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1.  Expression Analysis of the Transmembrane Mucin MUC20 in Human Corneal and Conjunctival Epithelia 
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.
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.
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.
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.
Elucidation of the mucin repertoire produced by the ocular surface epithelia is critical to understand how the eye is protected against environmental insult and infection. Here, we report on the expression, distribution, and regulation of a novel transmembrane mucin, MUC20, at the ocular surface.
PMCID: PMC4184386  PMID: 25168902
MUC20; transmembrane mucin; cornea; conjunctiva; epithelial cell
2.  Induction of Corneal Myofibroblasts by Lens-derived Transforming Growth Factor β1 (TGFβ1): A Transgenic Mouse Model 
Brain research bulletin  2009;81(2-3):287.
Transforming growth factor β(TGFβ) is an important cytokine in corneal development and wound healing. Transgenic mice that express an active form of human TGF β1 driven by a lens-specific promoter were used in the current study to determine the biological effects of lens-derived TGFβ1 on postnatal corneal development and homeostasis.
The postnatal corneal changes in the TGFβ1 transgenic mice were examined by fluorescein labeling and histology. Epithelial/endothelial-to-mesenchymal transition (E/EnMT) in the transgenic mouse cornea was demonstrated by immunostaining for α-smooth muscle actin (α-SMA) and cadherin-11. Expression of E- and N-cadherin in the corneal epithelial and endothelial cells, respectively, was analyzed by in situ hybridization.
Among the established TGF β1 transgenic lines, mice from line OVE853 and OVE917 had normal-sized eyeballs but developed a corneal haze after eyelid opening. Histological examination showed that prenatal corneal development appeared to be normal. However, after postnatal day 7 (P7), the corneal endothelial cells in transgenic line OVE853 began to lose normal cell-cell contact and basement membrane structure. The endothelial layer was eventually absent in the inner surface of the transgenic mouse cornea. The morphological changes in the cornea correlated with abnormal expression of α-SMA, a molecular marker of EMT, and stress fiber formation in myofibroblast-like cells, which initially appeared in the corneal endothelial layer and subsequently in the corneal epithelial and stromal layers. The E/EnMT in the transgenic mouse cornea was further demonstrated by loss of E- and N-cadherin expression in the corneal epithelial and endothelial cells respectively, and meanwhile increasing expression of cadherin-11 in both corneal epithelium and stroma.
Elevated levels of active TGF β1 in the anterior chamber can lead to myofibroblast formation in the corneal endothelial layer and subsequently in the corneal epithelial and stromal layers. Our data suggest that the levels of biologically active TGFβ in the aqueous humor must be under tight control to maintain corneal homeostasis. TGF β1 is the major cytokine during wound healing. Therefore, our findings also suggest a potential mechanism to explain the loss of corneal endothelial barrier and corneal opacification after intraocular surgery or trauma.
PMCID: PMC2814984  PMID: 19897021
3.  SSEA4 Is a Potential Negative Marker for the Enrichment of Human Corneal Epithelial Stem/Progenitor Cells 
This study shows the first evidence that SSEA4 is highly expressed in differentiated corneal epithelial cells and could be used as a negative marker to enrich the isolation of limbal stem cells.
To examine the expression of stage-specific embryonic antigen-4 (SSEA4) in the epithelium of the human ocular surface and characterize SSEA4+ and SSEA4− limbal epithelial cells.
SSEA4 expression in the human cornea and limbus was examined by RT-PCR and immunohistochemistry. SSEA4+ and SSEA4− cells were then separated by using magnetic beads. The phenotypes of these two cell populations were evaluated on the basis of cell size, clonogenic assay, and expression of putative limbal stem cell (LSC) and corneal epithelial differentiation markers.
SSEA4 was expressed in all layers of the corneal and anterior limbal epithelia. Discrete clusters of SSEA4+ cells were present in the central and posterior limbal epithelia. SSEA4+ cells accounted for an average of 40% of the total limbal epithelial cells. The SSEA4− population contained five times more small cells (≤11 μm in diameter) than did the SSEA4+ population. The expression levels of the putative LSC markers ABCG2, ΔNp63α, and cytokeratin (K)14 were significantly higher in the SSEA4− population than in the SSEA4+ population. The SSEA4− cells also expressed a significantly higher level of N-cadherin, but a lower level of the differentiation marker K12. The colony-forming efficiency in the SSEA4− population was 25.2% (P = 0.04) and 1.6-fold (P < 0.05) higher than in the unsorted population and the SSEA4+ population, respectively.
SSEA4 is highly expressed in differentiated corneal epithelial cells, and SSEA4− limbal epithelial cells contain a higher proportion of limbal stem/progenitor cells. SSEA4 could be used as a negative marker to enrich the isolation of LSCs.
PMCID: PMC3175983  PMID: 21685344
4.  Wakayama Symposium: Challenges of Future Research in Ocular Surface Cell Biology 
The ocular surface  2012;11(1):19-24.
During embryonic development, surface ectoderm differentiates to form corneal, conjunctival, and eyelid epidermal epithelia, and glandular epithelium (lacrimal and meibomian glands). Periocular mesenchymal cells of neural crest origin migrate and differentiate, leading to the formation of corneal endothelium and the stromas of the cornea, conjunctiva, eyelids, and trabecular meshwork. The formation of functional ocular surface tissues requires coordinated spatial and temporal expression of transcription factors and signaling molecules of various cytokines and signaling pathways, and the synthesis and remodeling of unique extracellular matrix. Although bidirectional interactions and signaling between mesenchyme and epithelium are considered necessary for embryonic formation of ocular surface tissues and homeostasis in adults, the molecular and cellular mechanisms that regulate such processes remain largely unknown. To investigate possible mechanisms, we have developed mouse models in which the gene functions of ocular surface epithelia and stromas can be altered by Doxycycline induction in spatial and temporal specific manners.
PMCID: PMC3551217  PMID: 23321356
Cre-LoxP system; gene targeting; genetically modified mouse lines; ocular surface tissues morphogenesis; Tet-On system; transgenesis
5.  Mucin Characteristics of Human Corneal-Limbal Epithelial Cells that Exclude the Rose Bengal Anionic Dye 
Rose bengal is an organic anionic dye used to assess damage of the ocular surface epithelium in ocular surface disease. It has been proposed that mucins have a protective role, preventing rose bengal staining of normal ocular surface epithelial cells. The current study was undertaken to evaluate rose bengal staining in a human corneal-limbal epithelial (HCLE) cell line known to produce and glycosylate membrane-associated mucins.
HCLE cells were grown to confluence in serum-free medium and switched to DMEM/F12 with 10% serum to promote differentiation. Immunolocalization of the membrane-associated mucins MUC1 and MUC16 and the T-antigen carbohydrate epitope was performed with the monoclonal antibodies HMFG-2 and OC125 and jacalin lectin, respectively. To assess dye uptake, cultures were incubated for 5 minutes with 0.1% rose bengal and photographed. To determine whether exclusion of negatively charged rose bengal requires a negative charge at the cell surface, cells were incubated with fluoresceinated cationized ferritin. The effect of hyperosmotic stress on rose bengal staining in vitro was evaluated by increasing the ion concentration (Ca+2 and Mg+2) in the rose bengal uptake assay.
The cytoplasm and nucleus of confluent HCLE cells cultured in media without serum, lacking the expression of MUC16 but not MUC1, as well as human corneal fibroblasts, which do not express mucins, stained with rose bengal. Culture of HCLE cells in medium containing serum resulted in the formation of islands of stratified cells that excluded rose bengal. Apical cells of the stratified islands produced MUC16 and the T-antigen carbohydrate epitope on their apical surfaces. Colocalization experiments demonstrated that fluoresceinated cationized ferritin did not bind to these stratified cells, indicating that rose bengal is excluded from cells that lack negative charges. Increasing the amounts of divalent cations in the media reduced the cellular area protected against rose bengal uptake.
These results indicate that stratification and differentiation of corneal epithelial cells, as measured by the capacity to produce the membrane-associated mucin MUC16 and the mucin-associated T-antigen carbohydrate on their apical surfaces provide protection against rose bengal penetrance in vitro and suggest a role for membrane-associated mucins and their oligosaccharides in the protection of ocular surface epithelia.
