Cornea

Chen Y, Chauhan SK, Lee SH, Saban DR, Dana R. Chronic dry eye disease is principally mediated by effector memory Th17 cells. Mucosal Immunol 2014;7(1):38-45.Abstract
Recent experimental and clinical data suggest that there is a link between dry eye disease (DED) and T-cell-mediated immunity. However, whether these immune responses are a consequence or cause of ocular surface inflammation remains to be determined. Thus far, only models of acute DED have been used to derive experimental data. This is in contrast to clinical DED which usually presents as a chronic disease. In the present study, using a murine model of chronic DED, it was established that the chronic phase of the disease is accompanied by T helper type 17 (Th17) responses at the ocular surface and that a significant memory T-cell population can be recovered from chronic DED. This memory response is predominantly mediated by Th17 cells. Moreover, adoptive transfer of this memory T-cell population was shown to induce more severe and rapidly progressing DED than did the adoptive transfer of its effector or naive counterparts. Not only do these results clearly demonstrate that effector memory Th17 cells are primarily responsible for maintaining the chronic and relapsing course of DED, but they also highlight a potentially novel therapeutic strategy for targeting memory immune responses in patients with DED.
Robert M-C, Arafat SN, Ciolino JB. Collagen cross-linking of the Boston keratoprosthesis donor carrier to prevent corneal melting in high-risk patients. Eye Contact Lens 2014;40(6):376-81.Abstract

OBJECTIVE: To examine the clinical relevance and pathophysiology of Boston keratoprosthesis (B-KPro)-related corneal keratolysis (cornea melt) and to describe a novel method of preventing corneal melt using ex vivo crosslinked cornea tissue carrier. METHODS: A review of B-KPro literature was performed to highlight cases of corneal melt. Studies examining the effect of corneal collagen cross-linking (CXL) on the biomechanical properties of corneal tissue are summarized. The use of crosslinked corneal tissue as a carrier to the B-KPro is illustrated with a case. RESULTS: Corneal melting after B-KPro is a relatively rare event, occurring in 3% of eyes during the first 3 years of postoperative follow-up. The risk of post-KPro corneal melting is heightened in eyes with chronic ocular surface inflammation such as eyes with Stevens-Johnson syndrome and mucous membrane pemphigoid. This chronic inflammation results in high tear levels of matrix metalloproteinases, the enzymes responsible for collagenolysis and corneal melt. Crosslinked corneal tissue has been shown to have stiffer biomechanical properties and to be more resistant to degradation by collagenolytic enzymes. We have previously optimized the technique for ex vivo corneal CXL and are currently studying its impact on the prevention of corneal melting after B-KPro surgery in high-risk eyes. Crosslinked carrier tissue was used in a 52-year-old man with familial aniridia and severe post-KPro corneal melt. The patient maintained his visual acuity and showed no evidence of corneal thinning or melt in the first postoperative year. CONCLUSION: Collagen crosslinking was previously shown to halt the enzymatic degradation of corneal buttons ex vivo. This study demonstrates the safety and potential benefit of using crosslinked corneal grafts as carriers for the B-KPro, especially in eyes at higher risk of postoperative melt.

Valentino MD, McGuire AM, Rosch JW, Bispo PJM, Burnham C, Sanfilippo CM, Carter RA, Zegans ME, Beall B, Earl AM, Tuomanen EI, Morris TW, Haas W, Gilmore MS. Unencapsulated Streptococcus pneumoniae from conjunctivitis encode variant traits and belong to a distinct phylogenetic cluster. Nat Commun 2014;5:5411.Abstract

Streptococcus pneumoniae, an inhabitant of the upper respiratory mucosa, causes respiratory and invasive infections as well as conjunctivitis. Strains that lack the capsule, a main virulence factor and the target of current vaccines, are often isolated from conjunctivitis cases. Here we perform a comparative genomic analysis of 271 strains of conjunctivitis-causing S. pneumoniae from 72 postal codes in the United States. We find that the vast majority of conjunctivitis strains are members of a distinct cluster of closely related unencapsulated strains. These strains possess divergent forms of pneumococcal virulence factors (such as CbpA and neuraminidases) that are not shared with other unencapsulated nasopharyngeal S. pneumoniae. They also possess putative adhesins that have not been described in encapsulated pneumococci. These findings suggest that the unencapsulated strains capable of causing conjunctivitis utilize a pathogenesis strategy substantially different from that described for S. pneumoniae at other infection sites.

