the dependence receptor properties of ALK. We will also try to make a non-exhaustive list of several alternative treatments targeting ALK-dependent and independent signaling pathways.

International audience; Iron is essential for retinal function but contributes to oxidative stress-mediated degeneration. Iron retinal homeostasis is highly regulated and transferrin (Tf), a potent iron chelator, is endogenously secreted by retinal cells. In this study, therapeutic potential of a local Tf delivery was evaluated in animal models of retinal degeneration. After intravitreal injection, Tf spread rapidly within the retina and accumulated in photoreceptors and retinal pigment epithelium, before reaching the blood circulation. Tf injected in the vitreous prior and, to a lesser extent, after light-induced retinal degeneration, efficiently protected the retina histology and function. We found an association between Tf treatment and the modulation of iron homeostasis resulting in a decrease of iron content and oxidative stress marker. The immunomodulation function of Tf could be seen through a reduction in macrophage/microglial activation as well as modulated inflammation responses. In a mouse model of hemochromatosis, Tf had the capacity to clear abnormal iron accumulation from retinas. And in the slow P23H rat model of retinal degeneration, a sustained release of Tf in the vitreous via non-viral gene therapy efficently slowed-down the photoreceptors death and preserved their function. These results clearly demonstrate the synergistic neuroprotective roles of Tf against retinal degeneration and allow identify Tf as an innovative and not toxic therapy for retinal diseases associated with oxidative stress.

International audience; The cornea represents the external part of the eye and consists of an epithelium, a stroma and an endothelium. Due to its curvature and transparency this structure makes up approximately 70% of the total refractive power of the eye. This function is partly made possible by the particular organization of the collagen extracellular matrix contained in the corneal stroma that allows a constant refractive power. The maintenance of such an organization involves other molecules such as type V collagen, FACITs (fibril-associated collagens with interrupted triple helices) and SLRPs (small leucine-rich proteoglycans). These components play crucial roles in the preservation of the correct organization and function of the cornea since their absence or modification leads to abnormalities such as corneal opacities. Thus, the aim of this review is to describe the different corneal collagens and proteoglycans by highlighting their importance in corneal transparency as well as their implication in corneal visual disorders.

The pathogenesis of keratoconus remains unknown. This review focuses on recent advances in the knowledge of keratoconus biology from a gene and protein expression. Biological hypotheses were investigated either on a candidate approach or on global approaches. Candidate approach relies on the knowledge of the disease pathology and biochemistry, or on association of keratoconus with other genetic or ophthalmologic diseases. Global approach aims to identify the origin of keratoconus without taking into account any preconceived ideas of the pathophysiology of the disease. Despite a great deal of research, the pathogenesis of keratoconus is still poorly understood. If genetic susceptibility is probably a key factor in the occurrence of keratoconus, as with many complex diseases, distinguishing between primary disease mechanisms from secondary effects is extremely challenging.

PURPOSE: To compare the efficacy and safety of topography-guided corneal collagen cross-linking (TG-CXL) to conventional corneal CXL (C-CXL) in progressive keratoconus. METHODS: In this prospective, nonrandomized clinical trial, 60 eyes of 60 patients were scheduled to receive either TG-CXL (30 eyes with deepithelialization focused on the cone, riboflavin application for 10 minutes, and 30 mW/cm 2 pulsed ultraviolet-A irradiance pattern according to topography) or C-CXL (30 eyes treated in accordance with the Dresden protocol). Patients were observed for 1 year postoperatively. Maximum keratometry (Kmax), mean keratometry in the inferior part of the cornea (I index), corrected distance visual acuity (CDVA), demarcation line observed in optical coherence tomography, and nerves and cell densities analyzed by confocal microscopy were compared preoperatively and at 1 year postoperatively. Results: The difference was significant for both Kmax ( P < .01) and I index ( P < .01) between the two groups. CDVA improved significantly in the TG-CXL (0.2162 ± 0.2495 logMAR, P < .05) versus the C-CXL (0.2648 ± 0.2574 logMAR, P = .104) group. A stromal demarcation line was observed in both treatment groups, with similar depth at the top of the cone ( P = .391), but it was shallower at the surrounding area in the TG-CXL group ( P < .0001). Stromal evaluation by confocal microscopy showed less damage and faster healing in the surrounding area than on the cone area in the TG-CXL group. CONCLUSIONS: At 1 year postoperatively, TG-CXL seems to be as safe as C-CXL with stronger flattening in Kmax and I index and better improvement in CDVA. TG-CXL induces a biological gradient between the cone and the surrounding area that facilitates nerve and cell recovery. [ J Refract Surg. 2017;33(5):290–297.]

Background: Apoptosis is a programmed cell death in multicellular organisms, found in a wide variety of conditions, including inflammatory process, everywhere in the body, including the cornea and conjunctiva. Aim: To evaluate the effect of a new topical formulation of sphingosine-1 phosphate on preventing apoptosis of the corneal epithelium. Setting: Medical University. Materials and Methods: We tested several formulations suitable for topical application. Twenty-five rabbits were distributed among five groups. Group 1 comprised the controls. In Group 2, 20% ethanol was applied topically for 20 seconds; in Group 3, 50 μM topical sphingosine-1 phosphate was applied 2 hours prior to 20% ethanol application. In Group 4, 200 μM topical sphingosine-1 phosphate was applied 2 hours before the 20% ethanol application. In Group 5, only 200 μM topical sphingosine-1 phosphate was applied. Apoptosis was evaluated using the terminal deoxynucleotidyl transferase biotin-dUTP Nick End Labeling (TUNEL) assay. Pairwise comparisons were performed using t-tests with Scheffe's correction. Data were analyzed using STATA 9.0 statistical software. Results: A suspension of sphingosine-1 phosphate in the presence of Montanox 80 was stable and could be formulated without sonication. Epithelial apoptosis was detected only in Groups 2 and 3. Conclusion: Sphingosine-1 phosphate can prevent ethanol-induced apoptosis in the corneal epithelium of rabbits.