PMCID: PMC1351157  PMID: 16384952
6.  Immune profile of squamous metaplasia development in autoimmune regulator-deficient dry eye 
Molecular Vision  2009;15:563-576.
Squamous metaplasia of the ocular surface epithelium in severe Sjögren syndrome (SS) dry eye has been implicated to be associated with chronic engagement of immune-mediated inflammation. While the detailed immunopathological mechanism underlying keratinization of the ocular muco-epithelium in this setting remains unclear, mice deficient in the autoimmune regulator gene (Aire) demonstrate SS-like pathological changes in the exocrine organs and ocular surface including squamous metaplasia. Using this murine model, we sought to determine the specific immune events that predict squamous metaplasia of the cornea in Aire deficiency.
Lissamine green staining, goblet cell density, and corneal small proline-rich protein 1B (SPRR1B) were compared in Aire-sufficient and -deficient mice at 4, 8, and 16 weeks of age. Corneal, limbal and conjunctival infiltration of CD4+ and CD8+ T cells as well as CD11c+ and MHC class II (I-Ad+) dendritic cells (DCs) were examined at the same time points. Ordinary least squares regression was used to model SPRR1B’s relationship with lissamine green staining, goblet cell density, and immune cell infiltration.
Lissamine green staining was present in Aire-deficient mice by four weeks of age and increased over time. Compared to Aire-sufficient controls, conjunctival goblet cell density (GCD) decreased and corneal SPRR1B increased in Aire-deficient mice with significant differences noted at both 8 and 16 weeks. Immune-mediated CD4+ T cell infiltration of the conjunctiva and limbus peaked at eight weeks and then decreased. In contrast, corneal T cell infiltration continued to increase over time, reaching a maximum cell number at 16 weeks. CD11c+ myeloid-derived DCs were found in the conjunctiva and limbus at all time points. As the mice aged, there was a notable increase in corneal CD11c+ cell counts. Interestingly, the dynamic of activated MHC class II+ DCs was nearly identical to that of CD4+ T cells, peaking first in the limbus at eight weeks with maximum infiltration of the cornea by 16 weeks. Regression analysis showed that squamous metaplasia biomarker, SPRR1B, is strongly related to the lissamine green staining of the ocular surface. Corneal infiltration of activated DCs was most prognostic of corneal SPRR1B expression while the presence of precursor DCs, activated DCs, and CD4+ T cells in the limbus were also significant predictors of SPRR1B.
Aire-deficient mice represent a useful model to study Sjögren-like autoimmune-mediated ocular surface disease. Results of the current study suggest that squamous cell precursor protein, SPRR1B, provides an important readout to evaluate ocular surface damage and specific events related to immune-mediated inflammation. Results also define an appropriate time frame for interventional studies to develop more effective therapies for keratinizing ocular surface disease.
PMCID: PMC2660375  PMID: 19365590
7.  Cleavage of functional IL-2 receptor alpha chain (CD25) from murine corneal and conjunctival epithelia by MMP-9 
IL-2 has classically been considered a cytokine that regulates T cell proliferation and differentiation, signaling through its heterotrimeric receptor (IL-2R) consisting of α (CD25), β (CD122), γ chains (CD132). Expression of IL-2R has also been detected in mucosal epithelial cells. Soluble IL-2Rα (CD25) has been reported as an inflammatory marker. We evaluated the expression of CD25 and CD122 in the ocular surface epithelium and investigated the mechanism of proteolytic cleavage of CD25 from these cells.
Desiccating stress (DS) was used as an inducer of matrix metalloproteinase 9 (MMP-9). DS was created by subjecting C57BL/6 and MMP-9 knockout (BKO) mice and their wild-type littermates (WT) mice to a low humidity and drafty environment for 5 days (DS5). A separate group of C57BL/6 mice was subjected to DS5 and treatment with topical 0.025% doxycycline, a MMP inhibitor, administered QID. The expression of CD25 and CD122 was evaluated in cryosections by dual-label laser scanning confocal microscopy. Western blot was used to measure relative levels of CD25 in epithelial lysates. Gelatinase activity was evaluated by in situ zymography. Soluble CD25 in tear fluid was measured by an immunobead assay.
CD25 and CD122 were abundantly expressed in cornea (all layers) and conjunctiva epithelia (apical and subapical layers) in nonstressed control mice. After desiccating stress, we found that immunoreactivity to CD25, but not CD122, decreased by the ocular surface epithelia and concentration of soluble CD25 in tears increased as MMP-9 staining increased. CD25 was preserved in C57BL/6 mice topically treated with an MMP-9 inhibitor and in MMP-9 knock-out mice. MMP-9 treatment of human cultured corneal epithelial cells decreased levels of CD25 protein in a concentration dependent fashion.
Our results indicate that functional IL-2R is produced by the ocular surface epithelia and that CD25 is proteolytic cleaved to its soluble form by MMP-9, which increases in desiccating stress. These findings provide new insight into IL-2 signaling in mucosal epithelia.
PMCID: PMC2777897  PMID: 19878594
8.  Conditional Disruption of Mouse Klf5 Results in Defective Eyelids with Malformed Meibomian Glands, Abnormal Cornea and Loss of Conjunctival Goblet Cells 
Developmental biology  2011;356(1):5-18.
Members of the Krüppel-like family of transcription factors regulate diverse developmental processes in various organs. Previously, we have demonstrated the role of Klf4 in the mouse ocular surface. Herein, we determined the role of the structurally related Klf5, using Klf5-conditional null (Klf5CN) mice derived by mating Klf5-LoxP and Le-Cre mice. Klf5 mRNA was detected as early as embryonic day 12 (E12) in the cornea, conjunctiva and eyelids, wherein its expression increased during development. Though the embryonic eye morphogenesis was unaltered in the Klf5CN mice, postnatal maturation was defective, resulting in smaller eyes with swollen eyelids that failed to separate properly. Klf5CN palpebral epidermis was hyperplastic with 7-9 layers of keratinocytes, compared with 2-3 in the wild type (WT). Klf5CN eyelid hair follicles and sebaceous glands were significantly enlarged, and the meibomian glands malformed. Klf5CN lacrimal glands displayed increased vasculature and large number of infiltrating cells. Klf5CN corneas were translucent, thicker with defective epithelial basement membrane and hypercellular stroma. Klf5CN conjunctiva lacked goblet cells, demonstrating that Klf5 is required for conjunctival goblet cell development. The number of Ki67-positive mitotic cells was more than doubled, consistent with the increased number of Klf5CN ocular surface epithelial cells. Co-ablation of Klf4 and Klf5 resulted in a more severe ocular surface phenotype compared with Klf4CN or Klf5CN, demonstrating that Klf4 and Klf5 share few if any, redundant functions. Thus, Klf5CN mice provide a useful model for investigating ocular surface pathologies involving meibomian gland dysfunction, blepharitis, corneal or conjunctival defects.
PMCID: PMC3130829  PMID: 21600198
Klf5; cornea; conjunctiva; meibomian glands; lacrimal glands; eyelids; goblet cells
9.  Eyelid Closure in Embryogenesis Is Required for Ocular Adnexa Development 
Mammalian eye development requires temporary fusion of the upper and lower eyelids in embryogenesis. Failure of lid closure in mice leads to an eye open at birth (EOB) phenotype. Many genetic mutant strains develop this phenotype and studies of the mutants lead to a better understanding of the signaling mechanisms of morphogenesis. The present study investigates the roles of lid closure in eye development.
Seven mutant mouse strains were generated by different gene ablation strategies that inactivated distinct signaling pathways. These mice, including systemic ablation of Map3k1 and Dkk2, ocular surface epithelium (OSE) knockout of c-Jun and Egfr, conditional knockout of Shp2 in stratified epithelium (SE), as well as the Map3k1/Jnk1 and Map3k1/Rhoa compound mutants, all exhibited defective eyelid closure. The embryonic and postnatal eyes in these mice were characterized by histology and immunohistochemistry.
