Cornea

Uchino Y, Woodward AM, Mauris J, Peterson K, Verma P, Nilsson UJ, Rajaiya J, Argüeso P. Galectin-3 is an amplifier of the interleukin-1β-mediated inflammatory response in corneal keratinocytes. Immunology 2018;154(3):490-499.Abstract
Interleukin-1β (IL-1β) is a potent mediator of innate immunity commonly up-regulated in a broad spectrum of inflammatory diseases. When bound to its cell surface receptor, IL-1β initiates a signalling cascade that cooperatively induces the expression of canonical IL-1 target genes such as IL-8 and IL-6. Here, we present galectin-3 as a novel regulator of IL-1β responses in corneal keratinocytes. Using the SNAP-tag system and digitonin semi-permeabilization, we show that recombinant exogenous galectin-3 binds to the plasma membrane of keratinocytes and is internalized into cytoplasmic compartments. We find that exogenous galectin-3, but not a dominant negative inhibitor of galectin-3 polymerization lacking the N-terminal domain, exacerbates the response to IL-1β by stimulating the secretion of inflammatory cytokines. The activity of galectin-3 could be reduced by a novel d-galactopyranoside derivative targeting the conserved galactoside-binding site of galectins and did not involve interaction with IL-1 receptor 1 or the induction of endogenous IL-1β. Consistent with these observations, we demonstrate that small interfering RNA-mediated suppression of endogenous galectin-3 expression is sufficient to impair the IL-1β-induced secretion of IL-8 and IL-6 in a p38 mitogen-activated protein kinase-independent manner. Collectively, our findings provide a novel role for galectin-3 as an amplifier of IL-1β responses during epithelial inflammation through an as yet unidentified mechanism.
Cruzat A, Gonzalez-Andrades M, Mauris J, AbuSamra DB, Chidambaram P, Kenyon KR, Chodosh J, Dohlman CH, Argüeso P. Colocalization of Galectin-3 With CD147 Is Associated With Increased Gelatinolytic Activity in Ulcerating Human Corneas. Invest Ophthalmol Vis Sci 2018;59(1):223-230.Abstract
Purpose: Galectin-3 is a carbohydrate-binding protein known to promote expression of matrix metalloproteinases, a hallmark of ulceration, through interaction with the extracellular matrix metalloproteinase inducer CD147. The aim of this study was to investigate the distribution of galectin-3 in corneas of patients with ulcerative keratitis and to determine its relationship to CD147 and the presence of gelatinolytic activity. Methods: This was an observational case series involving donor tissue from 13 patients with active corneal ulceration and 6 control corneas. Fixed-frozen sections of the corneas were processed to localize galectin-3 and CD147 by immunofluorescence microscopy. Gelatinolytic activity was detected by in situ zymography. Results: Tissue from patients with active corneal ulceration showed a greater galectin-3 immunoreactivity in basal epithelia and stroma compared with controls. Immunofluorescence grading scores revealed increased colocalization of galectin-3 and CD147 in corneal ulcers at the epithelial-stromal junction and within fibroblasts. Quantitative analysis using the Manders' colocalization coefficient demonstrated significant overlap in corneas from patients with ulcerative keratitis (M1 = 0.29; M2 = 0.22) as opposed to control corneas (M1 = 0.01, P < 0.01; M2 = 0.02, P < 0.05). In these experiments, there was a significant positive correlation between the degree of galectin-3 and CD147 colocalization and the presence of gelatinolytic activity. Conclusions: Our results indicate that concomitant stimulation and colocalization of galectin-3 with CD147 are associated with increased gelatinolytic activity in the actively ulcerating human cornea and suggest a mechanism by which galectin-3 may contribute to the degradation of extracellular matrix proteins during ulceration.
