Safety of an Intracameral Fixed Combination for Mydriasis and Intraocular Anaesthesia During Cataract Surgery.

Purpose: To compare the safety of a standardized, commercially available intracameral combination of mydriatics and anesthetic (ICMA) with a reference topical mydriatic regimen for cataract surgery. Patients and Methods: The safety results from two international, randomized, controlled clinical studies were combined to compare ICMA at the beginning of cataract surgery (ICMA group) to the reference topical mydriatic regimen (reference group). Data were collected on ocular and systemic adverse events, corneal and anterior chamber examination, endothelial cell density, retinal thickness and visual acuity. Analysis was performed on a pooled safety set from both studies, preoperatively and up to 1 month postoperatively. Results: 342 patients received ICMA and 318 the reference topical regimen. Ocular adverse events were reported in 17.0% of patients in the ICMA group and 18.6% in the reference group. No difference was shown between groups in endothelial cell density (2208 ± 498 cells/mm2 for ICMA group versus 2241 ± 513 cells/mm2 for the reference group; p=0.547) and retinal thickness (change from baseline less than 50 µm in 94.7% versus 95.0% of patients, respectively) at 1 month postoperatively. At 1-day post-surgery, less patients in the ICMA group had moderate or severe (Grades 2 and 3) superficial punctate corneal staining (3.9% versus 7.0% for the reference group; p=0.064). Postoperatively, some ocular symptoms were also less frequently reported in the ICMA group. Best-corrected visual acuity increased in 96.0% of patients in the ICMA group and 95.8% in the reference group at 1 month. Conclusion: ICMA injection at the beginning of cataract surgery was demonstrated to be safe and may also provide perioperative and postoperative advantages over the standard topical mydriatic regimen.

Multiple Sclerosis-Associated Uveitis: A Case Report of Refractory Bilateral Chronic Granulomatous Panuveitis Successfully Treated with Tocilizumab.

PURPOSE: To report a case of uveitis associated with multiple sclerosis (MS) that was refractory to multiple lines of therapy but achieved remission with tocilizumab. METHODS: We conducted a retrospective analysis of the patient's medical record including clinical, biological and imaging data. RESULTS: A 33-year-old female patient with a history of MS inactive for 5 years on teriflunomide, and no significant medical or ophthalmological history, presented with bilateral granulomatous panuveitis. Initial examination revealed a visual acuity of 0.4 logMAR and 1.3 logMAR in the right eye and the left eye, respectively, along with a significant anterior chamber flare in both eyes, posterior synechiae, large granulomatous keratic precipitates, bilateral vitritis, bilateral macular edema with foveolar pigment epithelial detachment, and significant bilateral venous and arterial vasculitis. The patient underwent several lines of treatment, all of which proved unsuccessful, including corticosteroids alone or in combination with azathioprine, methotrexate, and mycophenolate mofetil. As a final therapeutic option, tocilizumab was initiated, leading to the remission of uveitis. One year later, the uveitis remained inactive under a 5 mg/day prednisone regimen. CONCLUSIONS: Tocilizumab appears to be an efficient option for managing uveitis associated with MS and may be a valuable choice for clinicians dealing with such cases.

Inflammation of Dry Eye Syndrome: A Cellular Study of the Epithelial and Macrophagic Involvement of NFAT5 and RAGE.