Some eye abnormalities, such as smaller lens in the Map3k1-null mice and Harderian gland hypoplasia in the Dkk2-null mice, appeared to be mutant strain–specific, whereas other abnormalities were seen in all mutants examined. The common defects included corneal erosion/ulceration, meibomian gland hypoplasia, truncation of the eyelid tarsal muscles, failure of levator palpebrae superioris (LPS) extension into the upper eyelid and misplacement of the inferior oblique (IO) muscle and inferior rectus (IR) muscle. The muscle defects were traced to the prenatal fetuses.
In addition to providing a protective barrier for the ocular surface, eyelid closure in embryogenesis is required for the development of ocular adnexa, including eyelid and extraocular muscles.
Eyelid closure is required for tarsals, extraocular muscle and LPS development. Failure of eyelid closure does not affect corneal and conjunctival differentiation of the prenatal eye, but leads to exposure-driven remodeling changes of the postnatal eye.
PMCID: PMC4245069  PMID: 25377219
embryonic eyelid closure; ocular adnexa; extraocular muscles; tarsal muscles; LPS
10.  14-3-3σ Controls Corneal Epithelium Homeostasis and Wound Healing 
Stratified squamous epithelial differentiation is characterized by expression of 14-3-3σ. This study provides evidence that 14-3-3σ regulates corneal epithelial cell differentiation. Reduced 14-3-3σ activity led to age-dependent meibomian gland atrophy and corneal plaque formation.
To investigate the functional role of 14-3-3σ in regulation of the corneal epithelial proliferation, differentiation, and wound-healing response.
Corneal phenotypes were investigated in heterozygous repeated epilation (Er) mice carrying mutations in the sfn (14-3-3σ) gene. Immunohistochemistry was used to study the corneal morphogenesis of the Er/Er embryos at embryonic day (E)18.5. Corneal homeostasis and the wound-healing response were investigated macroscopically and microscopically in the adult heterozygous Er mice. Corneal epithelial cell proliferation and differentiation were assessed by BrdU incorporation and immunohistochemistry with specific antibodies for differentiation markers. Furthermore, corneal stroma neovascularization and meibomian gland degeneration were examined by immunohistochemistry. The healing of corneal wounds after debridement was monitored and visualized by fluorescent staining.
Homozygous mutation of 14-3-3σ led to defects in embryonic corneal epithelial development and differentiation, whereas young heterozygotes showed normal corneal development and homeostasis. However, older heterozygotes displayed a dramatic corneal wound-healing defect characterized by hyperplastic basal progenitor cells (some of which undergo a differentiation switch to express markers of keratinized epidermis); cornea stroma changes including neovascularization; and corneal opacity, leading to plaque formation. Aged heterozygotes also showed meibomian gland atrophy.
14-3-3σ is essential for corneal epithelium differentiation, and plays an important role in corneal epithelium development and daily renewal of the adult corneal epithelium.
PMCID: PMC3081250  PMID: 21228373
11.  Limbal Stem Cell Transplantation 
Executive Summary
The objective of this analysis is to systematically review limbal stem cell transplantation (LSCT) for the treatment of patients with limbal stem cell deficiency (LSCD). This evidence-based analysis reviews LSCT as a primary treatment for nonpterygium LSCD conditions, and LSCT as an adjuvant therapy to excision for the treatment of pterygium.
Clinical Need: Condition and Target Population
The outer surface of the eye is covered by 2 distinct cell layers: the corneal epithelial layer that overlies the cornea, and the conjunctival epithelial layer that overlies the sclera. These cell types are separated by a transitional zone known as the limbus. The corneal epithelial cells are renewed every 3 to 10 days by a population of stem cells located in the limbus.
Nonpterygium Limbal Stem Cell Deficiency
When the limbal stem cells are depleted or destroyed, LSCD develops. In LSCD, the conjunctival epithelium migrates onto the cornea (a process called conjunctivalization), resulting in a thickened, irregular, unstable corneal surface that is prone to defects, ulceration, corneal scarring, vascularization, and opacity. Patients experience symptoms including severe irritation, discomfort, photophobia, tearing, blepharospasm, chronic inflammation and redness, and severely decreased vision.
Depending on the degree of limbal stem cell loss, LSCD may be total (diffuse) or partial (local). In total LSCD, the limbal stem cell population is completed destroyed and conjunctival epithelium covers the entire cornea. In partial LSCD, some areas of the limbus are unharmed, and the corresponding areas on the cornea maintain phenotypically normal corneal epithelium.
Confirmation of the presence of conjunctivalization is necessary for LSCD diagnosis as the other characteristics and symptoms are nonspecific and indicate a variety of diseases. The definitive test for LSCD is impression cytology, which detects the presence of conjunctival epithelium and its goblet cells on the cornea. However, in the opinion of a corneal expert, diagnosis is often based on clinical assessment, and in the expert’s opinion, it is unclear whether impression cytology is more accurate and reliable than clinical assessment, especially for patients with severe LSCD.
The incidence of LSCD is not well understood. A variety of underlying disorders are associated with LSCD including chemical or thermal injuries, ultraviolet and ionizing radiation, Stevens-Johnson syndrome, multiple surgeries or cryotherapies, contact lens wear, extensive microbial infection, advanced ocular cicatricial pemphigoid, and aniridia. In addition, some LSCD cases are idiopathic. These conditions are uncommon (e.g., the prevalence of aniridia ranges from 1 in 40,000 to 1 in 100,000 people).
Pterygium is a wing-shaped fibrovascular tissue growth from the conjunctiva onto the cornea. Pterygium is the result of partial LSCD caused by localized ultraviolet damage to limbal stem cells. As the pterygium invades the cornea, it may cause irregular astigmatism, loss of visual acuity, chronic irritation, recurrent inflammation, double vision, and impaired ocular motility.
Pterygium occurs worldwide. Incidence and prevalence rates are highest in the “pterygium belt,” which ranges from 30 degrees north to 30 degrees south of the equator, and lower prevalence rates are found at latitudes greater than 40 degrees. The prevalence of pterygium for Caucasians residing in urban, temperate climates is estimated at 1.2%.
Existing Treatments Other Than Technology Being Reviewed
Nonpterygium Limbal Stem Cell Deficiency
In total LSCD, a patient’s limbal stem cells are completely depleted, so any successful treatment must include new stem cells. Autologous oral mucosal epithelium transplantation has been proposed as an alternative to LSCT. However, this procedure is investigational, and there is very limited level 4c evidence1 to support this technique (fewer than 20 eyes examined in 4 case series and 1 case report).
For patients with partial LSCD, treatment may not be necessary if their visual axis is not affected. However, if the visual axis is conjunctivalized, several disease management options exist including repeated mechanical debridement of the abnormal epithelium; intensive, nonpreserved lubrication; bandage contact lenses; autologous serum eye drops; other investigational medical treatments; and transplantation of an amniotic membrane inlay. However, these are all disease management treatments; LSCT is the only curative option.
The primary treatment for pterygium is surgical excision. However, recurrence is a common problem after excision using the bare sclera technique: reported recurrence rates range from 24% to 89%. Thus, a variety of adjuvant therapies have been used to reduce the risk of pterygium recurrence including LSCT, amniotic membrane transplantation (AMT), conjunctival autologous (CAU) transplantation, and mitomycin C (MMC, an antimetabolite drug).
New Technology Being Reviewed
To successfully treat LSCD, the limbal stem cell population must be repopulated. To achieve this, 4 LSCT procedures have been developed: conjunctival-limbal autologous (CLAU) transplantation; living-related conjunctival-limbal allogeneic (lr-CLAL) transplantation; keratolimbal allogeneic (KLAL) transplantation; and ex vivo expansion of limbal stem cells transplantation. Since the ex vivo expansion of limbal stem cells transplantation procedure is considered experimental, it has been excluded from the systematic review. These procedures vary by the source of donor cells and the amount of limbal tissue used. For CLAU transplants, limbal stem cells are obtained from the patient’s healthy eye. For lr-CLAL and KLAL transplants, stem cells are obtained from living-related and cadaveric donor eyes, respectively.
