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To investigate if human oral mucosal fibroblasts (HOMF) from patients with limbal stem cell deficiency (LSCD) can be used as an autologous feeder layer to support the culture of epithelial cells for potential clinical use.HOMF were isolated from oral mucosal biopsies obtained from the following groups of patients with LSCD: aniridia, mucous membrane pemphigoid (MMP), Stevens-Johnson syndrome (SJS), and ectodermal dysplasia (ED). The ability of these cells to support the culture of human limbal epithelial cells (HLE) was compared to that of HOMF from non-LSCD donors and 3T3s commonly used to culture epithelial cells for use in the clinic to treat LSCD.HOMF were successfully obtained by explant culture for 3/3 aniridia patients, 3/3 MMP patients, 1/3 SJS patients, and 1/1 ED patients. All HOMF cultured from these LSCD groups supported the expansion of HLE with epithelial culture times and total colony forming efficiency (CFE) comparable to those achieved on HOMF isolated from donors without LSCD. PCR showed that all HLE cultured on LSCD donor HOMF expressed p63α, CK15, PAX6, CK12, and MUC16 as did HLE cultured on the control non-LSCD donor HOMF and 3T3s. Western blotting detected CK15 and MUC16 protein expression in all groups.HOMF from patients with LSCD can be successfully used to support the expansion of epithelial cells. These cells may therefore be useful as autologous feeder fibroblasts for the expansion of epithelial cells for use in the clinic to treat LSCD.

To elucidate the microarchitecture of limbus and cornea in subjects with Vogt Koyanagi Haradas syndrome (VKH).From January 2019 to December 2019 a total of 17 VKH subjects, 5 acute and 12 chronic, 18 non VKH uveitis controls, and 6 healthy controls were recruited for this study . In vivo confocal microscopic (IVCM) analysis of the limbal basal epithelium, scleral side of the limbus, limbal niche, corneal keratocytes, and corneal nerves was carried out .Absence of pigmented cells in the limbal basal epithelium, presence of inflammatory cells on the scleral side of the limbus, fibrotic niche, degenerated keratocytes, thinning, and beading of corneal nerves were noted in VKH eyes as compared to controls.Presence of inflammatory cells and depigmentation of limbus in chronic VHK points to disease progression.Keratocyte and corneal nerve changes in Vogt Koyanagi Haradas syndrom are novel findings.

To investigate the corneal epithelial and limbal epithelial alterations in patients under topical glaucoma treatment using anterior segment-OCT (AS-OCT) and to determine the changes of the limbal region due to the preservatives and glaucoma drugs, that can progress to limbal stem cell deficiency (LSCD). Limbal thickness was measured by AS-OCT to evaluate limbal cell deficiency.Forty-seven patients using topical medication for glaucoma, and 48 control subjects were enrolled in this matched case-control study. The patients were divided into four groups according to the treatment regimens. Group 1: One-drug regimen, Group 2: Two-drug regimen, Group 3: Three-drug regimen, Group 4: Four-drug regimen For the ocular surface evaluation; tear break-up time with standard fluorescein sodium sterile strip application, Schirmer test-I, Ocular Surface Disease Index Questionnaire, and AS-OCT were performed.A total of 95 subjects were included: 47 eyes of 47 patients with glaucoma medication and 48 eyes of 48 healthy subjects. There was a statistically significant difference between patients and controls according to BUT, SCH, and OSDI (Using at least one glaucoma drug caused limbal area injury, changed ocular surface measurements, and significantly reduced the limbal epithelial thickness where the stem cells reside. The limbal epithelial thickness measurement by AS-OCT seems to be an innovative, non-invasive, and promising technique for detecting and staging corneal damage in topical glaucoma therapy.

