Cornea Publications

He M, Lippestad M, Li D, Hodges RR, Utheim TP, Dartt DA. Activation of the EGF Receptor by Histamine Receptor Subtypes Stimulates Mucin Secretion in Conjunctival Goblet Cells. Invest Ophthalmol Vis Sci 2018;59(8):3543-3553.Abstract
Purpose: The purpose of this study was to determine if histamine receptors interact with the epidermal growth factor receptor (EGFR) in cultured rat conjunctival goblet cells. Methods: Goblet cells from rat conjunctiva were grown in organ culture. First-passage goblet cells were used in all experiments. Phosphorylated (active) and total EGFR, AKT, and extracellular signal-regulated kinase (ERK)1/2 were measured by Western blot analysis. Cells were preincubated with the EGFR antagonist AG1478 for 30 minutes or small interfering RNA specific to the EGFR for 3 days prior to stimulation with histamine or agonists specific for histamine receptor subtypes for 2 hours. Goblet cell secretion was measured using an enzyme-linked lectin assay. Goblet cells were incubated for 1 hour with the calcium indicator molecule fura-2/AM, and intracellular [Ca2+] ([Ca2+]i) was determined. Data were collected in real time and presented as the actual [Ca2+]i with time and as the change in peak [Ca2+]i. Results: Histamine increased the phosphorylation of the EGFR. Mucin secretion and increase in [Ca2+]i stimulated by histamine, and agonists specific for each histamine receptor subtype were blocked by inhibition of the EGFR. Increase in [Ca2+]i stimulated by histamine and specific agonists for each histamine receptor was also inhibited by TAPI-1, a matrix metalloproteinase (MMP) inhibitor. The histamine-stimulated increase in activation of AKT, but not ERK1/2, was blocked by AG1478. Conclusions: In conjunctival goblet cells, histamine, using all four receptor subtypes, transactivates the EGFR via an MMP. This in turn phosphorylates AKT to increase [Ca2+]i and stimulate mucin secretion.
Tan X, Chen Y, Foulsham W, Amouzegar A, Inomata T, Liu Y, Chauhan SK, Dana R. The immunoregulatory role of corneal epithelium-derived thrombospondin-1 in dry eye disease. Ocul Surf 2018;Abstract
PURPOSE: In this study, we examine the expression of corneal epithelium-derived thrombospondin-1 (TSP-1) and its immunomodulatory functions in a validated murine model of dry eye disease (DED). METHODS: DED was induced in female C57BL/6 using a controlled environment chamber (CEC) for 14 days. mRNA and protein expression of TSP-1 by corneal epithelial cells was quantified using real-time PCR and flow cytometry. Corneal epithelial cells from either naïve or DED mice were cultured with bone marrow derived dendritic cells (BMDCs) in the presence of IFNγ for 48 h, and BMDC expression of MHC-II and CD86 was determined using flow cytometry. Next, either recombinant TSP-1 or anti-TSP-1 antibody was added to the co-culture, and BMDC expression of above activation markers was evaluated. Finally, either DED mice were topically treated with either recombinant TSP-1 or human serum albumin (HSA), and maturation of corneal DCs, expression of inflammatory cytokines, and DED severity were investigated. RESULTS: mRNA expression of TSP-1 by the corneal epithelium was upregulated in DED. Corneal epithelial cells derived from mice with DED demonstrated an enhanced capacity in suppressing BMDC expression of MHC-II and CD86 relative to wild type mice, and this effect was abrogated by TSP-1 blockade and potentiated by recombinant TSP-1. Finally, topical application of recombinant TSP-1 significantly suppressed corneal DC maturation and mRNA expression of pro-inflammatory cytokines, and ameliorated disease severity in mice with DED. CONCLUSIONS: Our study elucidates the function of epithelium-derived TSP-1 in inhibiting DC maturation and shows its translational potential to limit corneal epitheliopathy in DED.