Dry eye inflammation is a key step in a vicious circle and needs to be better understood in order to break it. The goals of this work were to, first, characterize alarmins and cytokines released by ocular surface cells in the hyperosmolar context and, second, study the role of NFAT5 in this process. Finally, we studied the potential action of these alarmins in ocular surface epithelial cells and macrophages via RAGE pathways. HCE and WKD cell lines were cultured in a NaCl-hyperosmolar medium and the expression of alarmins (S100A4, S100A8, S100A9, and HMGB1), cytokines (IL6, IL8, TNFα, and MCP1), and NFAT5 were assessed using RT-qPCR, ELISA and multiplex, Western blot, immunofluorescence, and luciferase assays. In selected experiments, an inhibitor of RAGE (RAP) or NFAT5 siRNAs were added before the hyperosmolar stimulations. HCE and WKD cells or macrophages were treated with recombinant proteins of alarmins (with or without RAP) and analyzed for cytokine expression and chemotaxis, respectively. Hyperosmolarity induced epithelial cell inflammation depending on cell type. NFAT5, but not RAGE or alarmins, participated in triggering epithelial inflammation. Furthermore, the release of alarmins induced macrophage migration through RAGE. These in vitro results suggest that NFAT5 and RAGE have a role in dry eye inflammation.

Efficacy of treatments for neurotrophic keratopathy: a systematic review and meta-analysis.

PURPOSE: Management of NK can be difficult, involving a range of treatments with variable efficacy. We conducted a systematic review and meta-analysis to evaluate the efficacy of medical and surgical treatments for neurotrophic keratitis (NK). METHOD: PubMed, Cochrane Library, Embase, ClinicalTrial.gov, and ScienceDirect were searched for studies assessing efficacy of NK treatments. We computed random-effect meta-analyses on corneal healing, time to complete healing, and visual acuity changes between baselines and after treatment, stratified on treatment classes. We followed the PRISMA guidelines (registration number CRD42021225721). RESULTS: We included 20 studies: 571 patients and 5 treatment classes (2 surgical and 3 non-surgical). The percentage of patients with complete corneal healing did not differ between specific treatments (nerve growth factor eyedrops (NGF), 75%, 95CI 46 to 104%; autologous serum (AS), 92%, 86 to 98%; neurotization, 99%, 95 to 103%; amniotic membrane transplantation (AMT), 86%, 78 to 94%). All specific treatments had better percentage of complete healing (p < 0.001) than non-specific treatment groups, i.e., mainly lubricants (23%, 14 to 32). Time to complete healing was 24.2 days (5.4 to 43.1) with NGF, 27.6 days (15.2 to 40.0) with AS, 117 days (28.8 to 205.2) with neurotization, and 16.4 days (11.1 to 21.7) with AMT. Only NGF and AMT improved visual acuity. Efficacy outcomes were not affected by sociodemographic (age, sex) nor severity of disease (Mackie stages). CONCLUSION: We confirmed the efficacy of specific treatments in NK. Further comparative trials are needed to investigate the medical and economic benefits of innovative therapies.

Complications of Adenoviral Keratoconjunctivitis in Ophthalmologists and Orthoptists: Epidemiology and Risk Factor, A Retrospective Questionnaire Analysis.

PURPOSE: The purpose of this study was to assess the medical history of adenoviral keratoconjunctivitis (AK) and subepithelial infiltrates (SEIs) among French ophthalmologists and orthoptists and the frequency of unreported occupational diseases. We also described short-term and long-term consequences of AK and evaluated associated factors. METHODS: The REDCap questionnaire was diffused online several times over 7 consecutive months, from October 2019 to May 2020, through mailing lists (French Society of Ophthalmology, residents, and hospital departments), social networks, and by word of mouth. RESULTS: Seven hundred ten participants were included with a response rate of 6.2% for ophthalmologists, 3.8% for orthoptists, and 28.3% for ophthalmology residents. The medical history of AK was found in 24.1% (95% confidence interval 21%-27.2%) of respondents and SEI in 43.9% (36.5%-51.3%) of the AK population. In total, 87.1% (82.1%-92.1%) of AK occupational diseases were not declared. In total, 57.7% of respondents took 9.4 ± 6.2 days of sick leave, mostly unofficial, and 95.7% stopped surgeries for 13.0 ± 6.6 days. Among the AK population, 39.8% had current sequelae, with 17.5% having persistent SEIs, 19.9% using current therapy, and 16.4% experiencing continuing discomfort. SEIs were associated with wearing contact lenses (odds ratio 3.31, 95% confidence interval 1.19-9.21) and smoking (4.07, 1.30-12.8). Corticosteroid therapy was associated with a greater number of sequelae (3.84, 1.51-9.75). CONCLUSIONS: AK and SEI affect a large proportion of ophthalmologists and orthoptists, possibly for years, with high morbidity leading to occupational discomfort. Few practitioners asked for either to be recognized as an occupational disease. Associated factors would require a dedicated study.