In CLAU and lr-CLAL transplants, 2 to 4 limbal grafts are removed from the superior and inferior limbus of the donor eye. In KLAL transplants, the entire limbus from the donor eye is used.
The recipient eye is prepared by removing the abnormal conjunctival and scar tissue. An incision is made into the conjunctival tissue into which the graft is placed, and the graft is then secured to the neighbouring limbal and scleral tissue with sutures. Some LSCT protocols include concurrent transplantation of an amniotic membrane onto the cornea.
Regulatory Status
Health Canada does not require premarket licensure for stem cells. However, they are subject to Health Canada’s clinical trial regulations until the procedure is considered accepted transplantation practice, at which time it will be covered by the Safety of Human Cells, Tissues and Organs for Transplantation Regulations (CTO Regulations).
Review Strategy
The Medical Advisory Secretariat systematically reviewed the literature to assess the effectiveness and safety of LSCT for the treatment of patients with nonpterygium LSCD and pterygium. A comprehensive search method was used to retrieve English-language journal articles from selected databases.
The GRADE approach was used to systematically and explicitly evaluate the quality of evidence and strength of recommendations.
Summary of Findings
Nonpterygium Limbal Stem Cell Deficiency
The search identified 873 citations published between January 1, 2000, and March 31, 2008. Nine studies met the inclusion criteria, and 1 additional citation was identified through a bibliography review. The review included 10 case series (3 prospective and 7 retrospective).
Patients who received autologous transplants (i.e., CLAU) achieved significantly better long-term corneal surface results compared with patients who received allogeneic transplants (lr-CLAL, P< .001; KLAL, P< .001). There was no significant difference in corneal surface outcomes between the allogeneic transplant options, lr-CLAL and KLAL (P = .328). However, human leukocyte antigen matching and systemic immunosuppression may improve the outcome of lr-CLAL compared with KLAL. Regardless of graft type, patients with Stevens-Johnson syndrome had poorer long-term corneal surface outcomes.
Concurrent AMT was associated with poorer long-term corneal surface improvements. When the effect of the AMT was removed, the difference between autologous and allogeneic transplants was much smaller.
Patients who received CLAU transplants had a significantly higher rate of visual acuity improvements compared with those who received lr-CLAL transplants (P = .002). However, to achieve adequate improvements in vision, patients with deep corneal scarring will require a corneal transplant several months after the LSCT.
No donor eye complications were observed.
Epithelial rejection and microbial keratitis were the most common long-term complications associated with LSCT (complications occurred in 6%–15% of transplantations). These complications can result in graft failure, so patients should be monitored regularly following LSCT.
The search yielded 152 citations published between January 1, 2000 and May 16, 2008. Six randomized controlled trials (RCTs) that evaluated LSCT as an adjuvant therapy for the treatment of pterygium met the inclusion criteria and were included in the review.
Limbal stem cell transplantation was compared with CAU, AMT, and MMC. The results showed that CLAU significantly reduced the risk of pterygium recurrence compared with CAU (relative risk [RR], 0.09; 95% confidence interval [CI], 0.01–0.69; P = .02). CLAU reduced the risk of pterygium recurrence for primary pterygium compared with MMC, but this comparison did not reach statistical significance (RR, 0.48; 95% CI, 0.21–1.10; P = .08). Both AMT and CLAU had similar low rates of recurrence (2 recurrences in 43 patients and 4 in 46, respectively), and the RR was not significant (RR, 1.88; 95% CI, 0.37–9.5; P = .45). Since sample sizes in the included studies were small, failure to detect a significant difference between LSCT and AMT or MMC could be the result of type II error. Limbal stem cell transplantation as an adjuvant to excision is a relatively safe procedure as long-term complications were rare (< 2%).
GRADE Quality of Evidence
Nonpterygium Limbal Stem Cell Deficiency
The evidence for the analyses related to nonpterygium LSCD was based on 3 prospective and 7 retrospective case series. Thus, the GRADE quality of evidence is very low, and any estimate of effect is very uncertain.
The analyses examining LSCT as an adjuvant treatment option for pterygium were based on 6 RCTs. The quality of evidence for the overall body of evidence for each treatment option comparison was assessed using the GRADE approach. In each of the comparisons, the quality of evidence was downgraded due to serious or very serious limitations in study quality (individual study quality was assessed using the Jadad scale, and an assessment of allocation concealment and the degree of loss to follow-up), which resulted in low- to moderate-quality GRADE evidence ratings (low-quality evidence for the CLAU and AMT and CLAU and MMC comparisons, and moderate-quality evidence for the CLAU and CAU comparison).
Ontario Health System Impact Analysis
Nonpterygium Limbal Stem Cell Deficiency
Since 1999, Ontario’s out-of-country (OOC) program has approved and reimbursed 8 patients for LSCTs and 1 patient for LSCT consultations. Similarly, most Canadian provinces have covered OOC or out-of-province LSCTs. Several corneal experts in Ontario have the expertise to perform LSCTs.
As there are no standard guidelines for LSCT, patients who receive transplants OOC may not receive care aligned with the best evidence. To date, many of the patients from Ontario who received OOC LSCTs received concurrent AMTs, and the evidence from this analysis questions the use of this procedure. In addition, 1 patient received a cultured LSCT, a procedure that is considered investigational. Many patients with LSCD have bilateral disease and therefore require allogeneic transplants. These patients will require systemic and topical immunosuppression for several years after the transplant, perhaps indefinitely. Thus, systemic side effects associated with immunosuppression are a potential concern, and patients must be monitored regularly.
Amniotic membrane transplantation is a common addition to many ocular surface reconstruction procedures, including LSCT. Amniotic membranes are recovered from human placentas from planned, uneventful caesarean sections. Before use, serological screening of the donor’s blood should be conducted. However, there is still a theoretical risk of disease transmission associated with this procedure.
Financial Impact
For the patients who were reimbursed for OOC LSCTs, the average cost of LSCT per eye was $18,735.20 Cdn (range, $8,219.54–$33,933.32). However, the actual cost per patient is much higher as these costs do not include consultations and follow-up visits, multiple LSCTs, and any additional procedures (e.g., corneal transplants) received during the course of treatment OOC. When these additional costs were considered, the average cost per patient was $57,583 Cdn (range, $8,219.54–$130,628.20).
The estimated average total cost per patient for performing LSCT in Ontario is $2,291.48 Cdn (range, $951.48–$4,538.48) including hospital and physician fees. This cost is based on the assumption that LSCT is technically similar to a corneal transplant, an assumption which needs to be verified. The cost does not include corneal transplantations, which some proportion of patients receiving a LSCT will require within several months of the limbal transplant.
Pterygium recurrence rates after surgical excision are high, ranging from 24% to 89%. However, according to clinical experts, the rate of recurrence is low in Ontario. While there is evidence that the prevalence of pterygium is higher in the “pterygium belt,” there was no evidence to suggest different recurrence rates or disease severity by location or climate.
Nonpterygium Limbal Stem Cell Deficiency
Successful LSCTs result in corneal re-epithelialization and improved vision in patients with LSCD. However, patients who received concurrent AMT had poorer long-term corneal surface improvements. Conjunctival-limbal autologous transplantation is the treatment option of choice, but if it is not possible, living-related or cadaveric allogeneic transplants can be used. The benefits of LSCT outweigh the risks and burdens, as shown in Executive Summary Table 1. According to GRADE, these recommendations are strong with low- to very low-quality evidence.
Benefits, Risks, and Burdens – Nonpterygium Limbal Stem Cell Deficiency
Short- and long-term improvement in corneal surface (stable, normal corneal epithelium and decreased vascularization and opacity)
Improvement in vision (visual acuity and functional vision)
Long-term complications are experienced by 8% to 16% of patients
Risks associated with long-term immunosuppression for recipients of allogeneic grafts
Potential risk of induced LSCD in donor eyes
High cost of treatment (average cost per patient via OOC program is $57,583; estimated cost of procedure in Ontario is $2,291.48)
Costs are expressed in Canadian dollars.