Guo X, Sriram S, Tran JA, Hutcheon AEK, Zieske JD. Inhibition of Human Corneal Myofibroblast Formation. Invest Ophthalmol Vis Sci 2018;59(8):3511-3520.Abstract
Purpose: Transforming growth factor-beta (TGF-β) isoform 1 (T1) is involved in corneal fibrotic wound healing by stimulating myofibroblast transformation and altering fibrotic gene expression. In this study, two specific inhibitors were used to dissect the relationship between myofibroblast generation and the TGF-β/Smad- or TGF-β/p38-signaling pathway in human corneal fibroblasts (HCF). Methods: In HCF, Trx-SARA (Smad-pathway inhibitor) was used to block the TGF-β/Smad-signaling pathway, and the p38 inhibitor (p38inh, SB202190) was used to inhibit p38MAPK, thus blocking the TGF-β/p38-signaling pathway. HCF ± Trx-SARA or Trx-GA (SARA control) were serum starved overnight in Eagle's minimum essential medium (EMEM) ± p38inh, grown in EMEM ± T1 ± p38inh for 24 hours, and then processed for indirect-immunofluorescence, Western blot, or quantitative real-time polymerase chain reaction to examine α-smooth muscle actin (αSMA) and other fibrotic genes, such as fibronectin, thrombospondin1, and type III collagen. In addition, the morphology and the effect of p38inh on myofibroblast phenotype after myofibroblast formation were examined. Results: We observed that Trx-SARA had little effect on αSMA expression, indicating that blocking the Smad pathway did not significantly inhibit myofibroblast formation. However, p38inh did significantly inhibit αSMA and other fibrotic genes, thus efficiently preventing the transition of HCFs to myofibroblasts. In addition, morphology changed and αSMA decreased in myofibroblasts exposed to p38inh medium, as compared with controls. Conclusions: HCF transition to myofibroblasts was mainly through the p38 pathway. Therefore, blocking the p38 pathway may be a potential therapeutic tool for human corneal fibrosis prevention/treatment, because it controls myofibroblast formation in human corneal cells, while leaving other functions of T1 unaffected.
Huang C-C, Yang W, Guo C, Jiang H, Li F, Xiao M, Davidson S, Yu G, Duan B, Huang T, Huang AJW, Liu Q. Anatomical and functional dichotomy of ocular itch and pain. Nat Med 2018;Abstract
Itch and pain are refractory symptoms of many ocular conditions. Ocular itch is generated mainly in the conjunctiva and is absent from the cornea. In contrast, most ocular pain arises from the cornea. However, the underlying mechanisms remain unknown. Using genetic axonal tracing approaches, we discover distinct sensory innervation patterns between the conjunctiva and cornea. Further genetic and functional analyses in rodent models show that a subset of conjunctival-selective sensory fibers marked by MrgprA3 expression, rather than corneal sensory fibers, mediates ocular itch. Importantly, the actions of both histamine and nonhistamine pruritogens converge onto this unique subset of conjunctiva sensory fibers and enable them to play a key role in mediating itch associated with allergic conjunctivitis. This is distinct from skin itch, in which discrete populations of sensory neurons cooperate to carry itch. Finally, we provide proof of concept that selective silencing of conjunctiva itch-sensing fibers by pruritogen-mediated entry of sodium channel blocker QX-314 is a feasible therapeutic strategy to treat ocular itch in mice. Itch-sensing fibers also innervate the human conjunctiva and allow pharmacological silencing using QX-314. Our results cast new light on the neural mechanisms of ocular itch and open a new avenue for developing therapeutic strategies.