The cornea is a transparent, avascular, and highly specialized connective tissue that provides the majority of light refraction in the optical system of the eye. The human cornea is composed of several layers interacting in a complex manner and possessing specific functions, like eye protection and optical clearness. Only few proteomic studies of mammalian cornea have been performed leading to the identification of less than 200 proteins in human corneas. The present study explores the proteome of the intact normal human cornea using a shot-gun nanoLC-MS/MS strategy and an LTQ Orbitrap mass spectrometer. A total of 2070 distinct corneal proteins were identified from five human cornea samples, which represents a 14-fold improvement in the number of proteins identified so far for human cornea. This enlarged dataset of human corneal proteins represents a valuable reference library for further studies on cornea homeostasis and pathophysiology. Network and gene ontology analyses were used to determine biological pathways specific of the human cornea. They allowed the identification of subnetworks of putative importance for corneal diseases, like a redox regulation and oxidative stress network constituted of aldehyde and alcohol dehydrogenases, most of them being described for the first time in human cornea.

INTRODUCTION The aim of the present study was to evaluate the clinical usefulness of riboflavin- and ultraviolet-A (UV-A)-induced collagen cross-linking (CXL) on progressive keratoconus. PATIENTS AND METHODS Twenty eyes of 20 patients with progressive keratoconus were included in a prospective, nonrandomized pilot study. The preoperative progression of keratoconus was documented by an increase in maximum keratometry (Kmax) on serial corneal topographies. Preoperative corneal thickness was at least 400 microm. The postoperative progression of keratoconus was evaluated from corneal topography, best-corrected visual acuity (BCVA) testing, and corneal biomechanical properties assessed with the Ocular Response Analyzer (ORA). The follow-up time was between 3 and 18 months. RESULTS The steepening decreased significantly by 1.68+/-2.18 diopters (D) in the first year (p

International audience; Purpose: We compared an iontophoresis riboflavin delivery technique for transepithelial corneal collagen crosslinking (I-CXL) with a conventional CXL (C-CXL).Methods: We designed three experimental sets using 152 New Zealand rabbits to study riboflavin application by iontophoresis using charged riboflavin solution (Ricrolin+) with a 1-mA current for 5 minutes. The first set was to compare riboflavin concentration measured by HPLC in corneas after iontophoresis or conventional riboflavin application. The second set was to analyze autofluorescence and stromal collagen modification immediately and 14 days after I-CXL or C-CXL, by using nonlinear two-photon microscopy (TP) and second harmonic generation (SHG). In the third set, physical modifications after I-CXL and C-CXL were evaluated by stress-strain measurements and by studying corneal resistance against collagenase digestion. Results: Based on HPLC analysis, we found that iontophoresis allowed riboflavin diffusion with 2-fold less riboflavin concentration than conventional application (936.2 ± 312.5 and 1708 ± 908.3 ng/mL, respectively, P < 0.05). Corneal TP and SHG imaging revealed that I-CXL and C-CXL resulted in a comparable increased anterior and median stromal autofluorescence and collagen packing. The stress at 10% strain showed a similar stiffness of corneas treated by I-CXL or C-CXL (631.9 ± 241.5 and 680.3 ± 216.4 kPa, respectively, P = 0.908). Moreover, we observed an increased resistance against corneal collagenase digestion after I-CXL and C-CXL (61.90% ± 5.28% and 72.21% ± 4.32% of remaining surface, respectively, P = 0.154).Conclusions: This experimental study suggests that I-CXL is a promising alternative methodology for riboflavin delivery in crosslinking treatments, preserving the epithelium.

International audience; In vivo, transforming growth factor (TGF)-beta1 and matrix metalloproteinases (MMPs) present at the site of airway injury are thought to contribute to epithelial wound repair. As TGF-beta1 can modulate MMP expression and MMPs play an important role in wound repair, we hypothesized that TGF-beta1 may enhance airway epithelial repair via MMPs secreted by epithelial cells. We evaluated the in vitro influence of TGF-beta1 on wound repair in human airway epithelial cells cultured under conditions allowing differentiation. The results showed that TGF-beta1 accelerated in vitro airway wound repair, whereas MMP inhibitors prevented this acceleration. In parallel, we examined the effect of TGF-beta1 on the expression of MMP-2 and MMP-9. TGF-beta1 induced a dramatic increase of MMP-2 expression with an increased steady-state level of MMP-2 mRNA, contrasting with a slight increase in MMP-9 expression. To confirm the role of MMP-2, we subsequently evaluated the effect of MMP-2 on in vitro airway wound repair and demonstrated that the addition of MMP-2 reproduced the acceleration of wound repair induced by TGF-beta1. These results strongly suggest that TGF-beta1 increases in vitro airway wound repair via MMP-2 upregulation. It also raises the issue of a different in vivo biological role of MMP-2 and MMP-9 depending on the cytokine microenvironment.

9 pages; International audience; Respiratory tract lesions induced by the chemical warfare agent sulfur mustard (SM) are characterized by epithelial damages associated with inflammatory cell infiltration. Here we evaluated the imbalance between gelatinase and tissue inhibitors of metalloproteinases (TIMPs), and we tested pretreatment with the protease inhibitor doxycycline. Guinea pigs were intoxicated intratracheally with SM and evaluated 24 h after exposure. Matrix metalloproteinase (MMP) gelatinase activity of bronchial lavage (BL) fluid from SM-exposed guinea pigs was high compared with controls, as shown by both zymography and biotinylated substrate degradation, whereas TIMP-1 and -2 levels by immunoblotting were similar. Extensive areas of lysis were evidenced by in situ zymography, indicating imbalance between gelatinases and inhibitors towards net proteolytic activity. Doxycycline pretreatment resulted in 1) decreased gelatinase activity (zymography, free gelatinase activity assay, and in situ zymography); 2) decreased inflammation (BL fluid cellularity and protein level); and 3) dramatic decrease in histological epithelial lesions. Our results suggest inadequate levels of TIMP to counteract increased gelatinase activity and further support a role for MMP gelatinases in SM-induced respiratory lesions. They also suggest that doxycycline may hold promise as a therapeutic tool.