A prospective multicentre study of intravitreal injections and ocular surface in 219 patients: IVIS study.

PURPOSE: To assess the impact of intravitreal injections (IVTI) on ocular surface of patients treated with multiple injections. METHODS: Prospective, tricentric study conducted in patients treated with unilateral IVTI. An asepsis protocol with povidone-iodine was used for all patients during IVTI. The primary endpoint was the difference between the pre-IVTI Ocular Surface Disease Index (OSDI 1) score and that measured on day one (D1) post-IVTI (OSDI 2). Secondary endpoints were the evaluation of predictive factors for OSDI scores, pain assessment on D1, and the Lacrydiag® analysis of tears from the injected eye versus contralateral eye before IVTI. RESULTS: Two hundred and nineteen patients with a mean age of 75.9 ± 10 years were included. The mean OSDI2-OSDI1 difference was 19.2 ± 20.6 (p < 0.001). The mean noninvasive tear break-up time was 6.41 ± 4.59 seconds in the injected eye versus 7.36 ± 4.36 seconds in the contralateral eye (p < 0.001). In the multivariate analysis, the factors significantly associated with the OSDI 2 score were the OSDI 1 score (p < 0.001), the pain score on D1 (p < 0.001) the number of instilled glaucoma eye drop (p = 0.01) and a centre effect (centres 2 and 3 versus centre 1, p < 0.001). CONCLUSION: Our results confirm the impairment of the ocular surface and quality of life immediately after an IVTI. These results suggest 3 levels of action to improve the immediate tolerance: improving the basal status of the ocular surface, reducing the contact time with povidone-iodine that might be toxic to the surface, and improving immediate post-IVTI treatment.

Safety and efficacy of a standardized intracameral combination of mydriatics and anesthetic for cataract surgery in type-2 diabetic patients.

BACKGROUND: Cataract surgery in diabetics is more technically challenging due to a number of factors including poor intraoperative pupil dilation and a higher risk of vision threatening complications. This study evaluates the safety and efficacy of an intracameral combination of 2 mydriatics and 1 anesthetic (ICMA, Mydrane) for cataract surgery in patients with well-controlled type-2 diabetes. METHODS: Post-hoc subgroup analysis of a phase 3 randomized study, comparing ICMA to a conventional topical regimen. Data were collected from 68 centers in Europe and Algeria. Only well-controlled type-2 diabetics, free of pre-proliferative retinopathy, were included. The results for non-diabetics are also reported. The primary efficacy variable was successful capsulorhexis without additional mydriatic treatment. Postoperative safety included adverse events, endothelial cell density and vision. RESULTS: Among 591 randomized patients, 57 (9.6%) had controlled type 2 diabetes [24 (42.1%) in the ICMA Group and 33 (57.9%) in the Topical Group; intention-to-treat (ITT) set]. Among diabetics, capsulorhexis was successfully performed without additional mydriatics in 24 (96.0%; modified-ITT set) patients in the ICMA Group and 26 (89.7%) in the Topical Group. These proportions were similar in non-diabetics. No diabetic patient [1 (0.5%) non-diabetics] in the ICMA Group had a significant decrease in pupil size (≥3 mm) intraoperatively compared to 4 (16.0%; modified-ITT set) diabetics [16 (7.3%) non-diabetics] in the Topical group. Ocular AE among diabetics occurred in 2 (8.0%; Safety set) patients in the ICMA Group and 5 (16.7%) in the Topical Group. Endothelial cell density at 1 month postoperatively was similar between groups in diabetics (P = 0.627) and non-diabetics (P = 0.368). CONCLUSIONS: ICMA is effective and can be safely used in patients with well-controlled diabetes, with potential advantages compared to a topical regimen including reduced systemic risk, better corneal integrity and reduced risk of ocular complications. TRIAL REGISTRATION: The trial was registered at (reference # NCT02101359) on April 2, 2014.