Utheim OA, Lyberg T, Eidet JR, Raeder S, Sehic A, Roald B, Messelt E, de la Paz MF, Dartt DA, Utheim TP. Effect of Transportation on Cultured Limbal Epithelial Sheets for Worldwide Treatment of Limbal Stem Cell Deficiency. Sci Rep 2018;8(1):10502.Abstract
Limbal stem cell deficiency can be treated with transplantation of cultured human limbal epithelial cells (LEC). It can be advantageous to produce LEC in centralized labs and thereafter ship them to eye clinics. The present study used transport simulations of LEC to determine if vigorous shaking during transport altered the viability, morphology and phenotype during a 4 day-long storage of LEC with a previously described serum-free storage method. Inserts with LEC cultured on amniotic membranes were sutured to caps inside air-tight containers with generous amounts of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-buffered minimal essential medium (MEM). The containers were distributed among the following testing conditions: 6 hours with full containers, 36 hours with full containers, 36 hours with container three quarters full of medium, and 36 hours with container full of medium containing a shear-protecting agent (Pluronic-F68). Compared to stored, but non-transported controls, no statistically significant changes in viability and immunohistochemical staining were observed. The epithelial sheets remained intact. However, an air-liquid interface in the containers reduced the number of desmosomes and hemi-desmosomes compared to the controls. In conclusion, cultured LEC sheets appear to endure vigorous shaking for at least 36 hours if the container is full.
Kumar S, Ichhpujani P, Thakur S, Singh RB. Traumatic corneal perforation with exteriorisation of Ahmed glaucoma valve tube. BMJ Case Rep 2018;2018Abstract
We report a rare case of traumatic corneal perforation with Ahmed glaucoma valve (AGV) tube. A 5-year-old female child, diagnosed with refractory glaucoma, had undergone AGV implantation, presented with the posterior migration of AGV tube after trauma to the eye. The detailed ocular history, ophthalmic findings, clinical course and surgical management are discussed.
Wang JC, Rudnisky CJ, Belin MW, Ciolino JB, Group BTKS1. Outcomes of Boston keratoprosthesis type 1 reimplantation: multicentre study results. Can J Ophthalmol 2018;53(3):284-290.Abstract
OBJECTIVE: To investigate the visual and anatomical outcomes of Boston keratoprosthesis (Kpro) type 1 reimplantation. DESIGN: Subgroup analysis of multicentre prospective cohort study. PARTICIPANTS: Of 303 eyes that underwent Kpro implantation between January 2003 and July 2008 by 1 of 19 surgeons at 18 medical centres, 13 eyes of 13 patients who underwent reimplantation of Boston Kpro type 1 were compared with 13 eyes of 13 diagnosis-matched patients who underwent initial implantation. METHODS: Forms reporting preoperative, intraoperative, and postoperative parameters were prospectively collected and analyzed. Main outcome measures were Kpro retention and logMAR visual acuity. RESULTS: After a mean follow-up time of 17.1 ± 17.6 months, the retention of both initial and repeat Kpro implantation was 92.3% (12/13 in both groups), and 62% of initial implantation and 58% of repeat implantation eyes achieved visual acuity better than 20/200. Vision worse than 20/200 was often due to glaucoma or posterior segment pathology. Best-recorded logMAR visual acuity was significantly improved postoperatively in both groups (p < 0.001), and there was no statistically significant difference in final logMAR visual acuity between the 2 groups (p = 0.89). Sterile keratolysis (n = 4) and fungal infection (n = 5) were the most common causes of initial Kpro failure in the repeat Kpro group. The single failure in the repeat Kpro implantation group was due to fungal keratitis, and in the control group it was related to Kpro extrusion. CONCLUSIONS: Repeat Kpro implantation is a viable option after failed initial Kpro, with visual and anatomical outcomes comparable to those of initial procedures.