GRADE of recommendation: Strong recommendation, low-quality or very low-quality evidence
benefits clearly outweigh risks and burdens
case series studies
strong, but may change if higher-quality evidence becomes available
Conjunctival-limbal autologous transplantations significantly reduced the risk of pterygium recurrence compared with CAU. No other comparison yielded statistically significant results, but CLAU reduced the risk of recurrence compared with MMC. However, the benefit of LSCT in Ontario is uncertain as the severity and recurrence of pterygium in Ontario is unknown. The complication rates suggest that CLAU is a safe treatment option to prevent the recurrence of pterygium. According to GRADE, given the balance of the benefits, risks, and burdens, the recommendations are very weak with moderate quality evidence, as shown in Executive Summary Table 2.
Benefits, Risks, and Burdens – Pterygium
Reduced recurrence; however, if recurrence is low in Ontario, this benefit might be minimal
Long-term complications rare
Increased cost
GRADE of recommendation: Very weak recommendations, moderate quality evidence.
uncertainty in the estimates of benefits, risks, and burden; benefits, risks, and burden may be closely balanced
very weak, other alternatives may be equally reasonable
PMCID: PMC3377549  PMID: 23074512
12.  Transcription, Translation, and Function of Lubricin, a Boundary Lubricant, at the Ocular Surface 
JAMA ophthalmology  2013;131(6):10.1001/jamaophthalmol.2013.2385.
Lubricin may be an important barrier to the development of corneal and conjunctival epitheliopathies that may occur in dry eye disease and contact lens wear.
To test the hypotheses that lubricin (ie, proteoglycan 4 [PRG4]), a boundary lubricant, is produced by ocular surface epithelia and acts to protect the cornea and conjunctiva against significant shear forces generated during an eyelid blink and that lubricin deficiency increases shear stress on the ocular surface and promotes corneal damage.
Design, Setting, and Participants
Human, porcine, and mouse tissues and cells were processed for molecular biological, immunohistochemical, and tribological studies, and wild-type and PRG4 knockout mice were evaluated for corneal damage.
Our findings demonstrate that lubricin is transcribed and translated by corneal and conjunctival epithelial cells. Lubricin messenger RNA is also present in lacrimal and meibomian glands, as well as in a number of other tissues. Absence of lubricin in PRG4 knockout mice is associated with a significant increase in corneal fluorescein staining. Our studies also show that lubricin functions as an effective friction-lowering boundary lubricant at the human cornea-eyelid interface. This effect is specific and cannot be duplicated by the use of hyaluronate or bovine serum albumin solutions.
Conclusions and Relevance
Our results show that lubricin is transcribed, translated, and expressed by ocular surface epithelia. Moreover, our findings demonstrate that lubricin presence significantly reduces friction between the cornea and conjunctiva and that lubricin deficiency may play a role in promoting corneal damage.
PMCID: PMC3887468  PMID: 23599181
13.  The Ocular Surface Phenotype of Muc5ac and Muc5b Null Mice 
Recent development of mice null for either Muc5ac or Muc5b mucin allows study of their specific roles at the mouse ocular surface. A recent report indicated that Muc5ac null mice show an ocular surface phenotype similar to that seen in dry eye syndrome. The purpose of our study was to determine the effect of lack of Muc5ac or Muc5b on the ocular surface, and to determine if environmental desiccating stress exacerbated a phenotype.
Muc5ac null and Muc5b null mice, and their wild-type controls were examined for ocular surface defects by fluorescein staining. The number of goblet cells per area of conjunctival epithelium was counted, and levels of mucin gene expression and genes associated with epithelial stress, keratinization, and differentiation, known to be altered in dry eye syndrome, were assayed. To determine if the null mice would respond more to desiccating stress than their wild-type controls, they were challenged in a controlled environment chamber (CEC) and assessed for changes in fluorescein staining, tear volume, and inflammatory cells within the conjunctival and corneal epithelia.
Unlike the previous study, we found no ocular surface phenotype in the Muc5ac null mice, even after exposure to desiccating environmental stress. Similarly, no ocular surface phenotype was present in the Muc5b null mice, either before or after exposure to a dry environment in the CEC.
Our results indicate that deleting either the Muc5ac or Muc5b gene is insufficient to create an observable dry eye phenotype on the ocular surface of these mice.
Mice null for either Muc5ac or Muc5b, the major secretory mucins on the ocular surface, were assessed for an ocular surface phenotype. Results indicate that deletion of either the Muc5ac or Muc5b gene is insufficient to create an observable dry eye phenotype on the ocular surface of these mice.
PMCID: PMC3894795  PMID: 24327612
mucins; dry eye; animal models
14.  Tear Film Mucins: Front Line Defenders of the Ocular Surface; Comparison with Airway and Gastrointestinal Tract Mucins 
Experimental eye research  2013;117:62-78.
The ocular surface including the cornea and conjunctiva and its overlying tear film are the first tissues of the eye to interact with the external environment. The tear film is complex containing multiple layers secreted by different glands and tissues. Each layer contains specific molecules and proteins that not only maintain the health of the cells on the ocular surface by providing nourishment and removal of waste products but also protect these cells from environment. A major protective mechanism that the corneal and conjunctival cells have developed is secretion of the innermost layer of the tear film, the mucous layer. Both the cornea and conjunctiva express membrane spanning mucins, whereas the conjunctiva also produces soluble mucins. The mucins present in the tear film serve to maintain the hydration of the ocular surface and to provide lubrication and anti-adhesive properties between the cells of the ocular surface and conjunctiva during the blink. A third function is to contribute to the epithelial barrier to prevent pathogens from binding to the ocular surface. This review will focus on the different types of mucins produced by the corneal and conjunctival epithelia. Also included in this review will be a presentation of the structure of mucins, regulation of mucin production, role of mucins in ocular surface diseases, and the differences in mucin production by the ocular surface, airways and gastrointestinal tract.
PMCID: PMC4222248  PMID: 23954166
Cornea; Conjunctiva; Mucin; Secretion; Proliferation; Tears
15.  A Role for Smoothened during Murine Lens and Cornea Development 
PLoS ONE  2014;9(9):e108037.
Various studies suggest that Hedgehog (Hh) signalling plays roles in human and zebrafish ocular development. Recent studies (Kerr et al., Invest Ophthalmol Vis Sci. 2012; 53, 3316–30) showed that conditionally activating Hh signals promotes murine lens epithelial cell proliferation and disrupts fibre differentiation. In this study we examined the expression of the Hh pathway and the requirement for the Smoothened gene in murine lens development. Expression of Hh pathway components in developing lens was examined by RT-PCR, immunofluorescence and in situ hybridisation. The requirement of Smo in lens development was determined by conditional loss-of-function mutations, using LeCre and MLR10 Cre transgenic mice. The phenotype of mutant mice was examined by immunofluorescence for various markers of cell cycle, lens and cornea differentiation. Hh pathway components (Ptch1, Smo, Gli2, Gli3) were detected in lens epithelium from E12.5. Gli2 was particularly localised to mitotic nuclei and, at E13.5, Gli3 exhibited a shift from cytosol to nucleus, suggesting distinct roles for these transcription factors. Conditional deletion of Smo, from ∼E12.5 (MLR10 Cre) did not affect ocular development, whereas deletion from ∼E9.5 (LeCre) resulted in lens and corneal defects from E14.5. Mutant lenses were smaller and showed normal expression of p57Kip2, c-Maf, E-cadherin and Pax6, reduced expression of FoxE3 and Ptch1 and decreased nuclear Hes1. There was normal G1-S phase but decreased G2-M phase transition at E16.5 and epithelial cell death from E14.5-E16.5. Mutant corneas were thicker due to aberrant migration of Nrp2+ cells from the extraocular mesenchyme, resulting in delayed corneal endothelial but normal epithelial differentiation. These results indicate the Hh pathway is required during a discrete period (E9.5–E12.5) in lens development to regulate lens epithelial cell proliferation, survival and FoxE3 expression. Defective corneal development occurs secondary to defects in lens and appears to be due to defective migration of peri-ocular Nrp2+ neural crest/mesenchymal cells.