Epidemiological and experimental studies suggest that diesel exhaust particles (DEPs) may be associated with increased respiratory mortality and morbidity. Several recent studies have also shown that DEPs increase the production of inflammatory cytokines by human bronchial epithelium (HBE) cells in vitro. The present study investigates the effects of DEPs on the interaction of l-HBE cells (16HBE14o-) with the cell and matrix microenvironment based on evaluation of integrin-type cell/matrix ligand expression, cytoskeleton (CSK) stiffness, and matrix remodeling via matrix metalloproteinase (MMP)-1, MMP-2, and MMP-9 expression. The results showed that DEP exposure induced: 1) a net dose-dependent decrease in CSK stiffness through actin fibers, 2) a concomitant specific reduction of both α3- and β1-integrin subunits extensively expressed on the HBE cell surface, 3) a decrease in the level of CD44, which is a major HBE cell-cell and HBE cell-matrix adhesion molecule; and 4) an isolated decrease in MMP-1 expression without any change in tissue inhibitor of matrix metalloproteinase (TIMP)-1 or TIMP-2 tissue inhibitors. Restrictive modulation of cell-matrix interaction, cell-cell connection, CSK stiffness, and fibrillary collagen remodeling results in a decreased wound closure capacity and an increased deadhesion capacity. In conclusion, on the basis of these results, we can propose that, in addition to their ability to increase the production of inflammatory cytokines, DEPs could also alter the links between actin CSK and the extracellular matrix, suggesting that they might facilitate HBE cell detachment in vivo.

International audience; Purpose: To characterize the expression of the bone morphogenetic protein-1 (BMP-1)/tolloid-like proteinases (collectively called BTPs), which include BMP-1, mammalian tolloid (mTLD), and mammalian tolloid-like 1 (mTLL-1) and 2 (mTLL-2), as well as the associated proteins procollagen C-proteinase enhancers (PCPE-1 and -2), in corneal scarring. Methods: Using a mouse full-thickness corneal excision model, wound healing was followed for up to 28 days by transmission electron microscopy, immunohistology (BMP-1/mTLD and PCPE-1), and quantitative PCR (Q-PCR: collagen III, BMP-1/mTLD, mTLL-1, mTLL-2, PCPE-1, PCPE-2). Bone morphogenetic protein-1/mTLD and PCPE-1 were also immunolocalized in cases of human corneal scarring following injuries. Results: In the mouse model, throughout the follow-up period, there was a large increase in collagen III mRNA expression in the stroma. By transmission electron microscopy, there was marked cellular infiltration into the wound as well as disorganization of collagen fibrils, but no significant difference in fibril diameter. In control corneas, by Q-PCR, BMP-1/mTLD showed the highest expression, compared to low levels of mTLL-1 and undetectable levels of mTLL-2, in both epithelium and stroma. Following wounding, both BMP-1/mTLD and PCPE-1 mRNA and protein increased, while PCPE-2 mRNA decreased. Finally, by immunofluorescence, BMP-1/mTLD and PCPE-1 were strongly expressed in the scar region in both mouse and human corneas. Conclusions: Bone morphogenetic protein-1/mTLD and PCPE-1 are upregulated in corneal scars. Both proteins may therefore contribute to the process of corneal scarring.

Purpose Very low-density lipoprotein receptor mutant mice (Vldlr(-/-)) demonstrate retinal neovascularisation. Monoclonal antibody CL1-R2 recognizes human membrane receptor CD160, which is expressed on activated endothelial cells of neovessels but not in vessels of healthy tissues. In this study, we evaluated the therapeutic potential of CL1R2 antibody in Vldlr(-/-) mice as a model for exudative age-related macular degeneration and macular telangiectasia. Methods Thirty Vldlr(-/-) mice benefitted at postnatal day (P) 42 from retinal angiography. Mice population was then divided in three groups (ten mice per group). According to the groups, mice underwent intravitreal injection of CL1-R2 or Bevacizumab or control antibody. At P56, mice benefitted from angiography and were sacrificed. Results At P42, Vldlr(-/-) retinas presented with neovascular lesions. At P56, we observed decrease of neovascular lesions in eyes treated with CL1R2 antibody and with bevacizumab. This decrease of neovascular lesions was not observed in control mice. Conclusion CL1R2 antibody, as well as Bevacizumab, promotes the regression of pathologic retinal neovascularization in the Vldlr knockout mouse. As CL1R2 is an activator of NK cells, our result suggest that the activation of NK cells inhibits the development of Vldlr(-/-) mice retinal neovascularisation.

International audience; Type II pneumocytes are essential for repair of the injured alveolar epithelium. The effect of two MMP collagenases, MMP-1 and MMP-13 on alveolar epithelial repair was studied in vitro. The A549 alveolar epithelial cell line and primary rat alveolar epithelial cell cultures were used. Cell adhesion and cell migration were measured with and without exogenous MMP-1. Wound healing of a cell monolayer of rat alveolar epithelial cell after a mechanical injury was evaluated by time lapse video analysis. Cell adhesion on type I collagen, as well as cytoskeleton stiffness, was decreased in the presence of exogenous collagenases. A similar decrease was observed when cell adhesion was tested on collagen that was first incubated with MMP-1 (versus control on intact collagen). Cell migration on type I collagen was promoted by collagenases. Wound healing of an alveolar epithelial cell monolayer was enhanced in the presence of exogenous collagenases. Our results suggest that collagenases could modulate the repair process by decreasing cell adhesion and cell stiffness, and by increasing cell migration on type I collagen. Collagen degradation could modify cell adhesion sites and collagen degradation peptides could induce alveolar type II pneumocyte migration. New insights regarding alveolar epithelial cell migration are particularly relevant to investigate early events during alveolar epithelial repair following lung injury.