Amparo F, Shikari H, Saboo U, Dana R. Corneal fluorescein staining and ocular symptoms but not Schirmer test are useful as indicators of response to treatment in chronic ocular GVHD. Ocul Surf 2018;16(3):377-381.Abstract
PURPOSE: To evaluate long-term ocular surface clinical signs and symptoms response to therapy in patients with chronic ocular GVHD. METHODS: Retrospective review and data modeling. We reviewed the records of post-bone marrow transplantation patients who were newly diagnosed with ocular GVHD and initiated therapy, and analyzed changes in symptoms (Ocular Surface Disease Index [OSDI]; Symptom Assessment in Dry Eye [SANDE]) and signs (corneal fluorescein staining [CFS]; Schirmer test). We used a LOESS technique to fit a model in function of data variations and obtain a predictive value of the scores progression over time. RESULTS: The records of 123 patients who were followed-up for over 2 years (up to 62 months) were reviewed. The median baseline scores recorded were: OSDI 52 units, SANDE 62.2 units, CFS 2.0 Oxford units, and Schirmer 4 mm. After six months of follow up, scores improved for OSDI (-18.6 units, p = 0.007), SANDE (23.7 units, p = 0.01), and CFS (-0.7 Oxford units, p < 0.001). Data analysis showed that after a 2-year follow up the three parameters continued to improve: OSDI -13.67 units (27% reduction), SANDE -17.55 units (28%), CFS -1.1 units (54%), but Schirmer test scores progressively worsened -1.2 mm (22%). CONCLUSION: In patients with ocular GVHD symptoms and corneal fluorescein staining improved after initiation of treatment, meanwhile Schirmer scores declined progressively. This indicates that appropriate treatment in chronic ocular GVHD can lead to mid- and long-term improvements in symptoms and corneal epitheliopathy; however, sustained reduction in Schirmer test scores suggests chronic tear production impairment.
Reshef ER, Wolkow N, Jakobiec FA, Yoon MK. Histopathologic Findings of Linear Scleroderma Displaying Focal Trichiasis Secondary to Tarsal Thinning. Ophthalmic Plast Reconstr Surg 2018;34(4):e124-e127.Abstract
Linear scleroderma en coup de sabre with ophthalmic findings has been previously described in the literature on numerous occasions. A 57-year-old woman presented with focal trichiasis secondary to tarsal thinning, adjacent to a linear brow and forehead deformity consistent with linear scleroderma en coup de sabre. Cases of linear scleroderma en coup de sabre involving the eyelids have been reported, most often with madarosis, ptosis, or skin atrophy; however, to the authors' knowledge, this is the first reported case of linear scleroderma associated with trichiasis and involvement of the deeper eyelid tissues, particularly the tarsus.
Fan BJ, Chen X, Sondhi N, Sharmila FP, Soumittra N, Sripriya S, Sacikala S, Asokan R, Friedman DS, Pasquale LR, Gao RX, Vijaya L, Bailey JC, Vitart V, Macgregor S, Hammond CJ, Khor CC, Haines JL, George R, Wiggs JL, and Consortium MAGGS; IGGC; NEIGHBORHOOD. Family-Based Genome-Wide Association Study of South Indian Pedigrees Supports WNT7B as a Central Corneal Thickness Locus. Invest Ophthalmol Vis Sci 2018;59(6):2495-2502.Abstract
Purpose: To identify genetic risk factors contributing to central corneal thickness (CCT) in individuals from South India, a population with a high prevalence of ocular disorders. Methods: One hundred ninety-five individuals from 15 large South Indian pedigrees were genotyped using the Omni2.5 bead array. Family-based association for CCT was conducted using the score test in MERLIN. Results: Genome-wide association study (GWAS) identified strongest association for single nucleotide polymorphisms (SNPs) in the first intron of WNT7B and CCT (top SNP rs9330813; β = -0.57, 95% confidence interval [CI]: -0.78 to -0.36; P = 1.7 × 10-7). We further investigated rs9330813 in a Latino cohort and four independent European cohorts. A meta-analysis of these data sets demonstrated statistically significant association between rs9330813 and CCT (β = -3.94, 95% CI: -5.23 to -2.66; P = 1.7 × 10-9). WNT7B SNPs located in the same genomic region that includes rs9330813 have previously been associated with CCT in Latinos but with other ocular quantitative traits related to myopia (corneal curvature and axial length) in a Japanese population (rs10453441 and rs200329677). To evaluate the specificity of the observed WNT7B association with CCT in the South Indian families, we completed an ocular phenome-wide association study (PheWAS) for the top WNT7B SNPs using 45 ocular traits measured in these same families including corneal curvature and axial length. The ocular PheWAS results indicate that in the South Indian families WNT7B SNPs are primarily associated with CCT. Conclusions: The results indicate robust evidence for association between WNT7B SNPs and CCT in South Indian pedigrees, and suggest that WNT7B SNPs can have population-specific effects on ocular quantitative traits.