Advanced Glycation End Products and Receptor (RAGE) Promote Wound Healing of Human Corneal Epithelial Cells.

Purpose: We used a human corneal epithelial cell (HCE) line to determine the involvement of the advanced glycation end products (AGEs) / receptor for AGEs (RAGE) couple in corneal epithelium wound healing. Methods: After wounding, HCE cells were exposed to two major RAGE ligands (HMGB1 and AGEs), and wound healing was evaluated using the in vitro scratch assay. Following wound healing, the HCE cells were used to study the influence of the RAGE ligands on HCE proliferation, invasion, and migration. Activation of the nuclear factor (NF)-κB signaling pathway by the AGEs/RAGE couple was tested using a luciferase reporter assay. Functional transcriptional regulation by this pathway was confirmed by quantification of expression of the connexin 43 target gene. For each experiment, specific RAGE involvement was confirmed by small interfering RNA treatments. Results: AGEs treatment at a dose of 100 µg/mL significantly improved the wound healing process in a RAGE-dependent manner by promoting cell migration, whereas HMGB1 had no effect. No significant influence of the AGEs/RAGE couple was observed on cell proliferation and invasion. However, this treatment induced an early activation of the NF-κB pathway and positively regulated the expression of the target gene, connexin 43, at both the mRNA and protein levels. Conclusions: Our results demonstrate that the RAGE pathway is activated by AGEs treatment and is involved in the promotion of corneal epithelial wound healing. This positive action is observed only during the early stages of wound healing, as illustrated by the quick activation of the NF-κB pathway and induction of connexin 43 expression.

Evaluating the cost-consequence of a standardized strategy for the etiological diagnosis of uveitis (ULISSE study).

MAIN OBJECTIVE: To prospectively assess the cost-consequence of a standardized diagnostic strategy as to compared to an open one for the etiological diagnosis of uveitis. DESIGN: This was a prospective, non-inferiority, multicentre, randomized controlled trial. METHODS: We included all consecutive patients with uveitis who had visited at least one of the Departments of Ophthalmology. In the standardized group, patients had a minimal work-up regardless of the type of uveitis (including evaluation of the CBC, ESR, C-reactive protein, tuberculin skin test, syphilis serology and chest X-ray). Depending on ophthalmological findings, further investigations could be performed. In the open strategy, ophthalmologists were free to order any kind of investigation. The main outcome was the mean cost per patient of each strategy. RESULTS: 903 uveitis patients were included from January, 2010 to May, 2013. The mean cost per patient of the standardized strategy was 182.97 euros [CI 95% (173.14; 192.80)], and the mean cost per patient of the open strategy was 251.75 euros [CI 95% (229.24; 274.25)]. Therefore, the mean cost per patient of the standardized strategy was significantly lower than the mean cost per patient of the open strategy (p<0.001). There were significantly fewer visits (p<0.001), fewer radiological procedures (p<0.004) and fewer laboratory investigations (p<0.001) in the standardized group. CONCLUSION: A standardized strategy is a cost-saving approach for the etiological diagnosis of uveitis.

Systemic exposure to intracameral vs topical mydriatic agents: in cataract surgery.