PMCID: PMC4182430  PMID: 25268479
16.  Desiccating Stress Promotion of Th17 Differentiation by Ocular Surface Tissues through a Dendritic Cell-Mediated Pathway 
This study explored the interesting phenomenon that desiccating stress creates an environment on the ocular surface that stimulates the production of Th17-inducing cytokines by corneal and conjunctival epithelia that promote Th17 differentiation through a dendritic cell-mediated pathway.
To explore the phenomenon that corneal and conjunctival tissues subjected to desiccating stress (DS) promote Th17 differentiation by stimulating the production of Th17-inducing cytokines through a dendritic cell (DC)–mediated pathway.
Experimental dry eye was created by subjecting C57BL/6 mice to desiccating environmental stress. Corneal and conjunctival explants from dry eye or control mice were cocultured with DCs for 24 hours before CD4+ T cells were added for an additional 4 to 7 days. Expression of Th17-associated genes in the cornea, conjunctiva, DCs, and CD4+ T cells was evaluated by real-time PCR. Cytokine concentrations in coculture supernatants were measured by immunobead assay. IL-17–producing T cells were identified by ELISPOT bioassay.
Higher levels of IL-17A, TGF-β1, TGF-β2, IL-6, IL-23, and IL-1β mRNA transcripts and TGF-β1, IL-6, and IL-1β protein were observed in corneal epithelium and conjunctiva from dry eye mice. DCs cocultured with epithelial explants from dry eye mice for 2 days produced higher levels of TGF-β1, IL-6, IL-23, and IL-1β mRNA transcripts and of TGF-β1, IL-6, and IL-1β protein. CD4+ T cells cocultured with DCs and epithelial explants from dry eye mice expressed increased levels of IL-17A, IL-17F, IL-22, CCL-20, and retinoic acid receptor–related orphan receptor-γt mRNA transcripts and increased IL-17A protein and number of IL-17–producing T cells (Th17 cells).
These findings demonstrate that DS creates an environment on the ocular surface that stimulates the production of Th17-inducing cytokines by corneal and conjunctival epithelia that promote Th17 differentiation through a dendritic cell–mediated pathway.
PMCID: PMC2891467  PMID: 20130281
17.  Notch Signaling Modulates MUC16 Biosynthesis in an In Vitro Model of Human Corneal and Conjunctival Epithelial Cell Differentiation 
Notch proteins are a family of transmembrane receptors that coordinate binary cell fate decisions and terminal differentiation. This study demonstrates that biosynthesis of the cell surface-associated mucin MUC16 is posttranscriptionally regulated by Notch signaling at early stages of epithelial cell differentiation, suggesting that Notch signaling plays an important role in maintaining a wet-surface phenotype at the ocular surface.
Notch proteins are a family of transmembrane receptors that coordinate binary cell fate decisions and differentiation in wet-surfaced epithelia. We sought to determine whether Notch signaling contributes to maintaining mucosal homeostasis by modulating the biosynthesis of cell surface-associated mucins in an in vitro model of human corneal (HCLE) and conjunctival (HCjE) epithelial cell differentiation.
HCLE and HCjE cells were grown at different stages of differentiation, representing nondifferentiated (preconfluent and confluent) and differentiated (stratified) epithelial cultures. Notch signaling was blocked with the γ-secretase inhibitor dibenzazepine (DBZ). The presence of Notch intracellular domains (Notch1 to Notch3) and mucin protein (MUC1, -4, -16) was evaluated by electrophoresis and Western blot analysis. Mucin gene expression was determined by TaqMan real-time polymerase chain reaction.
Here we demonstrate that Notch3 is highly expressed in undifferentiated and differentiated HCLE and HCjE cells, and that Notch1 and Notch2 biosynthesis is enhanced by induction of differentiation with serum-containing media. Inhibition of Notch signaling with DBZ impaired MUC16 biosynthesis in a concentration-dependent manner in undifferentiated cells at both preconfluent and confluent stages, but not in postmitotic stratified cells. In contrast to protein levels, the amount of MUC16 transcripts were not significantly reduced after DBZ treatment, suggesting that Notch regulates MUC16 posttranscriptionally. Immunoblots of DBZ-treated epithelial cells grown at different stages of differentiation revealed no differences in the levels of MUC1 and MUC4.
These results indicate that MUC16 biosynthesis is posttranscriptionally regulated by Notch signaling at early stages of epithelial cell differentiation, and suggest that Notch activation contributes to maintaining a mucosal phenotype at the ocular surface.
PMCID: PMC3176047  PMID: 21508102
18.  Conjunctival FOXP3 Expression in Trachoma: Do Regulatory T Cells Have a Role in Human Ocular Chlamydia trachomatis Infection? 
PLoS Medicine  2006;3(8):e266.
Trachoma, caused by ocular infection with Chlamydia trachomatis, remains the leading infectious cause of blindness and in 2002 was responsible for 3.6% of total global blindness. Although transmission can be successfully interrupted using antibiotics and improvements in public and personal hygiene, the long-term success of the control programmes advocated by the World Health Organization are still uncertain. For the complete control and prevention of trachoma, a vaccine would be highly desirable. Currently there are no licensed vaccines for trachoma, and no human vaccine trials have been conducted since the 1960s. A barrier to new attempts to design and introduce a vaccine is the identification of immunologic correlates of protective immunity or immunopathology. We studied important correlates of the immune response in a trachoma-endemic population in order to improve our knowledge of this disease. This is essential for the successful development of a vaccine against both ocular and genital C. trachomatis infection.
Methods and Findings
We used quantitative real-time PCR for C. trachomatis 16S rRNA to identify conjunctival infection. The expression of IFN-γ, IDO, IL-10, and FOXP3 mRNA transcripts was measured. We evaluated the role of immune effector and regulatory responses in the control of chlamydial infection and in the resolution of clinical signs of trachoma in endemic communities in Gambia. All host transcripts examined were detectable even in normal conjunctiva. The levels of these transcripts were increased, compared to normal uninfected conjunctiva, when infection was detected, with or without clinical disease signs. Interestingly, when clinical disease signs were present in the absence of infection, the expression of a regulatory T cell transcription factor, FOXP3, remained elevated.
There is evidence of an increase in the magnitude of the local anti-chlamydial cytokine immune responses with age. This increase is coupled to a decline in the prevalence of infection and active trachoma, suggesting that effective adaptive immunity is acquired over a number of years. The anti-chlamydial and inflammatory immune response at the conjunctival surface, which may control chlamydial replication, is closely matched by counter inflammatory or regulatory IL-10 expression. Differences in the level of FOXP3 expression in the conjunctiva may indicate a role for regulatory T cells in the resolution of the conjunctival immune response, which is important in protection from immunopathology. However, the expression of cytokines that control chlamydial replication and those that regulate the conjunctival immune response is not simply juxtaposed; the interaction between the infection and the clinical disease process is therefore more complex.
The immune response in a trachoma-endemic population showed an increase in local anti-chlamydial cytokine responses with age, associated with a decline in the prevalence of infection and active trachoma.
Editors' Summary
Trachoma is the leading infectious cause of blindness worldwide. Six million people—most of whom live in crowded, unhygienic conditions with limited water supplies—are blind because of repeated eye infections with Chlamydia trachomatis. This bacterium passes easily from person to person on hands or clothing and is also spread by flies. Successive infections starting in childhood cause progressive scarring of the inside of the eyelid. Eventually, the eyelashes turn inwards and rub painfully over the front of the eye (the cornea). This causes corneal scarring, loss of corneal transparency, and, finally, irreversible loss of sight, usually in adulthood. C. trachomatis infections can be prevented by improving personal hygiene and by reducing fly breeding sites, and they can be treated with antibiotics. In addition, early scarring of the eyelid and turned-in eyelashes can be treated surgically.
Why Was This Study Done?