Behlau I, Martin KV, Martin JN, Naumova EN, Cadorette JJ, Sforza TJ, Pineda R, Dohlman CH. Infectious endophthalmitis in Boston keratoprosthesis: incidence and prevention. Acta Ophthalmol 2014;92(7):e546-55.Abstract
PURPOSE: To determine the cumulative worldwide incidence of infectious endophthalmitis and associated vision loss after Boston keratoprosthesis (B-KPro) Type I/II implantation and to propose both safe and inexpensive prophylactic antibiotic regimens. METHODS: Two retrospective methods were used to determine the incidence, visual outcomes and aetiologies of infectious endophthalmitis associated with the B-KPro divided per decade: (i) systematic review of the literature from 1990 through January 2013 and (ii) a surveillance survey sent to all surgeons who implanted B-KPros through 2010 with 1-year minimum follow-up. In addition, a single-Boston surgeon 20-year experience was examined. RESULTS: From 1990 through 2010, there were 4729 B-KPros implanted worldwide by 209 U.S. surgeons and 159 international surgeons. The endophthalmitis cumulative mean incidence declined from 12% during its first decade of use to about 3% during its second decade in the Unites States and about 5% internationally during the second decade. There remains a large incidence range both in the United States (1-12.5%) and internationally (up to 17%). Poor compliance with daily topical antibiotics is an important risk factor. While Gram-positive organisms remained dominant, fungal infections emerged during the second decade. CONCLUSIONS: Daily prophylactic topical antibiotics have dramatically reduced the endophthalmitis incidence. Although Gram-positive organisms are the most common aetiology, antimicrobials must be inclusive of Gram-negative organisms. Selection of prophylactic regimens should be tailored to local antibiotic susceptibility patterns, be cost-effective, and should not promote the emergence of antimicrobial resistance. An example of a broad-spectrum, low-cost prophylactic option for non-autoimmune patients includes trimethoprim/polymyxinB once daily.
Kodati S, Chauhan SK, Chen Y, Dohlman TH, Karimian P, Saban D, Dana R. CCR7 is critical for the induction and maintenance of Th17 immunity in dry eye disease. Invest Ophthalmol Vis Sci 2014;55(9):5871-7.Abstract

PURPOSE: We characterized antigen-presenting cell (APC)-relevant chemokine receptor expression in dry eye disease (DED), and investigated the effect of topical CC chemokine receptor (CCR)-7 blockade specifically on Th17 cell immunity and dry eye disease severity. METHODS: We induced DED in female C57BL/6 mice. Chemokine receptor expression by corneal APCs was characterized using immunohistochemistry. To determine the functional role of CCR7 in DED, mice were treated topically with either anti-CCR7, a control isotype antibody, or left untreated, and clinical disease severity, Th17 responses, and molecular markers of DED were quantified. RESULTS: Frequencies of CD11b(+) cells and their chemokine expression were increased in the cornea of DED mice. Mice treated topically with anti-CCR7 antibody displayed a significant reduction in clinical disease severity and Th17 response compared to the isotype and untreated groups. Topical CCR7 blockade was effective in ameliorating DED in its acute and chronic stages. CONCLUSIONS: Our findings suggest that CCR7-mediated trafficking of APCs drives the induction and maintenance of Th17 immunity in DED and that CCR7 blockade is effective in suppressing the immunopathogenic mechanisms in DED.