OBJECTIVE: The objective of this study was to compare systemic exposure to tropicamide/phenylephrine following intracameral or topical administration before cataract surgery. PATIENTS AND METHODS: Mydriatics exposure was calculated in patients randomized to intracameral fixed combination of mydriatics and anesthetic ([ICMA]: tropicamide 0.02%, phenylephrine 0.31%, and lidocaine 1%, N=271) or mydriatic eye drops ([EDs]: tropicamide 0.5% and phenylephrine 10%, N=283). Additional doses were permitted if required. Mydriatic plasma levels were determined by mass spectrometric HPLC in 15 patients per group before and after administration. RESULTS: Most ICMA patients (73.6%) received a single dose (200 µL) representing an exposure to tropicamide of 0.04 mg and phenylephrine of 0.62 mg. None of these patients received additional mydriatics. In the control group (three administrations), the exposure was 0.45 (11.3-fold higher than ICMA) and 10.2 (16.5-fold higher) mg. When additional ED was used in this group (9.2% of patients), it was 37.5-fold higher for tropicamide (10 drops, 1.5 mg) and 54.8-fold higher for phenylephrine (10 drops, 34 mg) than the recommended ICMA dose. Tropicamide plasma levels were not detectable at any time point in ICMA patients while it was detectable in all ED patients at 12 and 30 minutes. Phenylephrine was detectable in 14.3% of ICMA patients compared to all ED patients at least at one time point. More ED patients experienced a meaningful increase in blood pressure and/or heart rate (11.2% vs 6.0% of ICMA patients; P=0.03). CONCLUSION: Systemic exposure to tropicamide/phenylephrine was lower and cardiovascular (CV) effects were less frequent with ICMA. This could be of particular significance in patients at CV risk.

Pupil dilation dynamics with an intracameral fixed combination of mydriatics and anesthetic during cataract surgery.

PURPOSE: To compare the pupil dynamics of an intracameral combination of 2 mydriatics and 1 anesthetic (Mydrane) to a standard topical regimen for cataract surgery. SETTING: Sixty-two centers in Europe and 6 centers in Algeria. DESIGN: Prospective case series. METHODS: Pupil size measurements were performed in 2 randomized studies (phase 2 and phase 3) under masked conditions (recorded videography, masked reading center). The outcomes in the phase 2 study supported evaluation of the timeframe to obtain pupil dilation and the phase 3 study provided results on mydriasis stability. RESULTS: Phase 2 and phase 3 comprised 139 patients and 591 patients, respectively. After intracameral combination administration, 95% of the pupil dilation was achieved within a mean of 28.6 seconds ± 4.6 (SD). At the beginning of capsulorhexis creation, the mean pupil diameter was larger than 7.0 mm in both groups. The intraoperative pupil diameter remained stable in the intracameral combination group and decreased in the topical group. The mean change in pupil size just before capsulorhexis to the end of surgery (just before cefuroxime injection) was -0.22 ± 0.72 mm and -1.67 ± 0.98 mm, respectively. No clinically significant change in pupil diameter (change <1.0 mm) occurred in the majority of the intracameral combination group (89.3%) compared with the topical group (26.8%). CONCLUSIONS: Intracameral combination of 2 mydriatics and 1 anesthetic is an alternative to topical mydriatics for cataract surgery. The prompt onset of pupil dilation and the stable mydriasis induced by this drug combination improved the intraoperative conditions during crucial steps, such as intraocular lens implantation.

Retinoic Acid Engineered Amniotic Membrane Used as Graft or Homogenate: Positive Effects on Corneal Alkali Burns.