Background Corneal intraepithelial dyskeratosis is an extremely rare condition. The classical form, affecting Native American Haliwa-Saponi tribe members, is called hereditary benign intraepithelial dyskeratosis (HBID). Herein, we present a new form of corneal intraepithelial dyskeratosis for which we identified the causative gene by using deep sequencing technology. Methods and results A seven member Caucasian French family with two corneal intraepithelial dyskeratosis affected individuals (6-year-old proband and his mother) was ascertained. The proband presented with bilateral complete corneal opacification and dyskeratosis. Palmoplantar hyperkeratosis and laryngeal dyskeratosis were associated with the phenotype. Histopathology studies of cornea and vocal cord biopsies showed dyskeratotic keratinisation. Quantitative PCR ruled out 4q35 duplication, classically described in HBID cases. Next generation sequencing with mean coverage of 50× using the Illumina Hi Seq and whole exome capture processing was performed. Sequence reads were aligned, and screened for single nucleotide variants and insertion/deletion calls. In-house pipeline filtering analyses and comparisons with available databases were performed. A novel missense mutation M77T was discovered for the gene NLRP1 which maps to chromosome 17p13.2. This was a de novo mutation in the proband9s mother, following segregation in the family, and not found in 738 control DNA samples. NLRP1 expression was determined in adult corneal epithelium. The amino acid change was found to destabilise significantly the protein structure. Conclusions We describe a new corneal intraepithelial dyskeratosis and how we identified its causative gene. The NLRP1 gene product is implicated in inflammation, autoimmune disorders, and caspase mediated apoptosis. NLRP1 polymorphisms are associated with various diseases.

PURPOSE: To investigate type XII collagen expression in corneal scars in vivo. METHODS: Type XII collagen protein expression was evaluated by immunohistochemistry in human corneal scars and in a mouse model of corneal scarring at several time points (from day 7 to day 210) after full-thickness excision. Alternative splice variants of the NC3 and NC1 domains of type XII collagen were investigated in the mouse wound-healing model using RT-PCR. RESULTS: Type XII collagen was overexpressed in human corneal scars in areas that were also positive for alpha-smooth muscle actin staining. In a mouse model of corneal wound injury we found that at 14 and 21 days postexcision, type XII collagen was largely concentrated in the subepithelial region of the cornea, especially in and near the wound bed. By 28 days postexcision, expression of type XII collagen decreased but remained higher than that in controls. NC3 short form is the main form expressed in the cornea during the wound-healing process. After injury, the NC1 long splice variant mRNA was the most highly overexpressed variant in the cornea, especially in the epithelium (×2.7, 3.72, and 5.57 at days 7, 14, and 21, respectively, P < 0.01 to 0.001 compared with uninjured samples). Corneal scars from a 7-month-old mouse revealed an overexpression of type XII collagen in the wound area similar to what we observed in human corneal scars. CONCLUSIONS: Type XII collagen is overexpressed in permanent human and mouse corneal scars and could represent a new target to treat corneal scarring.

PURPOSE: To decipher the biological pathways involved in keratoconus pathophysiology by determining the patterns of differential gene expression between keratoconus and control corneas. METHODS: RNA was extracted from surgically removed corneas of 10 keratoconus patients and from normal corneas of 10 control patients who had undergone enucleation of an eye for ocular melanoma. Several hundred thousand RNA transcripts were assessed using exon microarrays. Statistical comparison and identification of differentially regulated and differentially spliced RNA transcripts was performed by comparing keratoconus cases and controls. In addition, relevant biological pathways were identified by information extraction using network biology. RESULTS: Eighty-seven genes showed significant differences in expression levels. Among these, 69 were downregulated in keratoconus patients, particularly partners of the transcription factor AP-1. The 18 overexpressed genes include mucins, keratins, and genes involved in fibroblast proliferation. In addition, 36 genes were shown to be differentially spliced, including 9 among those that were differentially expressed. Network biology and analysis using Gene Ontology descriptors suggest that many members of both groups belong to pathways of apoptosis and regulation of the balance between cellular differentiation and proliferation. CONCLUSIONS: This work constitutes the first genome-wide transcriptome analysis of keratoconus patient corneas that include all currently known genes and exons. Differential expression suggests that mechanisms of cell loss resulting from antiproliferative and hyperapoptotic phenotypes may be responsible for the pathogenesis of keratoconus. Array information, experimental design, raw intensities, and processed log(2) ratios were deposited at the European Bioinformatic Institute's ArrayExpress database (http://www.ebi.ac.uk/arrayexpress/). The accession number is E-MEXP-2777.

A monoclonal anti-CD160 antibody inhibits the growth of new vessels in pathological ocular and tumor neoangiogenesis but not in healthy tissues., Angiogenesis plays an essential role in several diseases of the eye and in the growth of solid tumors, but existing antiangiogenic therapies have limited benefits in several cases. We report the antiangiogenic effects of a monoclonal antibody, CL1-R2, in several animal models of neovascularization. CL1-R2 recognizes human CD160, a membrane receptor which is conserved in various mammal species. We show that CD160 is expressed on the endothelial cells of newly formed blood vessels in human colon carcinoma and mouse B16 melanoma but not in vessels of healthy tissues. CL1-R2 reduced fibroblast growth factor 2–induced neovascularization in the rabbit cornea, in a mouse model of oxygen-induced retinopathy, and in a mouse Matrigel plug assay. Treatment of B16 melanoma-bearing mice with CL1-R2 combined with cyclophosphamide chemotherapy caused regression of the tumor vasculature and normalization of the remaining vessels as shown by Doppler ultrasonography, intravital microscopy, and histology. These studies validate CD160 as a potential new target in cases of human pathological ocular and tumor neoangiogenesis that do not respond or become resistant to existing antiangiogenic drugs.