Woodward AM, Argüeso P. Expression analysis of the transmembrane mucin MUC20 in human corneal and conjunctival epithelia. Invest Ophthalmol Vis Sci 2014;55(10):6132-8.Abstract
PURPOSE: Cell surface mucins are a group of highly O-glycosylated transmembrane glycoproteins responsible for the protection of epithelial cells on mucosal surfaces. The aim of this study was to investigate the localization and regulation of mucin 20 (MUC20) at the ocular surface. METHODS: Localization of MUC20 in human corneal and conjunctival epithelia was evaluated by immunofluorescence microscopy. Immortalized corneal (HCLE) and conjunctival (HCjE) cell lines were grown at different stages of differentiation and subjected to quantitative PCR and Western blot analyses. Cell surface proteins on apical cell membranes were biotinylated and isolated by neutravidin chromatography. RESULTS: The MUC20 was detected throughout the entire human ocular surface epithelia, predominantly in cell membranes within intermediate cell layers. In conjunctiva, MUC20 also was observed in the cytoplasm of apical cells within the stratified squamous epithelium, but not in goblet cells. Quantitative PCR and immunoblotting demonstrated expression of MUC20 in HCLE and HCjE cells. Induction of differentiation with serum-containing medium resulted in upregulation of MUC20 mRNA and protein. Biotin labeling of the surface of stratified cultures revealed low levels of MUC20 protein on apical glycocalyces. Further, MUC20 was not detected in the cell culture media or in human tears, suggesting that the extracellular domain of MUC20 is not released from the ocular surface as described previously for other cell surface mucins. CONCLUSIONS: Our results indicate that MUC20 is a novel transmembrane mucin expressed by the human corneal and conjunctival epithelia, and suggest that differential expression of MUC20 during differentiation has a role in maintaining ocular surface homeostasis.
Emami-Naeini P, Dohlman TH, Omoto M, Hattori T, Chen Y, Lee HS, Chauhan SK, Dana R. Soluble vascular endothelial growth factor receptor-3 suppresses allosensitization and promotes corneal allograft survival. Graefes Arch Clin Exp Ophthalmol 2014;252(11):1755-62.Abstract
PURPOSE: To investigate the effect of VEGF-C and VEGF-D blockade via soluble VEGFR-3 (sVEGFR-3) on T cell allosensitization, corneal neovascularization, and transplant survival. METHODS: Corneal intrastromal suture placement and allogeneic transplantation were performed on BALB/c mice to evaluate the effect of sVEGFR-3 on corneal neovascularization. Soluble VEGFR-3 trap was injected intraperitoneally to block VEGF-C/D (every other day starting the day of surgery). Immunohistochemical staining of corneal whole mounts was performed using anti-CD31 (PECAM-1) and anti-LYVE-1 antibodies to quantify the levels of hem- and lymphangiogenesis, respectively. Mixed lymphocyte reaction (MLR) was performed to assess indirect and direct host T cell allosensitization and the frequencies of IFN-γ-producing T cells in the draining lymph nodes were assessed using flow cytometry. Graft opacity and survival was evaluated by slit-lamp biomicroscopy. RESULTS: Treatment with sVEGFR-3 resulted in a significant blockade of lymphangiogenesis 2 weeks post-transplantation and significantly prolonged corneal allograft survival compared to the control group at 8 weeks post-transplantation (87.5 % vs. 50 %), and this was associated with significant reduction in the frequencies of allosensitized T cells and decreased frequencies of IFN-γ-producing CD4 T cells. CONCLUSIONS: Soluble VEGFR-3 suppresses corneal lymphangiogenesis and allograft rejection and may offer a viable therapeutic modality for corneal neovascularization and corneal transplantation.
Sriram S, Gibson DJ, Robinson P, Pi L, Tuli S, Lewin AS, Schultz G. Assessment of anti-scarring therapies in ex vivo organ cultured rabbit corneas. Exp Eye Res 2014;125:173-82.Abstract
The effects of a triple combination of siRNAs targeting key scarring genes were assessed using an ex vivo organ culture model of excimer ablated rabbit corneas. The central 6 mm diameter region of fresh rabbit globes was ablated to a depth of 155 microns with an excimer laser. Corneas were excised, cultured at the air-liquid interface in defined culture medium supplemented with transforming growth factor beta 1 (TGFB1), and treated with either 1% prednisolone acetate or with 22.5 μM cationic nanoparticles complexed with a triple combination of siRNAs (NP-siRNA) targeting TGFB1, TGFB Receptor (TGFBR2) and connective tissue growth factor (CTGF). Scar formation was measured using image analysis of digital images and levels of smooth muscle actin (SMA) were assessed in ablated region of corneas using qRT-PCR and immunostaining. Ex vivo cultured corneas developed intense haze-like scar in the wounded areas and levels of mRNAs for pro-fibrotic genes were significantly elevated 3-8 fold in wounded tissue compared to unablated corneas. Treatment with NP-siRNA or steroid significantly reduced quantitative haze levels by 55% and 68%, respectively, and reduced SMA mRNA and immunohistostaining. This ex vivo corneal culture system reproduced key molecular patterns of corneal scarring and haze formation generated in rabbits. Treatment with NP-siRNAs targeting key scarring genes or an anti-inflammatory steroid reduced corneal haze and SMA mRNA and protein.
Ksander BR, Kolovou PE, Wilson BJ, Saab KR, Guo Q, Ma J, McGuire SP, Gregory MS, Vincent WJB, Perez VL, Cruz-Guilloty F, Kao WWY, Call MK, Tucker BA, Zhan Q, Murphy GF, Lathrop KL, Alt C, Mortensen LJ, Lin CP, Zieske JD, Frank MH, Frank NY. ABCB5 is a limbal stem cell gene required for corneal development and repair. Nature 2014;511(7509):353-7.Abstract