Purpose: Alkali burns are the most common, severe chemical ocular injuries, their functional prognosis depending on corneal wound healing efficiency. The purpose of our study was to compare the benefits of amniotic membrane (AM) grafts and homogenates for wound healing in the presence or absence of previous all-trans retinoic acid (atRA) treatment. Methods: Fifty male CD1 mice with reproducible corneal chemical burn were divided into five groups, as follows: group 1 was treated with saline solution; groups 2 and 3 received untreated AM grafts or grafts treated with atRA, respectively; and groups 4 and 5 received untreated AM homogenates or homogenates treated with atRA, respectively. After 7 days of treatment, ulcer area and depth were measured, and vascular endothelial growth factor (VEGF) and matrix metalloproteinase 9 (MMP-9) were quantified. Results: AM induction by atRA was confirmed via quantification of retinoic acid receptor ß (RARß), a well-established retinoic acid-induced gene. Significant improvements of corneal wound healing in terms of ulcer area and depth were obtained with both strategies. No major differences were found between the efficiency of AM homogenates and grafts. This positive action was increased when AM was pretreated with atRA. Furthermore, AM induced a decrease in VEGF and MMP-9 levels during the wound healing process. The atRA treatment led to an even greater decrease in the expression of both proteins. Conclusions: Amnion homogenate is as effective as AM grafts in promoting corneal wound healing in a mouse model. A higher positive effect was obtained with atRA treatment.

Pharmacokinetic evaluation of diquafosol tetrasodium for the treatment of Sjögren's syndrome.

INTRODUCTION: Dry eye is a multifactorial disease of the ocular surface causing ocular discomfort and visual impairment for the patient. A variety of topical and systemic drugs are available to treat dry eye. Conventional treatments are limited to tear supplementation or improvement of ocular surface inflammation by the use of corticosteroids or cyclosporine A. Treatment of severe dry eye associated with Sjögren's syndrome (SS) is even more challenging and is designed to improve the quality and quantity of tear fluid. Diquafosol tetrasodium , a P2Y2 purinergic receptor agonist, acts via a novel mechanism by activating P2Y2 receptors of the ocular surface. AREAS COVERED: The aim of this review is to summarize the pharmacokinetics, and pharmacological and clinical data of 3% diquafosol tetrasodium ophthalmic solution in patients with dry eye, particularly SS. The mechanisms of impaired ocular surface due to severe dry eye, as defined by the International Dry Eye Workshop, are analyzed. EXPERT OPINION: Diquafosol tetrasodium provides a novel mode of action in dry eye syndrome, including SS, by stimulating the quantity and quality of tear fluid secretion via various mechanisms. In clinical trials, 3% Diquafosol tetrasodium ophthalmic solution demonstrated a good safety profile and exhibited efficacy with clinical improvement of the ocular surface in dry eye including SS.

Antioxidants intake and dry eye syndrome: a crossover, placebo-controlled, randomized trial.

PURPOSE: To assess whether an orally administered antioxidant dietary supplement could improve the objective clinical signs and alleviate the subjective symptoms of dry eye syndrome. METHODS: Twenty-four subjects diagnosed with dry eye syndrome were randomized in a crossover, double-blind, controlled, randomized study to receive a placebo or an antioxidants combination (Oxybiane) for 12 weeks. In all subjects, break-up time (BUT) test, Schirmer test, ocular symptoms (sore eyes, burning, itching, sensation of foreign object in the eye, photophobia, sticky eyes, and redness), visual comfort, and general well-being were evaluated weekly. RESULTS: After 12 weeks of supplementation with Oxybiane, both the BUT scores (27.3%+/-8.4% with Oxybiane versus 3.61%+/-4.3% with the placebo, p=0.017) and the Schirmer scores (26.9%+/-14.2% with Oxybiane versus -4.7%+/-3.4% with the placebo, p=0.037) were significantly increased. A significantly improvement was also observed considering subjective clinical symptoms such as burning (p=0.031), itching (p=0.027), sensation of foreign body in eye (p=0.030), and redness (p=0.043). CONCLUSIONS. Supplementation with oral antioxidants can improve both tear stability and quantity but also subjective clinical signs.

Molecular and metabolic retinoid pathways in the human ocular surface.