Once a corneal scar develops, surgical management remains the only option for visual rehabilitation. Corneal transplantation is the definitive treatment for a corneal scar. In addition to the challenges posed by graft rejections and other postoperative complications, the lack of high-quality donor corneas can limit the benefits possible with keratoplasty. The purpose of our study was to evaluate a new therapeutic strategy for treating corneal scarring by targeting collagen deposition. We overexpressed a fibril collagenase (matrix metalloproteinase 14 (MMP14)) to prevent collagen deposition in the scar tissue. We demonstrated that a single and simple direct injection of recombinant adeno-associated virus-based vector expressing murine MMP14 can modulate gene expression of murine stromal keratocytes. This tool opens new possibilities with regard to treatment. In a mouse model of corneal full-thickness incision, we observed that MMP14 overexpression reduced corneal opacity and expression of the major genes involved in corneal scarring, especially type III collagen and α-smooth muscle actin. These results represent proof of concept that gene transfer of MMP14 can reduce scar formation, which could have therapeutic applications after corneal trauma.

Purpose Corneal wound healing is an everyday preoccupation for ophthalmologists.Corneal transparency depends on the scarring quality after a traumatic corneal wound, but also after refractive corneal surgery. Cicatrisation and fibrosis formation involve epithelial/fibroblast interactions via paracrin signals inducing extracellular matrix (ECM) remodelling. The major event is fibroblast activation and differentiation into myofibroblasts. These cells have a key role in the fibrotic response. They acquire contractile properties, and synthetise a new ECM, mainly composed of type III collagen. This scar tissue is less organised than the regular stroma, thus explaining corneal opacity. ECM remodelling is a critical step which aims to digest the excess of ECM by proteolysis of type III collagen. MMP14 is a membrane-bound fibrillar collagenase from the Matrix Metalloprotease family. We hypothesised that its overexpression in the corneal stroma during wound repair will increase ECM remodelling and thus prevent collagen deposition in the scar tissue. Methods We developed an adeno-associated virus-based vector expressing murine MMP14 under the control of the CMV promoter. We evaluated MMP14 overexpression after viral transfection in a murine model of corneal wound healing. We characterised several parameters: clinical observation, histology, and wound healing markers. Results Our preliminary results showed a decreased in oedema and corneal scar formation, associated with a decreased expression of alpha smooth actin and type III collagen. Conclusion These results represent proof of concept that gene transfer of MMP14 can reduce scar formation, which could have therapeutic applications after corneal trauma.

International audience; We have experimentally studied the fluorescence resonance energy transfer (FRET) between green fluorescent protein (GFP) molecules by inserting folded or intrinsically unstructured proteins between CyPet and Ypet. We discovered that most of the enhanced FRET signal previously reported for this pair was due to enhanced dimerization, so we engineered a monomerizing mutation into each. An insert containing a single fibronectin type III domain (3.7 nm end-to-end) gave a moderate FRET signal while a two-domain insert (7.0 nm) gave no FRET. We then tested unstructured proteins of various lengths, including the charged-plus-PQ domain of ZipA, the tail domain of alpha-adducin, and the C-terminal tail domain of FtsZ. The structures of these FRET constructs were also studied by electron microscopy and sedimentation. A 12 amino acid linker and the N-terminal 33 amino acids of the charged domain of the ZipA gave strong FRET signals. The C-terminal 33 amino acids of the PQ domain of the ZipA and several unstructured proteins with 66-68 amino acids gave moderate FRET signals. The 150 amino acid charged-plus-PQ construct gave a barely detectable FRET signal. FRET efficiency was calculated from the decreased donor emission to estimate the distance between donor and acceptor. The donor-acceptor distance varied for unstructured inserts of the same length, suggesting that they had variable stiffness (persistence length). We conclude that GFP-based FRET can be useful for studying intrinsically unstructured proteins, and we present a range of calibrated protein inserts to experimentally determine the distances that can be studied.

ALK is a receptor tyrosine kinase with an oncogenic role in various types of human malignancies. Despite constitutive activation of the kinase through gene alterations, such as chromosomal translocation, gene amplification or mutation, treatments with kinase inhibitors invariably lead to the development of resistance. Aiming to develop new tools for ALK targeting, we took advantage of our previous demonstration identifying ALK as a dependence receptor, implying that in the absence of ligand the kinase-inactive ALK triggers or enhances apoptosis. Here, we synthesized peptides mimicking the proapoptotic domain of ALK and investigated their biological effects on tumor cells. We found that an ALK-derived peptide of 36 amino acids (P36) was cytotoxic for ALK-positive anaplastic large-cell lymphoma and neuroblastoma cell lines. In contrast, ALK-negative tumor cells and normal peripheral blood mononuclear cells were insensitive to P36. The cytotoxic effect was due to caspase-dependent apoptosis and required N-myristoylation of the peptide. Two P36-derived shorter peptides as well as a cyclic peptide also induced apoptosis. Surface plasmon resonance and mass spectrometry analysis of P36-interacting proteins from two responsive cell lines, Cost lymphoma and SH-SY5Y neuroblastoma, uncovered partners that could involve p53-dependent signaling and pre-mRNA splicing. Furthermore, siRNA-mediated knockdown of p53 rescued these cells from P36-induced apoptosis. Finally, we observed that a treatment combining P36 with the ALK-specific inhibitor crizotinib resulted in additive cytotoxicity. Therefore, ALK-derived peptides could represent a novel targeted therapy for ALK-positive tumors.