Corneal epithelial homeostasis and regeneration are sustained by limbal stem cells (LSCs), and LSC deficiency is a major cause of blindness worldwide. Transplantation is often the only therapeutic option available to patients with LSC deficiency. However, while transplant success depends foremost on LSC frequency within grafts, a gene allowing for prospective LSC enrichment has not been identified so far. Here we show that ATP-binding cassette, sub-family B, member 5 (ABCB5) marks LSCs and is required for LSC maintenance, corneal development and repair. Furthermore, we demonstrate that prospectively isolated human or murine ABCB5-positive LSCs possess the exclusive capacity to fully restore the cornea upon grafting to LSC-deficient mice in xenogeneic or syngeneic transplantation models. ABCB5 is preferentially expressed on label-retaining LSCs in mice and p63α-positive LSCs in humans. Consistent with these findings, ABCB5-positive LSC frequency is reduced in LSC-deficient patients. Abcb5 loss of function in Abcb5 knockout mice causes depletion of quiescent LSCs due to enhanced proliferation and apoptosis, and results in defective corneal differentiation and wound healing. Our results from gene knockout studies, LSC tracing and transplantation models, as well as phenotypic and functional analyses of human biopsy specimens, provide converging lines of evidence that ABCB5 identifies mammalian LSCs. Identification and prospective isolation of molecularly defined LSCs with essential functions in corneal development and repair has important implications for the treatment of corneal disease, particularly corneal blindness due to LSC deficiency.
Lin YB, Gardiner MF. Fingernail-induced corneal abrasions: case series from an ophthalmology emergency department. Cornea 2014;33(7):691-5.Abstract
PURPOSE: Fingernail-induced corneal abrasions are one of the most common eye injuries that present to the emergency department, and yet there is little literature available to offer guidelines for management. We analyzed the treatment used in cases of fingernail-induced corneal abrasions that presented to the Massachusetts Eye and Ear Infirmary Emergency Department and studied its relationship to the development of complications such as recurrent erosion syndrome and infection. METHODS: We performed a retrospective review of 99 patients who presented to the Massachusetts Eye and Ear Infirmary Emergency Department with fingernail-induced corneal abrasions between January 1, 2009 and December 31, 2009. We followed the patients for 12 months and documented demographics, nature of the injury, treatment, and complications. RESULTS: The average age was 29.4 (range, 2-89) years. Forty-four percent (n = 44) were female and 56% (n = 55) were male. Of the 99 subjects, 39 had a full 12 month follow-up, and 7 developed a complication from the injury. Compared with the 32 subjects without complications, there was no difference in age or gender. However, there was a significant difference in that adults scratched by another adult were more highly represented in the group with complications (43%, n = 3/7 vs. 3%, n = 1/32; P = 0.0017). There was no significant difference in outcome by treatment used. CONCLUSIONS: This is the largest fingernail-induced corneal abrasion study completed to date. Patients are at risk of developing complications, but there is scant evidenced-based literature available for treating this common injury. Prospective trials should be performed to better optimize and standardize treatments.
Contreras-Ruiz L, Ryan DS, Sia RK, Bower KS, Dartt DA, Masli S. Polymorphism in THBS1 gene is associated with post-refractive surgery chronic ocular surface inflammation. Ophthalmology 2014;121(7):1389-97.Abstract
PURPOSE: To determine the association of single nucleotide polymorphisms (SNPs) of the thrombospondin 1 (THBS1) gene with development of chronic ocular surface inflammation (keratoconjunctivitis) after refractive surgery. DESIGN: Retrospective cohort study. PARTICIPANTS: Active duty U.S. Army soldiers (n = 143) who opted for refractive surgery. METHODS: Conjunctival impression cytology samples collected from participants before the surgery were used to harvest DNA for genotyping 5 THBS1 SNPs (rs1478604, rs2228262, rs2292305, rs2228262, and rs3743125) using the Sequenom iPLEX Gold platform (Sequenom, San Diego, CA). Samples collected after surgery were used to harvest RNA for gene expression analysis by real-time polymerase chain reaction (PCR). Participants were followed for 1 year after surgery to monitor the status of keratoconjunctivitis. MAIN OUTCOME MEASURES: Genetic basis of the development of chronic keratoconjunctivitis after refractive surgery. RESULTS: Carriers of minor alleles of 3 SNPs each were found to be more susceptible to developing chronic keratoconjunctivitis (rs1478604: odds ratio [OR], 2.5; 95% confidence interval [CI], 1.41-4.47; P = 2.5 × 10(-3); rs2228262 and rs2292305: OR, 1.9; 95% CI, 1.05-3.51; P = 4.8 × 10(-2)). Carriers of the rs1478604 minor allele expressed significantly reduced levels of thrombospondin 1 (TSP1) (P = 0.042) and increased levels of an inflammatory cytokine associated with keratoconjunctivitis, interleukin-1β (P = 0.025), in their ocular surface epithelial cells compared with homozygous major allele controls. CONCLUSIONS: Genetic variation in the THBS1 gene that results in decreased expression of the encoded glycoprotein TSP1 in ocular surface epithelial cells significantly increases the susceptibility to develop chronic ocular surface inflammation after refractive surgery. Further investigation of THBS1 SNPs in a larger sample size is warranted.
Grob SR, Gonzalez-Gonzalez LA, Daly MK. Management of mydriasis and pain in cataract and intraocular lens surgery: review of current medications and future directions. Clin Ophthalmol 2014;8:1281-9.Abstract