Kheirkhah A, Satitpitakul V, Syed ZA, Müller R, Goyal S, Tu EY, Dana R. Factors Influencing the Diagnostic Accuracy of Laser-Scanning In Vivo Confocal Microscopy for Acanthamoeba Keratitis. Cornea 2018;37(7):818-823.Abstract
PURPOSE: To determine the factors that influence the sensitivity and specificity of laser-scanning in vivo confocal microscopy (IVCM) for diagnosing Acanthamoeba keratitis (AK). METHODS: This retrospective, controlled study included 28 eyes of 27 patients with AK and 34 eyes of 34 patients with bacterial keratitis (as the control group). All patients had undergone corneal imaging with a laser-scanning IVCM (Heidelberg Retina Tomograph 3 with the Rostock Cornea Module). The IVCM images were independently evaluated by 2 experienced and 2 inexperienced masked observers. Sensitivity and specificity of IVCM for diagnosing AK and the effects of various clinical and imaging parameters on the sensitivity were then investigated. RESULTS: Overall, IVCM had average sensitivity and specificity of 69.7% ± 2.5% and 97.1% ± 4.2% for experienced observers and 59.0% ± 7.6% and 92.7% ± 10.4% for inexperienced observers, respectively. However, the sensitivity did not show any significant association with the duration of disease, size of ulcer, depth of involvement, culture results, or cyst morphology. Although interobserver agreement was good (κ = 0.60, P < 0.001) for the experienced observers, it was only at a moderate level (κ = 0.48, P < 0.001) for the inexperienced observers. CONCLUSIONS: IVCM has a moderate sensitivity and a high specificity for diagnosis of AK. Although clinical parameters do not affect this diagnostic accuracy, a higher sensitivity is seen when images are interpreted by experienced observers.
Liu Y, Kam WR, Fernandes P, Sullivan DA. The Effect of Solithromycin, a Cationic Amphiphilic Drug, on the Proliferation and Differentiation of Human Meibomian Gland Epithelial Cells. Curr Eye Res 2018;43(6):683-688.Abstract
PURPOSE: We previously discovered that azithromycin (AZM) acts directly on immortalized human meibomian gland epithelial cells (IHMGECs) to stimulate their lipid and lysosome accumulation and overall differentiation. We hypothesize that this phospholipidosis-like effect is due to AZM's cationic amphiphilic drug (CAD) nature. If our hypothesis is correct, then other CADs (e.g., solithromycin [SOL]) should be able to duplicate AZM's action on IHMGECs. Our purpose was to test this hypothesis. MATERIALS AND METHODS: IHMGECs were cultured in the presence of vehicle or SOL (2, 10, or 20 µg/ml) for up to 7 days under proliferating or differentiating conditions. Positive (epidermal growth factor and bovine pituitary extract for proliferation; AZM for differentiation) and negative (vehicle) controls were included with the experiments. IHMGECs were evaluated for cell number, neutral lipid content, and lysosome accumulation. RESULTS: Our results demonstrate that SOL induces a rapid and dose-dependent increase in the accumulation of neutral lipids and lysosomes in HMGECs. The lysosomal effects were most prominent with the 10 and 20 µg/ml doses, and occurred earlier (i.e., 1 day) with SOL than with the AZM (10 µg/ml) control. The effects of SOL and AZM on IHMGEC differentiation were essentially the same after 3 days of culture. SOL did not influence the proliferation of HMGECs during a 7-day time period. CONCLUSIONS: Our results support our hypothesis that SOL, a CAD, is able to reproduce AZM's impact on lysosome and lipid accumulation, as well as the differentiation, of HMGECs. The effect of SOL on lysosome appearance was faster than that of AZM.
Kaufman AR, Cruzat A, Colby KA. Clinical Outcomes Using Oversized Back Plates in Type I Boston Keratoprosthesis. Eye Contact Lens 2018;44(6):399-404.Abstract
OBJECTIVES: To examine clinical outcomes of oversized titanium back plates in type I Boston keratoprosthesis (KPro) implantation. METHODS: Retrospective study of 22 sequential eyes (20 patients) undergoing type I KPro implantation with an oversized titanium back plate (larger than trephined wound diameter by 1.0 mm or more), performed by a single surgeon (K.A.C.) from June 2010 to November 2014. Data were collected regarding preoperative eye characteristics, surgical details, and postoperative clinical outcomes. RESULTS: Mean follow-up time per eye was 24.1±14.9 months. All eyes had improved vision after surgery; 13 eyes (59.1%) maintained visual acuity improvement at last follow-up. Initial KPro's were retained in 19 eyes (86.4%); one eye required KPro replacement. Primary retroprosthetic membrane (RPM) developed in three eyes (13.6%), with similar occurrence in aniridic (14.3%) and nonaniridic eyes (13.3%). Secondary RPM's developed in two eyes (9.1%) after vitritis (one eye) and retinal and choroidal detachment (one eye). Glaucoma was a common comorbidity: 2 of 14 eyes (14.3%) with preoperative glaucoma had glaucoma progression, and 4 of 8 eyes (50.0%) without preoperative glaucoma developed glaucoma postoperatively. Other postoperative complications included retinal detachment (5 eyes, 22.7%) and idiopathic vitritis (3 eyes, 13.6%). CONCLUSIONS: Oversized titanium KPro back plates are associated with a low rate of primary RPM formation and may have particular utility in reducing primary RPM formation in aniridic eyes. Glaucoma remains a challenge in postoperative KPro management. Complex eyes, at increased risk of postoperative complications, require careful management.