International audience; Epithelium repair, crucial for restoration of alveolo-capillary barrier integrity, is orchestrated by various cytokines and growth factors. Among them keratinocyte growth factor plays a pivotal role in both cell proliferation and migration. The urokinase plasminogen activator (uPA) system also influences cell migration through proteolysis during epithelial repair. In addition, the complex formed by uPAR-uPA and matrix-bound plasminogen activator inhibitor type-1 (PAI-1) exerts nonproteolytic roles in various cell types. Here we present new evidence about the dual role of PAI-1 under keratinocyte growth factor stimulation using an in vitro repair model of rat alveolar epithelial cells. Besides proteolytic involvement of the uPA system, the availability of matrix-bound-PAI-1 is also required for an efficient healing. An unexpected decrease of healing was shown when PAI-1 activity was blocked. However, the proteolytic action of uPA and plasmin were still required. Moreover, immediately after wounding, PAI-1 was dramatically increased in the newly deposited matrix at the leading edge of wounds. We thus propose a dual role for PAI-1 in epithelial cell wound healing, both as a soluble inhibitor of proteolysis and also as a matrix-bound regulator of cell migration. Matrix-bound PAI-1 could thus be considered as a new member of the matricellular protein family.

In order to understand the sensitivity of alveolar macrophages (AMs) to substrate properties, we have developed a new model of macrophages cultured on substrates of increasing Young's modulus: (i) a monolayer of alveolar epithelial cells representing the supple (approximately 0.1 kPa) physiological substrate, (ii) polyacrylamide gels with two concentrations of bis-acrylamide representing low and high intermediate stiffness (respectively 40 kPa and 160 kPa) and, (iii) a highly rigid surface of plastic or glass (respectively 3 MPa and 70 MPa), the two latter being or not functionalized with type I-collagen. The macrophage response was studied through their shape (characterized by 3D-reconstructions of F-actin structure) and their cytoskeletal stiffness (estimated by transient twisting of magnetic RGD-coated beads and corrected for actual bead immersion). Macrophage shape dramatically changed from rounded to flattened as substrate stiffness increased from soft ((i) and (ii)) to rigid (iii) substrates, indicating a net sensitivity of alveolar macrophages to substrate stiffness but without generating F-actin stress fibers. Macrophage stiffness was also increased by large substrate stiffness increase but this increase was not due to an increase in internal tension assessed by the negligible effect of a F-actin depolymerizing drug (cytochalasine D) on bead twisting. The mechanical sensitivity of AMs could be partly explained by an idealized numerical model describing how low cell height enhances the substrate-stiffness-dependence of the apparent (measured) AM stiffness. Altogether, these results suggest that macrophages are able to probe their physical environment but the mechanosensitive mechanism behind appears quite different from tissue cells, since it occurs at no significant cell-scale prestress, shape changes through minimal actin remodeling and finally an AMs stiffness not affected by the loss in F-actin integrity.

Using Magnetic Twisting Cytometry (MTC) technique, we attempted to characterize in vitro the rigidity of the lining tissue covering the lung alveolar wall from its apical face. We purposely used a cellular model constituted by a monolayer of human alveolar epithelial cell (A549) over which microbeads, fixed to InterCellular Adhesion Molecule (ICAM-1), exert a controlled mechanical stress. ICAM-1 expression was induced by Tumor Necrosis Factor-alpha (TNF-alpha). Rigidity measurements, performed in the course of cytochalasin D depolymerization, reveal the force transmitter role of the transmembrane receptor ICAM-1 and demonstrate that ICAM-1 and F-actin linkages confers mechanical rigidity to the apical face of the epithelial cell monolayer resembling that provided by integrins. These results confirm the ability of MTC in identifying transmembrane mechanoreceptors in relation with F-actin. Molecular linkages between ICAM-1 and F-actin were observed by spatial visualisations of the structure after double staining of F-actin and anti ICAM-1 antibody through confocal microscopy.

Human bronchial epithelial (HBE) cells adhere to underlying extracellular matrix (ECM) via integrin-type transmembrane receptors. Integrins link the ECM to the cytoskeleton (CSK), establishing a mechanical continuum by which forces are transmitted between the outside and the inside of the cells. The present study investigates the time course of global and actin CSK stiffness of HBE cells (16HBE14o-) growing on various matrix substrates as a function of culture time until confluence, and the concomitant time course of F-actin and adhesion molecule distribution. Our results showed a progressive increase in actin CSK stiffness from cell seeding to confluence, related to acquisition of highly polymerized cortical and cytosolic F-actin organization and up-regulation of certain matrix ligands, such as beta 1-, alpha 5-, and alpha v-integrin subunit expression. Moreover, compared to fibrillar type I collagen, reticular type IV collagen used as matrix substrate, appeared to amplify actin CSK stiffness of HBE confluent cells probably in relation to up-regulation of alpha 3-integrin subunit. Taken together, these results support the concept of a close interaction among actin CSK stiffness, structural actin organization, specific integrin molecule involvement, cell spreading, and extracellular matrix.

International audience; Epithelia play a key role as protective barriers, and mechanisms of repair are crucial for restoring epithelial barrier integrity, especially in the lung. Cell spreading and migration are the first steps of reepithelialization. Keratinocyte growth factor (KGF) plays a key role in lung epithelial repair and protects against various injuries. We hypothesized that KGF may protect the lung not only by inducing proliferation but also by promoting epithelial repair via enhanced epithelial cell migration. In an in vitro wound-healing model, we found that KGF enhanced wound closure by 33%. KGF acted primarily by inducing lamellipodia emission (73.2 +/- 3.9% of KGF-treated cells had lamellipodia vs 61.3 +/- 3.4% of control cells) and increasing their relative surface area (59 +/- 2.7% with KGF vs 48 +/- 2.0% in controls). KGF reduced cytoskeleton stiffness as measured by magnetic twisting cytometry and increased cell motility (5.8 +/- 0.42 microm/h with KGF vs 3.7 +/- 0.41 microm/h in controls). KGF-increased cell motility was associated with increased fibronectin deposition during wound closure and with fibronectin reorganization into fibrils at the rear of the cells. Taken together, our findings strongly suggest that KGF may promote epithelial repair through several mechanisms involved in cell migration.