The maintenance of mydriasis and the control of postoperative pain and inflammation are critical to the safety and success of cataract and intraocular lens replacement surgery. Appropriate mydriasis is usually achieved by topical and/or intracameral administration of anticholinergic agents, sympathomimetic agents, or both, with the most commonly used being cyclopentolate, tropicamide, and phenylephrine. Ocular inflammation is common after cataract surgery. Topical steroids and nonsteroidal anti-inflammatory drugs are widely used because they have been proved effective to control postsurgical inflammation and decrease pain. Topical nonsteroidal anti-inflammatory drugs have also been shown to help maintain dilation. However, use of multiple preoperative drops for pupil dilation, inflammation, and pain control have been shown to be time consuming, resulting in delays to the operating room, and they cause dissatisfaction among perioperative personnel; their use can also be associated with systemic side effects. Therefore, ophthalmologists have been in search of new options to streamline this process. This article will review the current medications commonly used for intraoperative mydriasis, as well as pain and inflammation control. In addition, a new combination of ketorolac, an anti-inflammatory agent, and phenylephrine, a mydriatic agent has recently been designed to maintain intraoperative mydriasis and to reduce postoperative pain and irritation from intraocular lens replacement surgery. Two Phase III clinical trials evaluating this combination have demonstrated statistically significant differences when compared to placebo in maintaining intraoperative mydriasis (P<0.00001) and in reducing pain in the early postoperative period (P=0.0002). This medication may be of benefit for use in cataract and lens replacement surgery in the near future.