Shanbhag SS, Saeed HN, Paschalis EI, Chodosh J. Keratolimbal allograft for limbal stem cell deficiency after severe corneal chemical injury: a systematic review. Br J Ophthalmol 2018;102(8):1114-1121.Abstract
PURPOSE: To review the published literature on outcomes of keratolimbal allograft (KLAL) for the surgical treatment of limbal stem cell deficiency (LSCD) and corneal blindness after severe corneal chemical injury. METHODS: Literature searches were conducted in the following electronic databases: MEDLINE, EMBASE, Science Citation Index, CINAHL, LILACS and the Cochrane Library. Standard systematic review methodology was applied. The main outcome measure was the proportion of eyes with best-corrected visual acuity (BCVA) ≥20/200 at last follow-up. Other measures of allograft success were also collected. RESULTS: We identified six reports in which KLAL outcomes in the eyes after chemical injury could be distinguished. There were no randomised controlled studies. The outcomes of KLAL in 36 eyes of 33 patients were analysed. One study with seven eyes did not specify KLAL follow-up specific to chemical injury. Median postoperative follow-up for the other 29 eyes in 26 patients was 42 months (range 6.2-114 months). In the same 29 eyes, 69% (20/29) had BCVA ≥20/200 at the last follow-up examination. Eighty-nine per cent of all eyes (32/36) underwent penetrating keratoplasty simultaneous or subsequent to KLAL. CONCLUSIONS: The number of studies where outcomes of KLAL in eyes with severe corneal chemical injury could be discerned was limited, and variability was observed in outcome reporting. The quality of evidence to support the use of KLAL in LSCD in severe chemical corneal burns was low. Standardisation and longer follow-up are needed to better define evidence-based best practice when contemplating surgical intervention for blindness after corneal chemical injury. PROSPERO REGISTRATION NUMBER: CRD42017054733.
Alageel SA, Arafat SN, Salvador-Culla B, Kolovou PE, Jahanseir K, Kozak A, Braithwaite GJC, Ciolino JB. Corneal Cross-Linking With Verteporfin and Nonthermal Laser Therapy. Cornea 2018;37(3):362-368.Abstract
PURPOSE: To test whether verteporfin with a nonthermal laser increases corneal mechanical stiffness and resistance to enzymatic degradation ex vivo. METHODS: Thirty human corneas (n = 5 per group) were treated with verteporfin alone (V), irradiated with nonthermal laser therapy (689 nm) alone (NTL), or received combined treatment of verteporfin with nonthermal laser therapy for 1 sequence (V+NTL1) or 6 sequences (V+NTL6) of 1 minute of NTL exposure. Positive controls were pretreated with 0.1% riboflavin/20% dextran every 3 to 5 minutes for 30 minutes and irradiated with ultraviolet light type A (λ = 370 nm, irradiance = 3 mW/cm) for 30 minutes using the Dresden protocol (R+UVA). Untreated corneas were used as negative controls. The corneal biomechanical properties were measured with enzymatic digestion, compression, creep, and tensile strength testing. RESULTS: V+NTL6- and R+UVA-treated corneas acquired higher rigidity and more pronounced curvature than untreated corneas. The stress-strain tests showed that V+NTL6 and R+UVA corneas became significantly stiffer than controls (P < 0.005). The V+NTL6 group seemed to be slightly stiffer than the R+UVA group, although the differences were not statistically significant. V+NTL6 corneas were found to have a significantly lower absolute creep rate (-1.87 vs. -3.46, P < 0.05) and significantly higher maximum stress values (7.67 vs. 3.02 P < 0.05) compared with untreated corneas. CONCLUSIONS: Verteporfin-NTL (V+NTL6) increases corneal mechanical stiffness and resistance to enzymatic collagenase degradation. Although a clinical study is needed, our results suggest that V+NTL6 induces corneal cross-linking and corneal biomechanical changes that are similar to those induced by standard corneal collagen cross-linking.