Through the above interventions, the World Health Organization hopes to eliminate trachoma by 2020, but a vaccine might also be necessary. To develop a vaccine, the human immune response to C. trachomatis needs to be better understood. As with other diseases, the immune response to C. trachomatis includes a pro-inflammatory side, which activates immune cells to attack the bacteria, and a regulatory side, which keeps the pro-inflammatory responses in check. The balance between these two sides is not perfect, however. Although the immune response deals with C. trachomatis infections efficiently, it also causes some of the tissue damage that leads to scarring and loss of sight. In this study, the researchers have investigated the human immune response to C. trachomatis to provide immunological information that might help vaccine development.
What Did the Researchers Do and Find?
The researchers examined school children living in Gambia, where trachoma is very common, for clinical signs of active trachoma (for example, red or swollen eyelids). To find out which children were infected with C. trachomatis, the researchers collected a few cells from the surface of their eyes and looked for a ribonucleic acid (RNA) molecule that is only made by C. trachomatis. The researchers also looked in these samples for human messenger RNA (mRNA) molecules that are made during pro-inflammatory and regulatory immune responses.
The children formed four groups based on infection with C. trachomatis and clinical signs. Some children—particularly the older ones—were uninfected and had no clinical signs. Others were infected but showed no clinical signs—these children were incubating the bacteria. Some were infected and had clinical disease; these children had the highest bacterial loads. Finally, children recovering from an infection carried no bacteria but still had some clinical signs.
The researchers detected different types of immune response in each of these groups. Children incubating the bacteria had a strong pro-inflammatory response—their immune systems were trying to fight off infection. The pro-inflammatory response was even stronger in the infected children with clinical signs, but now the regulatory response had also increased, presumably to limit inflammation. In children in the recovery phase, only regulatory immune cells, which were making an mRNA from a gene called FOXP3, remained active.
What Do These Findings Mean?
The relative rarity of infections and active disease in older children together with indications of a more active immune response to infection indicates that protective immunity to C. trachomatis is acquired through repeated exposure to it. This bodes well for the development of a vaccine, which would speed up the acquisition of this natural immunity. Furthermore, the new information about immune responses at different stages of infection with C. trachomatis should help in vaccine design. The findings need to be confirmed by tracking immune responses in individual children during episodes of infection, but could then be used to help design vaccines that produce good protective immunity against C. trachomatis without causing too much collateral tissue damage. The current results suggest, for example, that regulatory immune cells are important in limiting the inflammatory response, so vaccine developers may need to ensure that their vaccines stimulate the production of this sort of cell as well as of the pro-inflammatory cells needed to clear the infection.
Additional Information.
Please access these Web sites via the online version of this summary at
• NHS Direct Online patient information on trachoma
• World Health Organization information on trachoma and its elimination
• US Centers for Disease Control and Prevention general information on trachoma
• MedlinePlus encyclopedia entry on trachoma
PMCID: PMC1526769  PMID: 16881731
19.  Expression analysis of human pterygium shows a predominance of conjunctival and limbal markers and genes associated with cell migration 
Molecular Vision  2009;15:2421-2434.
Pterygium is a vision-impairing fibrovascular lesion that grows across the corneal surface and is associated with sunlight exposure. To increase our understanding of the cells types involved in pterygium, we have used expressed sequence tag analysis to examine the transcriptional repertoire of isolated pterygium and to identify marker genes for tissue origin and cell migration.
An unnormalized unamplified cDNA library was prepared from 15 pooled specimens of surgically removed pterygia as part of the NEIBank project. Gene expression patterns were compared with existing data for human cornea, limbus, and conjunctiva, and expression of selected genes was verified by immunofluorescence localization in normal eye ocular surface and in pterygium.
Sequence analysis of 2,976 randomly selected clones produced over 1,800 unique clusters, potentially representing single genes. The most abundant complementary DNAs from pterygium include clusterin, keratins 13 (Krt13) and 4 (Krt4), S100A9/calgranulin B, and spermidine/spermine N1-acetyltransferase (SAT1). Markers for both conjunctiva (such as keratin 13/4 and AQP3) and corneal epithelium (such as keratin 12/3 and AQP5) were present. Immunofluorescence of Krt12 and 13 in the normal ocular surface showed specificity of Krt12 in cornea and Krt13 in conjunctival and limbal epithelia, with a fairly sharp boundary at the limbal–corneal border. In the pterygium there was a patchy distribution of both Krt12 and 13 up to a normal corneal epithelial region specific for Krt12. Immunoglobulins were also among the prominently expressed transcripts. Several of the genes expressed most abundantly in excised pterygium, particularly S100A9 and SAT1, have roles in cell migration. SAT1 exerts its effects through control of polyamine levels. IPENSpm, a polyamine analogue, showed a significant ability to reduce migration in primary cultures of pterygium. A number of genes highly expressed in cornea were not found in pterygium (several small leucine-rich proteoglycan family members) or were expressed at considerably lower levels (ALDH3A1 and decorin).
The expression pattern of keratins and other markers in pterygium most closely resemble those of conjunctival and limbal cells; some corneal markers are present, notably Krt12, but at lower levels than equivalent conjunctival markers. Our data are consistent with the model of pterygium developing from the migration of conjunctival- and limbal-like cells into corneal epithelium. Identification of genes with roles in cell migration suggests potential therapeutic targets. In particular, the ability of polyamine analogues to reduce migration in primary cultures of pterygium presents a possible approach to slowing pterygium growth.
PMCID: PMC2785720  PMID: 19956562
20.  Targeted Deletion of AP-2α Leads to Disruption in Corneal Epithelial Cell Integrity and Defects in the Corneal Stroma 
The present study was undertaken to create a conditional knockout of AP-2α in the corneal epithelium.
A line of mice expressing Cre-recombinase specifically in the early lens placode was crossed with mice in which the AP-2α allele is flanked by two loxP sites. The resultant Le-AP-2α mutants exhibited a targeted deletion of AP-2α in lens placode derivatives, including the differentiating corneal epithelium.
The Le-AP-2α mutant mice were viable and had a normal lifespan. The adult corneal epithelium exhibited a variation in the number of stratified epithelial layers, ranging from 2 to 10 cell layers. A substantial decrease in expression of the cell–cell adhesion molecule, E-cadherin, was observed in all layers of the Le-AP-2α mutant corneal epithelium. The basement membrane, or Bowman's layer, was thinner in the mutant cornea and in many regions was discontinuous. These defects corresponded with altered distribution of laminin and entactin, and to a lesser degree, type IV collagen. The Le-AP-2α mutant cornea also exhibited stromal defects, including disrupted organization of the collagen lamellae and accumulation of fibroblasts beneath the epithelium that showed increased immunoreactivity for proliferating cell nuclear antigen (PCNA), α-smooth muscle actin (α-SMA), p-Smad2, and TGF-β2.
In the absence of AP-2α, the corneal epithelium exhibits altered cell adhesion and integrity and defects in its underlying basement membrane. These defects likely caused the alterations in the corneal stroma.
PMCID: PMC2517422  PMID: 16186342
21.  A Reconstituted Telomerase-Immortalized Human Corneal Epithelium In Vivo: A Pilot Study 
Current eye research  2011;36(8):706-712.
Telomerase-immortalized human corneal epithelial cells have been reported to stratify and differentiate in vitro similar to native tissue. The purpose of this study was to assess the ability of a telomerase-immortalized human corneal epithelial cell line to generate a full thickness epithelium in vivo in athymic mice.
Telomerized corneal epithelial cells were transduced with a retroviral vector encoding the herpes simplex thymidine kinase gene. Efficacy of the thymidine kinase suicide gene was confirmed using a live/dead assay. The epithelium was mechanically removed from athymic nude mice and remaining cells were treated with mitomycin C to prevent re-epithelialization. Telomerized corneal epithelial cells were seeded onto the denuded cornea and allowed to adhere for 4 and 24 hours. Cellular attachment was assessed using a fluorescent cell tracker. Stratification and differentiation were assessed after 7 days using phalloidin and a mouse monoclonal antibody to K3
Telomerized corneal epithelial cells were visualized across the denuded stromal surface at 4 and 24 hours, with multi-layering evident at the latter time point. No epithelium was present in the non-treated eye. 7 days post-transplantation cells stratified into a multilayered epithelium, with positive K3 expression in basal and suprabasal cells. Treatment with ganciclovir induced significant loss of viability in vitro.