Iglesias AI, Mishra A, Vitart V, Bykhovskaya Y, Höhn R, Springelkamp H, Cuellar-Partida G, Gharahkhani P, Bailey JCN, Willoughby CE, Li X, Yazar S, Nag A, Khawaja AP, Polašek O, Siscovick D, Mitchell P, Tham YC, Haines JL, Kearns LS, Hayward C, Shi Y, van Leeuwen EM, Taylor KD, Taylor KD, Bonnemaijer P, Rotter JI, Martin NG, Zeller T, Mills RA, Staffieri SE, Jonas JB, Schmidtmann I, Boutin T, Kang JH, Lucas SEM, Wong TY, Beutel ME, Wilson JF, Wilson JF, Wilson JF, Uitterlinden AG, Vithana EN, Foster PJ, Hysi PG, Hewitt AW, Khor CC, Pasquale LR, Montgomery GW, Klaver CCW, Aung T, Pfeiffer N, Mackey DA, Hammond CJ, Cheng C-Y, Craig JE, Rabinowitz YS, Wiggs JL, Burdon KP, van Duijn CM, Macgregor S. Cross-ancestry genome-wide association analysis of corneal thickness strengthens link between complex and Mendelian eye diseases. Nat Commun 2018;9(1):1864.Abstract
Central corneal thickness (CCT) is a highly heritable trait associated with complex eye diseases such as keratoconus and glaucoma. We perform a genome-wide association meta-analysis of CCT and identify 19 novel regions. In addition to adding support for known connective tissue-related pathways, pathway analyses uncover previously unreported gene sets. Remarkably, >20% of the CCT-loci are near or within Mendelian disorder genes. These included FBN1, ADAMTS2 and TGFB2 which associate with connective tissue disorders (Marfan, Ehlers-Danlos and Loeys-Dietz syndromes), and the LUM-DCN-KERA gene complex involved in myopia, corneal dystrophies and cornea plana. Using index CCT-increasing variants, we find a significant inverse correlation in effect sizes between CCT and keratoconus (r = -0.62, P = 5.30 × 10) but not between CCT and primary open-angle glaucoma (r = -0.17, P = 0.2). Our findings provide evidence for shared genetic influences between CCT and keratoconus, and implicate candidate genes acting in collagen and extracellular matrix regulation.
McDermott AM, Baidouri H, Woodward AM, Kam WR, Liu Y, Chen X, Ziemanski JF, Vistisen K, Hazlett LD, Nichols KK, Argüeso P, Sullivan DA. Short Tandem Repeat (STR) Profiles of Commonly Used Human Ocular Surface Cell Lines. Curr Eye Res 2018;:1-5.Abstract
PURPOSE: The purpose of this study is to establish the short tandem repeat (STR) profiles of several human cell lines commonly used in ocular surface research. MATERIALS AND METHODS: Independently DNA was extracted from multiple passages of three human corneal epithelial cell lines, two human conjunctival epithelial cell lines and one meibomian gland cell line, from different laboratories actively involved in ocular surface research. The samples were then subjected to STR analysis on a fee-for-service basis in an academic setting and the data compared against that in available databases. RESULTS: The STR profiles for the human corneal epithelial cells were different among the three cell lines studied and for each line the profiles were identical across the samples provided by three laboratories. Profiles for the human conjunctival epithelial cells were different among the two cell lines studied. Profiles for the meibomian gland cell line were identical across the samples provided by three laboratories. No samples were contaminated by elements of other cell lines such as HeLa. CONCLUSIONS: This comprehensive study provides verification of STR profiles for commonly used human ocular surface cell lines that can now be used as a reference by others in the field to authenticate the cell lines in use in their own laboratories.