The corneal epithelium and epidermis form a complex protective barrier against environmental aggressions. In the epidermis, the terminal differentiation program of keratinocytes is well known and largely provided by the stratum corneum, developed in the final stage of keratinocyte differentiation. In contrast, the differentiation of the corneal epithelial cells is yet poorly studied. Despite that, in some corneal disorders, the first manifestations appear in this tissue. Therefore, further research on the differentiation process of the corneal epithelial cells would significantly improve the early diagnosis and management of patients affected by corneal-related disorders like keratoconus. Keratoconus is a multifactorial corneal ectasia associated with numerous surface changes causing a gradual and irreversible degradation of vision. The aim of my work was to characterize the terminal differentiation program of the corneal epithelium in controls versus patients with keratoconus. Firstly, I analyzed the expression of three groups of genes involved in various steps of the corneal epidermal differentiation: 1) genes that encoded keratins, 2) genes that encoded junctional complex proteins and 3) genes that coded for components of the cornified envelope, a pericellular and highly resistant structure that replaces the plasma membrane of keratinocytes during the last step of their differentiation, by means of RT-qPCR, Western Blot and immunohistology. Previous studies demonstrated that many of these genes were shown to be expressed in the cornea; however no data was available for those encoding corneodesmosin, filaggrin, hornerin and keratins 1/10. In parallel, I investigated the differential expression of the selected genes in the corneal epithelium in two cohorts of keratoconus patients (mild versus severe). I showed that the expression of keratin 16 was absent in the corneas of patients with keratoconus in comparison to controls. In line with this, I discovered that the expression and localization of several genes encoding cornified envelope components (such as involucrin, the Small Proline-Rich Proteins (SPRRs) and proteins of desmosomal plaques) was down-regulated and abnormal, respectively, in keratoconus. Surprisingly, cornified envelopes are not produced by the differentiated corneal epithelial cells. SPRRs have been reported to quench the reactive oxygen species in vitro, therefore we hypothesized that they might play a role in the corneal epithelium. Subsequently, I analyzed the expression of the main genes involved in the NRF2-pathway of oxidative stress protection such as the heme oxygenases 1 and 2, the nuclear factor erythroid 2-related factor 2, the lysyl oxidase, KEAP1, among others. Many of them were under-expressed in the keratoconus cornea epitheliums, finding that supports the involvement of oxidative stress in the progression of the disease. My findings allowed, on one hand, the characterization of the differentiation program of corneal epithelial cells and, on the other hand, the better understanding of the pathophysiology of keratoconus by proposing the involvement of mechanical weakness and a deficit in the oxidative stress response.This could improve the management of the keratoconic patients and may provide new insights for its prevention and treatment in the future., L'épithélium cornéen, tout comme l'épiderme, forme une barrière complexe qui protège l'organisme contre les agressions de l'environnement. Dans l'épiderme, cette fonction est assurée en grande partie par la couche cornée qui correspond à l'étape finale, de mieux en mieux caractérisée, de la différenciation du kératinocyte. Par contre, le programme de différenciation de l'épithélium de la cornée est peu étudié. Pourtant, les premières manifestations de diverses pathologies cornéennes apparaissent au niveau de l'épithélium, et leur dépistage précoce pourrait modifier notablement leur prise en charge. Cela pourrait être le cas du kératocône, une ectasie cornéenne plurifactorielle et rare, associée à de nombreux remaniements de surface engendrant une dégradation progressive et irréversible de la vision. L'objectif de mon travail de thèse a été de caractériser le programme de différenciation terminale de l'épithélium cornéen en situation normale et dans le cas du kératocône en prenant comme référence les gènes de différenciation tardive de l' épiderme. Tout d'abord, j'ai analysé l'expression de 3 groupes de gènes représentatifs des étapes de la différenciation épidermique (gènes des kératines, des protéines des complexes jonctionnels et des composants de l'enveloppe cornée, une structure péricellulaire résistante qui remplace la membrane plasmique des kératinocytes épidermiques lors de l'étape ultime de leur différenciation) dans l'épithélium cornéen contrôle en utilisant des techniques de RT-qPCR, Western Blot et immunohistologie. J'ai montré que l'épithélium de la cornée exprime un grand nombre des gènes de l'épiderme, mais pas ceux de la cornéodesmosine, la filaggrine, l'hornerine et des kératines 1/10. En parallèle, j'ai recherché les mêmes gènes dans l'épithélium de la cornée de deux cohortes de patients atteints de kératocône. J'ai montré que la kératine 16 est exprimée dans les cornées contrôles et disparaît dans le kératocône. Curieusement, de nombreux gènes codant pour les composants de l'enveloppe cornée sont exprimés dans l'épithélium cornéen comme ceux de l'involucrine, des Small Proline- Rich Proteins (SPRRs) et des protéines de la plaque desmosomale. De plus leur expression diminue dans l'épithélium cornéen kératoconique, avec une localisation subcellulaire différente pour l'involucrine. Pourtant, l'enveloppe cornée n'est pas produite par les cellules épithéliales différenciées de la cornée. Les protéines SPRRs étant aussi impliquées dans la détoxification des espèces réactives de l'oxygène in vitro, nous avons supposé qu'elles jouent ce rôle dans l'épithélium cornéen. Enfin, j'ai complété mes analyses avec l'étude des principaux éléments de la voie NRF2 de protection au stress oxydatif : heme oxygenases 1 et 2, nuclear factor erythroid 2-related factor 2, lysyl oxydase, KEAP1, etc. Plusieurs de ces gènes sont exprimés plus faiblement dans les cornées du kératocône par rapport aux cornées contrôles. Ces résultats ont permis, d'une part, de caractériser le programme de différenciation des cellules épithéliales de la cornée et, d'autre part, de mieux comprendre la physiopathologie du kératocône en proposant l'implication d'un déficit de la réponse au stress oxydatif.