PURPOSE: To maintain its integrity, the human ocular surface (cornea and conjunctiva) has an absolute requirement for vitamin A and its active derivatives, the retinoic acids. These retinoids regulate transcriptional levels of target genes through the activation of members of a super-family of ligand-dependant nuclear receptors that feature retinoic acid receptors (RAR) alpha, beta, and gamma as well as retinoid X receptors (RXR) alpha, beta, and gamma. The expression patterns of these receptors have been partial characterized in rabbit, mouse, and human cornea and conjunctiva, but systematic tissue and cellular expression of the three RARs and three RXRs had to be completed at the adult human ocular surface. The first objective of our work was to define their expression patterns in term of genes and proteins for total human conjunctiva, cornea, and the major cell types comprising the adult human ocular surface. The second objective was to demonstrate the presence of different enzymes transforming vitamin A to retinoic acid and the functionality of this metabolic pathway in the corneal epithelium. METHODS: Total mRNA was extracted from human total cornea, conjunctiva, corneal epithelial cells (primary culture and established cell line), corneal keratocytes (primary culture), corneal endothelial cells (established cell line), and conjunctival epithelial cells (established cell line) and was submitted to reverse transcription-polymerase chain reaction (RT-PCR) analysis to determine the expression patterns of the RARs and RXRs using specific primers. Immunological staining (via histochemistry and cellular chemistry) experiments were performed to better localize RAR and RXR proteins in the ocular surface at tissue and cellular levels. We also checked mRNA expression of cellular retinol binding proteins (CRBPs) and cellular retinoic acid binding proteins (CRABPs) with the enzymes involved in retinoic acid generation, i.e., alcohol dehydrogenases (ADHs) and retinal dehydrogenases (RALDHs) or degradation (Cyp26 family members). The enzymatic generation of functional retinoids was confirmed using epithelial corneal cells treated with specific inhibitors of retinol metabolism. RESULTS: RAR alpha, RAR gamma, and RXR alpha are expressed in the cornea, conjunctiva, and all of their constitutive cells, whereas RXR gamma and RXR beta were never detected in the cornea or conjunctiva. RAR beta was absent in primary cultures of corneal keratinocytes. ADH3, ADH4, dehydrogenase/reductase (SDR family) 4 (DHRS4), dehydrogenase/reductase (SDR family) 9 (DHRS9), RALDH1, and RALDH3 are expressed in the ocular surface, as were the retinoid-binding proteins CRBP1, CRABP1, and CRABP2. Retinol dehydrogenase 4 (RODH4) was only detected in the conjunctiva. Corneal epithelial cells convert retinol into retinoic acid using an enzymatic pathway. CONCLUSIONS: For the first time, we have established an exhaustive description of the expressions patterns of RARs, RXRs, ADHs, RALDHs, CRBP, and CRABPs in the human ocular surface. Our results for the human ocular surface demonstrated the presence of all the metabolic and molecular actors of the retinoic acid signaling pathway. We also demonstrated the enzymatic conversion of retinol into active retinoids in the corneal environment.

Kruppel-like factor 6 (KLF6) affects the promoter activity of the alpha1-proteinase inhibitor gene.

PURPOSE: Keratoconus is a progressive disease that thins and scars the cornea. In keratoconus corneas, levels of degradative enzymes, including lysosomal acid phosphatase (LAP) and cathepsin B, are elevated, and those of inhibitors alpha1-proteinase inhibitor (alpha1-PI) and alpha2-macroglobulin (alpha2-M) are reduced. The present study explored the possible involvement in keratoconus of Krüppel-like factor 6 (KLF6), a transcription factor previously described to be essential for the integrity of the corneal epithelium. The transcript and proteins level of KLF6 and its action in regulating the genes affected in keratoconus were examined in this study. METHODS: Semiquantitative RT-PCR, Western blot analysis, immunofluorescence and in situ hybridization were used to investigate the expression of KLF6 mRNA and protein in normal and keratoconus corneas. Modulation by KLF6 of the promoter activity of alpha1-PI, LAP, cathepsin B, and alpha2-M genes was studied after transient transfection of KLF6 expression plasmid into corneal epithelial cells using promoter-reporter gene assays. Chromatin immunoprecipitation (ChIP) assays were performed to confirm the interactions between KLF6 and promoters of the genes affected in keratoconus. RESULTS: A global increased expression of the transcription factor KLF6 in terms of mRNAs and proteins was observed in total cornea and/or the epithelium in a substantial number of the keratoconus specimens. The promoter activity of the human alpha1-PI gene was suppressed by expression of KLF6 in corneal epithelial cells. The ChIP assay confirmed a physical interaction between KLF6 and the alpha1-PI promoter. CONCLUSIONS: Transcription factor KLF6 downregulates the alpha1-PI gene in corneal epithelial cells and may thereby be involved in keratoconus.