Chen D, Sahin A, Kam WR, Liu Y, Rahimi Darabad R, Sullivan DA. Influence of lipopolysaccharide on proinflammatory gene expression in human corneal, conjunctival and meibomian gland epithelial cells. Ocul Surf 2018;16(3):382-389.Abstract
PURPOSE: Lipopolysaccharide (LPS), a bacterial endotoxin, is known to stimulate leuokotriene B4 (LTB4) secretion by human corneal (HCECs), conjunctival (HConjECs) and meibomian gland (HMGECs) epithelial cells. We hypothesize that this LTB4 effect represents an overall induction of proinflammatory gene expression in these cells. Our objective was to test this hypothesis. METHODS: Immortalized HCECs, HConjECs and HMGECs were cultured in the presence or absence of LPS (15 μg/ml) and ligand binding protein (LBP; 150 ng/ml). Cells were then processed for RNA isolation and the analysis of gene expression by using Illumina BeadChips, background subtraction, cubic spline normalization and GeneSifter software. RESULTS: Our findings show that LPS induces a striking increase in proinflammatory gene expression in HCECs and HConjECs. These cellular reactions are associated with a significant up-regulation of genes associated with inflammatory and immune responses (e.g. IL-1β, IL-8, and tumor necrosis factor), including those related to chemokine and Toll-like receptor signaling pathways, cytokine-cytokine receptor interactions, and chemotaxis. In contrast, with the exception of Toll-like signaling and associated innate immunity pathways, almost no proinflammatory ontologies were upregulated by LPS in HMGECs. CONCLUSIONS: Our results support our hypothesis that LPS stimulates proinflammatory gene expression in HCECs and HConjECs. However, our findings also show that LPS does not elicit such proinflammatory responses in HMGECs.
Sié A, Diarra A, Millogo O, Zongo A, Lebas E, Bärnighausen T, Chodosh J, Porco TC, Deiner MS, Lietman TM, Keenan JD, Oldenburg CE. Seasonal and Temporal Trends in Childhood Conjunctivitis in Burkina Faso. Am J Trop Med Hyg 2018;Abstract
Acute conjunctivitis follows a seasonal pattern. Although its clinical course is typically self-limited, conjunctivitis epidemics incur a substantial economic burden because of missed school and work days. This study investigated seasonal and temporal trends of childhood conjunctivitis in the entire country of Burkina Faso from 2013 to 2016, using routine monthly surveillance from 2,444 government health facilities. A total of 783,314 cases were reported over the 4-year period. Conjunctivitis followed a seasonal pattern throughout the country, with a peak in April. A nationwide conjunctivitis outbreak with a peak in September 2016 was noted ( < 0.001), with an excess number of cases first detected in June 2016. Nationwide passive surveillance was able to detect an epidemic 3 months before its peak, which may aide in allocation of resources for containment and mitigation of transmission in future outbreaks.
Hua J, Inomata T, Chen Y, Foulsham W, Stevenson W, Shiang T, Bluestone JA, Dana R. Pathological conversion of regulatory T cells is associated with loss of allotolerance. Sci Rep 2018;8(1):7059.Abstract
CD4CD25Foxp3 Regulatory T cells (Tregs) play a critical role in immune tolerance. The plasticity and functional adaptability of Tregs in an inflammatory microenvironment has been demonstrated in autoimmunity. Here, using a double transgenic mouse model that permits Foxp3 lineage tracing, we investigated the phenotypic plasticity of Foxp3 Tregs in a well-characterized murine model of corneal transplantation. In order to subvert the normal immune privilege of the cornea and foster an inflammatory milieu, host mice were exposed to desiccating stress prior to transplantation. Treg frequencies and function were decreased following desiccating stress, and this corresponded to decreased graft survival. A fraction of Tregs converted to IL-17 or IFNγ 'exFoxp3' T cells that were phenotypically indistinguishable from effector Th17 or Th1 cells, respectively. We investigated how Foxp3 expression is modulated in different Treg subsets, demonstrating that neuropilin-1 peripherally-derived Tregs are particularly susceptible to conversion to IL-17/IFNγ exFoxp3 cells in response to cues from their microenvironment. Finally, we show that IL-6 and IL-23 are implicated in the conversion of Tregs to exFoxp3 cells. This report demonstrates that the pathological conversion of Tregs contributes to the loss of corneal immune privilege.