The corneal epithelium and epidermis form a complex protective barrier against environmental aggressions. In the epidermis, the terminal differentiation program of keratinocytes is well known and largely provided by the stratum corneum, developed in the final stage of keratinocyte differentiation. In contrast, the differentiation of the corneal epithelial cells is yet poorly studied. Despite that, in some corneal disorders, the first manifestations appear in this tissue. Therefore, further research on the differentiation process of the corneal epithelial cells would significantly improve the early diagnosis and management of patients affected by corneal-related disorders like keratoconus. Keratoconus is a multifactorial corneal ectasia associated with numerous surface changes causing a gradual and irreversible degradation of vision. The aim of my work was to characterize the terminal differentiation program of the corneal epithelium in controls versus patients with keratoconus. Firstly, I analyzed the expression of three groups of genes involved in various steps of the corneal epidermal differentiation: 1) genes that encoded keratins, 2) genes that encoded junctional complex proteins and 3) genes that coded for components of the cornified envelope, a pericellular and highly resistant structure that replaces the plasma membrane of keratinocytes during the last step of their differentiation, by means of RT-qPCR, Western Blot and immunohistology. Previous studies demonstrated that many of these genes were shown to be expressed in the cornea; however no data was available for those encoding corneodesmosin, filaggrin, hornerin and keratins 1/10. In parallel, I investigated the differential expression of the selected genes in the corneal epithelium in two cohorts of keratoconus patients (mild versus severe). I showed that the expression of keratin 16 was absent in the corneas of patients with keratoconus in comparison to controls. In line with this, I discovered that the expression and localization of several genes encoding cornified envelope components (such as involucrin, the Small Proline-Rich Proteins (SPRRs) and proteins of desmosomal plaques) was down-regulated and abnormal, respectively, in keratoconus. Surprisingly, cornified envelopes are not produced by the differentiated corneal epithelial cells. SPRRs have been reported to quench the reactive oxygen species in vitro, therefore we hypothesized that they might play a role in the corneal epithelium. Subsequently, I analyzed the expression of the main genes involved in the NRF2-pathway of oxidative stress protection such as the heme oxygenases 1 and 2, the nuclear factor erythroid 2-related factor 2, the lysyl oxidase, KEAP1, among others. Many of them were under-expressed in the keratoconus cornea epitheliums, finding that supports the involvement of oxidative stress in the progression of the disease. My findings allowed, on one hand, the characterization of the differentiation program of corneal epithelial cells and, on the other hand, the better understanding of the pathophysiology of keratoconus by proposing the involvement of mechanical weakness and a deficit in the oxidative stress response.This could improve the management of the keratoconic patients and may provide new insights for its prevention and treatment in the future.; L'épithélium cornéen, tout comme l'épiderme, forme une barrière complexe qui protège l'organisme contre les agressions de l'environnement. Dans l'épiderme, cette fonction est assurée en grande partie par la couche cornée qui correspond à l'étape finale, de mieux en mieux caractérisée, de la différenciation du kératinocyte. Par contre, le programme de différenciation de l'épithélium de la cornée est peu étudié. Pourtant, les premières manifestations de diverses pathologies cornéennes apparaissent au niveau de l'épithélium, et leur dépistage précoce pourrait modifier notablement leur prise en charge. Cela pourrait être le cas du kératocône, une ectasie cornéenne plurifactorielle et rare, associée à de nombreux remaniements de surface engendrant une dégradation progressive et irréversible de la vision. L'objectif de mon travail de thèse a été de caractériser le programme de différenciation terminale de l'épithélium cornéen en situation normale et dans le cas du kératocône en prenant comme référence les gènes de différenciation tardive de l' épiderme. Tout d'abord, j'ai analysé l'expression de 3 groupes de gènes représentatifs des étapes de la différenciation épidermique (gènes des kératines, des protéines des complexes jonctionnels et des composants de l'enveloppe cornée, une structure péricellulaire résistante qui remplace la membrane plasmique des kératinocytes épidermiques lors de l'étape ultime de leur différenciation) dans l'épithélium cornéen contrôle en utilisant des techniques de RT-qPCR, Western Blot et immunohistologie. J'ai montré que l'épithélium de la cornée exprime un grand nombre des gènes de l'épiderme, mais pas ceux de la cornéodesmosine, la filaggrine, l'hornerine et des kératines 1/10. En parallèle, j'ai recherché les mêmes gènes dans l'épithélium de la cornée de deux cohortes de patients atteints de kératocône. J'ai montré que la kératine 16 est exprimée dans les cornées contrôles et disparaît dans le kératocône. Curieusement, de nombreux gènes codant pour les composants de l'enveloppe cornée sont exprimés dans l'épithélium cornéen comme ceux de l'involucrine, des Small Proline- Rich Proteins (SPRRs) et des protéines de la plaque desmosomale. De plus leur expression diminue dans l'épithélium cornéen kératoconique, avec une localisation subcellulaire différente pour l'involucrine. Pourtant, l'enveloppe cornée n'est pas produite par les cellules épithéliales différenciées de la cornée. Les protéines SPRRs étant aussi impliquées dans la détoxification des espèces réactives de l'oxygène in vitro, nous avons supposé qu'elles jouent ce rôle dans l'épithélium cornéen. Enfin, j'ai complété mes analyses avec l'étude des principaux éléments de la voie NRF2 de protection au stress oxydatif : heme oxygenases 1 et 2, nuclear factor erythroid 2-related factor 2, lysyl oxydase, KEAP1, etc. Plusieurs de ces gènes sont exprimés plus faiblement dans les cornées du kératocône par rapport aux cornées contrôles. Ces résultats ont permis, d'une part, de caractériser le programme de différenciation des cellules épithéliales de la cornée et, d'autre part, de mieux comprendre la physiopathologie du kératocône en proposant l'implication d'un déficit de la réponse au stress oxydatif.