Cherfan DG, Melki SA. Corneal perforation by an astigmatic keratotomy performed with an optical coherence tomography-guided femtosecond laser. J Cataract Refract Surg 2014;40(7):1224-7.Abstract

UNLABELLED: We present a case of corneal perforation secondary to an intrastromal astigmatic keratotomy performed with an optical coherence tomography-guided femtosecond laser. The keratotomy was concomitant with cataract surgery and resulted in a flat anterior chamber prior to the start of lens extraction. Interrupted nylon sutures were placed to seal the keratotomy prior to phacoemulsification. Escape of cavitation bubbles into the anterior chamber or the liquid interface can alert the surgeon to the possibility of unintended perforation of the endothelium or the epithelium, respectively. FINANCIAL DISCLOSURE: Neither author has a financial or proprietary interest in any material or method mentioned.

Karamichos D, Hutcheon AEK, Zieske JD. Reversal of fibrosis by TGF-β3 in a 3D in vitro model. Exp Eye Res 2014;124:31-6.Abstract
Corneal scarring following moderate to severe injury is inevitable. Despite significant advancements in the field, current treatments following these types of injuries are limited, and often, the visual recovery is poor. One of the problems and limitations is that corneal wound healing is a complex process, involving corneal cells, extracellular matrix components and growth factors. Therefore, further understanding is required, along with new treatments and techniques to reduce or prevent corneal scarring following injury. Two isoforms of transforming growth factor-beta (TGF-β), TGF-β1 and -β3 (T1 and T3, respectively), are associated with corneal wound healing. T1 has been shown to drive the corneal keratocytes to differentiate into myofibroblasts; whereas, T3 has been found to inhibit fibrotic markers. In the current study, we examined whether the fibrotic characteristics expressed by human corneal fibroblasts (HCF) in our 3-dimensional (3D) construct following T1 stimulation could be reversed by introducing T3 to the in vitro system. To do this, HCF were isolated and cultured in 10% serum, and when they reached confluence, the cells were stimulated with a stable Vitamin C (VitC) derivative for 4 weeks, which allowed them to secrete a self-assembled matrix. Three conditions were tested: (1) CONTROL: 10% serum (S) only, (2) T1: 10%S + T1, or (3) Rescue: 10%S + T1 for two weeks and then switched to 10%S + T3 for another two weeks. At the end of 4 weeks, the constructs were processed for analysis by indirect-immunofluorescence (IF) and transmission electron microscopy (TEM). Different collagens that are normally present in healthy corneas in vivo, such as Type I and V, as well as Type III, which is a fibrotic indicator, were examined. In addition, we examined smooth muscle actin (SMA), a marker of myofibroblasts, and thrombospondin-1 (TSP-1), a multifunctional matrix protein known to activate the latent complex of TGF-β and appear upon wounding in vivo. Our data showed high expression of collagens type I and V under all conditions throughout the 3D constructs; however, type III and SMA expression were higher in the constructs that were stimulated with T1 and reduced to almost nothing in the Rescue samples. A similar pattern was seen with TSP-1, where TSP-1 expression following "rescue" was decreased considerably. Overall, this data is in agreement with our previous observations that T3 has a significant non-fibrotic effect on HCFs, and presents a novel model for the "rescue" of both cellular and matrix fibrotic components with a single growth factor.

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