Rodriguez JD, Lane KJ, Ousler GW, Angjeli E, Smith LM, Abelson MB. Blink: Characteristics, Controls, and Relation to Dry Eyes. Curr Eye Res 2018;43(1):52-66.Abstract
Blink is a complex phenomenon that is profoundly affected by diverse endogenous and exogenous stimuli. It has been studied in the context of cognition, emotional, and psychological states, as an indicator of fatigue and sleepiness, particularly in the automobile and transportation industry, in visual tasking, and finally, as it relates to tear film stability and ocular surface health. The fact that it is highly variable and has input from so many sources makes it very difficult to study. In the present review, the behavior of blink in many of these systems is discussed, ultimately returning in each instance to a discussion of how these factors affect blink in the context of dry eyes. Blink is important to ocular surface health and to an individual's optimal functioning and quality of life. Disturbances in blink, as cause or effect, result in a breakdown of tear film stability, optical clarity, and visual function.
Inomata T, Hua J, Nakao T, Shiang T, Chiang H, Amouzegar A, Dana R. Corneal Tissue From Dry Eye Donors Leads to Enhanced Graft Rejection. Cornea 2018;37(1):95-101.Abstract
PURPOSE: To assess the effect of dry eye disease (DED) in graft donors on dendritic cell (DC) maturation, host T-cell sensitization, and corneal allograft rejection. METHODS: Corneas of control (healthy donor) and DED mice (C57BL/6) were transplanted onto fully allogeneic naive BALB/c recipients (n = 10 mice/group). Long-term allograft survival was evaluated for 8 weeks. Corneas and draining lymph nodes (dLNs) were harvested at posttransplantation day 14 (n = 5 mice/group). The frequencies of MHCII CD11c DCs in the donor corneas and host dLNs and the frequencies of interferon (IFN)-γ and IL-17 CD4 T cells and Foxp3 expression by Tregs in host dLNs were investigated using flow cytometry. The enzyme-linked immunospot assay was used to assess host T-cell allosensitization through direct and indirect pathways (n = 3/group). RESULTS: Recipients of DED donor corneas showed significantly reduced graft survival (10%) compared with control mice (50% survival, P = 0.022), and had significantly increased frequencies of mature DCs in the grafted cornea (DED donor 44.0% ± 0.36% vs. healthy donor 35.4 ± 0.5%; P < 0.0001) and host dLNs (DED donor 25.1% ± 0.66% vs. healthy donor 13.7% ± 1.6%; P = 0.005). Frequencies of IFN-γ and IL-17 T cells were increased in the dLNs of recipients of DED corneas, whereas the expression (mean fluorescence intensity) of Foxp3 in Tregs was decreased significantly in these mice (DED donor 6004 ± 193 vs. healthy donor 6806 ± 81; P = 0.0002). Enzyme-linked immunospot analysis showed that the direct pathway of allosensitization was significantly amplified in recipients of grafts with DED (P = 0.0146). CONCLUSIONS: Our results indicate that DED in the donor is a significant risk factor for subsequent corneal allograft rejection.
Cavalcanti BM, Cruzat A, Sahin A, Pavan-Langston D, Samayoa E, Hamrah P. In vivo confocal microscopy detects bilateral changes of corneal immune cells and nerves in unilateral herpes zoster ophthalmicus. Ocul Surf 2018;16(1):101-111.Abstract
PURPOSE: To analyze bilateral corneal immune cell and nerve alterations in patients with unilateral herpes zoster ophthalmicus (HZO) by laser in vivo confocal microscopy (IVCM) and their correlation with corneal sensation and clinical findings. MATERIALS AND METHODS: This is a prospective, cross-sectional, controlled, single-center study. Twenty-four eyes of 24 HZO patients and their contralateral clinically unaffected eyes and normal controls (n = 24) were included. Laser IVCM (Heidelberg Retina Tomograph/Rostock Cornea Module), corneal esthesiometry (Cochet-Bonnet) were performed. Changes in corneal dendritiform cell (DC) density and morphology, number and length of subbasal nerve fibers and their correlation to corneal sensation, pain, lesion location, disease duration, and number of episodes were analyzed. RESULTS: HZO-affected and contralateral eyes showed a significant increase in DC influx of the central cornea as compared to controls (147.4 ± 33.9, 120.1 ± 21.2, and 23.0 ± 3.6 cells/mm2; p < 0.0001). In HZO eyes DCs were larger in area (319.4 ± 59.8 μm2; p < 0.001) and number of dendrites (3.5 ± 0.4 n/cell; p = 0.01) as compared to controls (52.2 ± 11.7, and 2.3 ± 0.5). DC density and size showed moderate negative correlation with total nerve length (R = -0.43 and R = -0.57, respectively; all p < 0.001). A higher frequency of nerve beading and activated DCs close to nerve fibers were detected specifically in pain patients. CONCLUSIONS: Chronic unilateral HZO causes significant bilateral increase in corneal DC density and decrease of the corneal subbasal nerves as compared to controls. Negative correlation was observed for DC density and size to nerve parameters, suggesting interplay between the immune and nervous systems. Patients with chronic pain also showed increased nerve beading and activated DCs.