Developmentally regulated expression of KLF6 in the mouse cornea and lens.

PURPOSE: To examine the expression of transcription factor Kruppel-like factor-6 (KLF6) in the cornea and lens of mouse eyes throughout developmental stages. METHODS: C57BL/6 mice were set up for timed mating. Embryos on embryonic day (E)10.5, E12.5, E15.5, and E18.5 and eyes from mice on postnatal day (P)0, P7, P11, P15, P30, and P60 were collected for immunofluorescence staining. Transcript of KLF6 was detected by in situ hybridization with digoxigenin-labeled RNA probes. Relative quantitative reverse transcription (RT)-PCR was performed to measure the KLF6 transcript level in eyes enucleated at embryonic stages, and the corneas and lenses isolated at postnatal stages. RESULTS: Staining for the KLF6 protein was observed at E10.5 in the lens pit and at E12.5 in the ectoderm, the mesenchyme, and the lens epithelium. Nuclear staining for KLF6 protein was evident at E15.5 in the corneal epithelium and the stroma. The staining was abundant between E18.5 and P60. In the lens epithelium, nuclear staining was detected at P0, P7, and P11. The staining intensity declined subsequently. The KLF6 transcript was found in the lens pit at E10.5 and in the ectoderm and mesenchyme at E12.5. The KLF6 mRNA level in corneal layers remained relatively constant until P60. In the lens epithelium, it was high during embryonic stages but decreased with development. CONCLUSIONS: KLF6 is expressed in both the cornea and the lens in mouse eyes. Its expression in the lens is temporally regulated, suggesting that it has a central role in lens development.

Regulation of corneal keratin-12 gene expression by the human Krüppel-like transcription factor 6.

PURPOSE: The keratin-12 (K12) protein is essential for the integrity of the corneal epithelium. This study was conducted to investigate the possible involvement of Krüppel-like factor 6 (KLF6) in the corneal regulation of K12 gene expression, in view of the presence of one KLF6 potential binding site in the human K12 promoter and the known role of KLF6 in regulating keratin gene expression. METHODS: RT-PCR, Western blot analysis, and immunolocalization experiments were used to investigate the expression of KLF6 mRNA and protein in five human total corneas. The same experimental design was used to explore human corneal epithelial (HCE) cells in 20 patients and a HCE cell line. The ability of the KLF6 protein to modulate K12 promoter activity was studied in the HCE cell line, by transient transfections with a KLF6 expression plasmid and promoter-reporter gene assays. Gel-shift assays were performed to confirm the interactions between the KLF6 protein and specific sequences of the K12 promoter. RESULTS: The presence of KLF6 transcripts and proteins in human total corneal extracts was demonstrated. Immunohistofluorescence experiments showed positive staining specifically present in the corneal epithelial layer. KLF6 transcripts and proteins were also present in corneal epithelial cells in 20 patients and the HCE cell line. Transient transfections of KLF6 showed statistical transactivation of the K12 promoter in HCE cells. The gel-shift assay showed a physical interaction between KLF6 and the K12 promoter. CONCLUSIONS: The expression of KLF6 in HCE cells and its role in the regulation of K12 gene expression were demonstrated.