The findings in this pilot study demonstrate that telomerized corneal epithelial cells possess the capacity to reconstitute a stratified corneal epithelium in vivo. The introduction of thymidine kinase allowed for the successful induction of cell death in proliferating cells in vitro. Collectively, these data suggest that a telomerase-immortalized corneal epithelial cell line transduced with thymidine kinase represents a potential model for studying differentiation and epithelial-niche interactions in vivo with potential applications in tissue engineering.
PMCID: PMC3149847  PMID: 21780919
cornea; epithelium; telomerase; differentiation; limbal niche
22.  Regeneration of the corneal epithelium with conjunctival epithelial equivalents generated in serum- and feeder-cell–free media 
Molecular Vision  2013;19:2542-2550.
Purpose: An alternative autologous tissue for ocular surface reconstruction is a potential treatment for the patients with bilateral limbal stem cell deficiency. For the purpose of regenerative procedures in patients, it is desirable to eliminate the involvement of xenogeneic components, such as nonhuman sera and feeder cells. In the present study, we examined the behavior and phenotypic features of cultured conjunctival epithelial sheets generated in serum- and 3T3-free culture conditions when transplanted into the de-epithelialized limbal corneal surface.
Methods: Epithelial cells from normal conjunctiva obtained by neutral protease digestion were expanded by culture in a serum-free low-calcium medium and set in an air-liquid interface culture for 14 days. The resulting multilayered epithelial sheets were grafted onto rabbit ocular surfaces made epithelial-free by alkali treatment. Pre-grafted and post-grafted epithelia were analyzed by electron microscopy and immunohistochemistry.
Results: At graft time the cultured epithelial sheet consisted of 6–8 layers of properly stratified epithelium that displayed a CK19+/MUC5AC+/ CK3 -/CK12- phenotype, consistent with the conjunctival epithelial lineage. Two weeks after xeno-grafting the in vivo epithelium consisted of 5-6 well compacted layers expressing the precursor cell-related protein p63, the proliferation marker Ki67, desmosomes, hemidesmosomes and its integrin (β4), and the corneal specific cytokeratins CK3, and CK12. Conjunctival goblet cell mucin (MUC5AC) was not visible. The engrafted epithelium stained positively for the anti-human nuclei antibody, confirming that the epithelial cells on the rabbit corneas were of human origin.
Conclusions: Our results suggest that conjunctival epithelial sheets generated in serum- and 3T3-free culture conditions can acquire the corneal epithelial phenotype when transferred to the in vivo corneal stromal environment.
PMCID: PMC3867160  PMID: 24357922
23.  Expression and Regulation of Cornified Envelope Proteins in Human Corneal Epithelium 
Stratified squamous epithelial cells assemble a specialized protective barrier structure on their periphery, termed the cornified envelope. The purpose of this study was to evaluate the presence and distribution of cornified envelope precursors in human corneal epithelium, their expression in human corneal epithelial cell cultures, and the effect of ultraviolet radiation (UVB) and transglutaminase (TG) inhibition on their expression.
Tissue distribution of small proline-rich proteins (SPRRs) and filaggrin and involucrin was studied in human cornea sections by immunofluorescence staining. Primary human corneal epithelial cells (HCECs) from limbal explants were used in cell culture experiments. A single dose of UVB at 20 mJ/cm2 was used to stimulate these cells, in the presence or absence of mono-dansyl cadaverine (MDC), a TG inhibitor. SPRR2 and involucrin protein levels were studied by immunofluorescence staining and Western blot analysis. Gene expression of 12 proteins was investigated by semiquantitative reverse transcription–polymerase chain reaction.
In human cornea tissue, SPRR1, SPRR2, filaggrin, and involucrin protein expression were detected in the central and peripheral corneal and limbal epithelium. In HCECs, SPRR2 and involucrin proteins were detected in the cytosolic fraction, and involucrin levels increased after UVB. Both SPRR2 and involucrin levels accumulated in the presence of MDC. Nine genes including involucrin, SPRR (types 1A, 1B, 2A, 2B, and 3), late envelope protein (LEP) 1 and 16, and filaggrin were expressed by HCECs. SPRR 4, loricrin, and LEP 6 transcripts were not detected. UVB downregulated SPRR (2A, 2B) and LEP 1 transcripts.
Various envelope precursors are expressed in human corneal epithelium and in HCECs, acute UVB stress differentially alters their expression in HCECs. The expression of envelope precursors and their rapid modulation by UVB supports the role of these proteins in the regulation of ocular surface stress. TG function may be relevant in the regulation of soluble precursors in UVB-stimulated corneal epithelium.
PMCID: PMC2906387  PMID: 16639001
24.  A role for chromosomal protein HMGN1 in corneal maturation 
Corneal differentiation and maturation are associated with major changes in the expression levels of numerous genes, including those coding for the chromatin-binding high-mobility group (HMG) proteins. Here we report that HMGN1, a nucleosome-binding protein that alters the structure and activity of chromatin, affects the development of the corneal epithelium in mice. The corneal epithelium of Hmgn1−/− mice is thin, has a reduced number of cells, is poorly stratified, is depleted of supra-basal wing cells, and its most superficial cell layer blisters. In mature Hmgn1−/− mice, the basal cells retain the ovoid shape of immature cells, and rest directly on the basal membrane which is disorganized. Gene expression was modified in Hmgn1−/− corneas: glutathione-S-transferase (GST)α 4and GST ω 1, epithelial layer-specific markers, were selectively reduced while E-cadherin and α-, β-, and γ-catenin, components of adherens junctions, were increased. Immunofluorescence analysis reveals a complete co-localization of HMGN1 and p63 in small clusters of basal corneal epithelial cells of wild-type mice, and an absence of p63 expressing cells in the central region of the Hmgn1−/− cornea. We suggest that interaction of HMGN1 with chromatin modulates the fidelity of gene expression and affects corneal development and maturation.
PMCID: PMC3730489  PMID: 16466397
HMG proteins; eye differentiation; corneal epithelium; chromatin
25.  Identification of a basic protein of Mr 75,000 as an accessory desmosomal plaque protein in stratified and complex epithelia 
The Journal of Cell Biology  1988;106(5):1679-1691.
Desmosomes are intercellular adhering junctions characterized by a special structure and certain obligatory constituent proteins such as the cytoplasmic protein, desmoglein. Desmosomal fractions from bovine muzzle epidermis contain, in addition, a major polypeptide of Mr approximately 75,000 ("band 6 protein") which differs from all other desmosomal proteins so far identified by its positive charge (isoelectric at pH approximately 8.5 in the denatured state) and its avidity to bind certain type I cytokeratins under stringent conditions. We purified this protein from bovine muzzle epidermis and raised antibodies to it. Using affinity-purified antibodies, we identified a protein of identical SDS-PAGE mobility and isoelectric pH in all epithelia of higher complexity, including representatives of stratified, complex (pseudostratified) and transitional epithelia as well as benign and malignant human tumors derived from such epithelia. Immunolocalization studies revealed the location of this protein along cell boundaries in stratified and complex epithelia, often resolved into punctate arrays. In some epithelia it seemed to be restricted to certain cell types and layers; in rat cornea, for example, it was only detected in upper strata. Electron microscopic immunolocalization showed that this protein is a component of the desmosomal plaque. However, it was not found in the desmosomes of all simple epithelia examined, in the tumors and cultured cells derived thereof, in myocardiac and Purkinje fiber cells, in arachnoideal cells and meningiomas, and in dendritic reticulum cells of lymphoid tissue, i.e., all cells containing typical desmosomes. The protein was also absent in all nondesmosomal adhering junctions. From these results we conclude that this basic protein is not an obligatory desmosomal plaque constituent but an accessory component specific to the desmosomes of certain kinds of epithelial cells with stratified tissue architecture. This suggests that the Mr 75,000 basic protein does not serve general desmosomal functions but rather cell type-specific ones and that the composition of the desmosomal plaque can be different in different cell types. The possible diagnostic value of this protein as a marker in cell typing is discussed.
PMCID: PMC2115063  PMID: 3131348

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