Yin J, Jurkunas U. Limbal Stem Cell Transplantation and Complications. Semin Ophthalmol 2017;:1-8.Abstract
Corneal epithelial stem cells are adult somatic stem cells located at the limbus and represent the ultimate source of transparent corneal epithelium. When these limbal stem cells become dysfunctional or deficient, limbal stem cell deficiency (LSCD) develops. LSCD is a major cause of corneal scarring and is particularly prevalent in chemical and thermal burns of the ocular surface. LSCD leads to conjunctivalization of the corneal surface, neovascularization, recurrent or persistent epithelial defects, ocular surface inflammation, and scarring that, in turn, lead to decreased vision, pain, and impaired quality of life. Several techniques have been reported for limbal stem cell transplantation (LSCT). We introduce the surgical techniques, examine the success rate, and discuss the postoperative complications of conjunctival limbal autograft (CLAU), cultivated limbal stem cell transplantation (CLET), simple limbal epithelial transplantation (SLET), and limbal allograft, including keratolimbal allografts (KLAL) and living-related conjunctival allograft (LR-CLAL).
Riau AK, Venkatraman SS, Dohlman CH, Mehta JS. Surface Modifications of the PMMA Optic of a Keratoprosthesis to Improve Biointegration. Cornea 2017;36 Suppl 1:S15-S25.Abstract
Biointegration of a keratoprosthesis (KPro) is critical for the mitigation of various long-term postoperative complications. Biointegration of a KPro occurs between the haptic skirt (corneal graft) and the central optic [poly(methyl methacrylate) (PMMA)]. Various studies have highlighted common problems associated with poor bonding and biointegration between these 2 incompatible biomaterials. Resolution of these issues could be achieved by surface modification of the inert material (PMMA). A calcium phosphate (CaP) coating deposited on dopamine-activated PMMA sheets by simulated body fluid incubation (d-CaP coating) was shown to improve adhesion to collagen type I (main component of corneal stroma) compared with untreated PMMA and PMMA with other surface modifications. However, the d-CaP coating could easily undergo delamination, thereby reducing its potential for modification of KPro optical cylinders. In addition, the coating did not resemble the Ca and P composition of hydroxyapatite (HAp). A novel dip-coating method that involves the creation of cavities to trap and immobilize HAp nanoparticles on the PMMA surface was introduced to address the problems associated with the d-CaP coating. The newly obtained coating offered high hydrophilicity, resistance to delamination, and preservation of the Ca and P composition of HAp. These advantages resulted in improved adhesion strength by more than 1 order of magnitude compared with untreated PMMA. With respect to biointegration, human corneal stromal fibroblasts were able to adhere strongly and proliferate on HAp-coated PMMA. Furthermore, the new coating technique could be extended to immobilization of HAp nanoparticles on 3-mm-diameter PMMA cylinders, bringing it closer to clinical application.
Inomata T, Mashaghi A, Hong J, Nakao T, Dana R. Scaling and maintenance of corneal thickness during aging. PLoS One 2017;12(10):e0185694.Abstract
Corneal thickness is tightly regulated by its boundary endothelial and epithelial layers. The regulated set-point of corneal thickness likely shows inter-individual variations, changes by age, and response to stress. Using anterior segment-optical coherence tomography, we measure murine central corneal thickness and report on body size scaling of murine central corneal thickness during aging. For aged-matched mice, we find that corneal thickness depends on sex and strain. To shed mechanistic insights into these anatomical changes, we measure epithelial layer integrity and endothelial cell density during the life span of the mice using corneal fluorescein staining and in vivo confocal microscopy, respectively and compare their trends with that of the corneal thickness. Cornea thickness increases initially (1 month: 114.7 ± 3.0 μm, 6 months: 126.3 ± 1.6 μm), reaches a maximum (9 months: 129.3 ± 4.4 μm) and then reduces (12 months: 127 ± 2.9 μm, 13 months: 119.5 ± 7.6 μm, 14 months: 110.6 ± 10.6 μm), while the body size (weight) increases with age. We find that endothelial cell density reduces from 2 months old to 8 months old as the mice age and epithelial layer accumulates damages within this time frame. Finally, we compare murine corneal thickness with those of several other mammals including humans and show that corneal thickness has an allometric scaling with body size. Our results have relevance for organ size regulation, translational pharmacology, and veterinary medicine.

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