Aim: Chemical injuries can potentially lead to the necrosis anterior segment of the eye, and cornea in particular. Inflammatory cytokines are the first factors produced after chemical ocular injuries. Inflammation via promoting the angiogenesis factor tries to implement the wound healing mechanism in the epithelial and stromal layer of the cornea. Methods: Narrative review.Results: In our review, we described the patterns of chemical injuries in the cornea and their molecular mechanisms associated with the expression of inflammatory cytokines. Moreover, the effects of inflammation signals on angiogenesis factors and CNV were explained. Conclusion: The contribution of inflammation and angiogenesis causes de novo formation of blood vessels that is known as the corneal neovascularization (CNV). The new vascularity interrupts cornea clarity and visual acuity. Inflammation also depleted the Limbal stem cells (LSCs) in the limbus causing the failure of normal corneal epithelial healing and conjunctivalization of the cornea.

1) To elucidate the role of collector channels in the aqueous humor outflow pathway 2) To suggest anatomic and functional methods of imaging collector channels in-vitro and in-vivo and 3) To discuss the role of such imaging modalities in the surgical management of glaucoma.A thorough literature search was conducted on databases for studies published in English regarding the available methods to determine the role of collecting channels in normal and glaucomatous patients and to assess their patency.Intraocular pressure (IOP) exists as a balance between aqueous humor production and aqueous humor outflow. Collector channels are an essential anatomical constituent of the distal portion of the conventional aqueous humor outflow pathway. There are different surgical options for glaucoma management and with the recent advances in Schlemm's canal-based surgeries, collector channel's patency became a key factor in determining the optimum management for the glaucomatous eye. The advent of anatomic imaging methods has improved the ability to visualize collector channel morphology in-vitro, including swept-source optical coherence tomography (SS-OCT), spectral domain optical coherence tomography (SD-OCT), micro-computed tomography (micro CT), new immunohistochemistry techniques and scanning electron microscopy. The recent advent of real-time assessment of collector channel patency (including evaluation of episcleral venous outflow, observation of episcleral venous fluid wave, and tracer studies utilizing fluorescein, indocyanine green, and trypan blue) has been validated by the aforementioned anatomic imaging modalities.New modalities of in-vitro and in-vivo studies of collector channels provide promise to aid in the assessment of collector channel patency and individualization of surgical management for glaucoma patients.

OBJECTIVES: The conditioned-medium derived from corneal mesenchymal stromal cells (cMSCs) has been shown to have wound healing and immunomodulatory effects in corneal injury models. Here, the therapeutic effects of lyophilized cMSC conditioned-medium were compared with fresh conditioned-medium. METHODS: The epithelial wound healing effects of fresh and lyophilized cMSC conditioned-medium were compared with conditioned-medium from non-MSC cells (corneal epithelial cells) using scratch assay. To evaluate the anti-inflammatory effects of fresh and lyophilized cMSC conditioned-media, macrophages were stimulated by a Toll-Like Receptor (TLR) ligand followed by treatment with the conditioned-media and measuring the expression of inflammatory genes. In vivo wound healing effects of fresh and lyophilized cMSC conditioned-media were assessed in a murine model of cornea epithelial injury. RESULTS: Both fresh and lyophilized cMSCs-derived conditioned-medium induced significantly faster closure of in vitro epithelial wounds compared to conditioned-medium from non-MSC cells (P

Purpose The effects of lithium (Li) and Valproic Acid (VA) drugs have been recently revealed to improve the Mesenchymal stem cells')MSCs (migration and proliferation processes. The aim of this study is to determine the expression of the genes involved in the proliferation and migration of limbal epithelial stem/progenitor cells (LESPCs) after treatment with Li and VA. Methods After extraction of LSCs from human Corneoscleral tissue, cells were subcultured three times. The cell culture media were divided into four separate groups including groups treated with VA, Li, combination, and control groups after determining the non-toxic concentration of drugs (64mml) Li and (28mml) VA based on MTT assay, and then cells cultures were treated for 3 hours. A real-time polymerase chain reaction was performed to detect the expression levels of CD44, Ki67, CXCR4, CXCR7, MMP-2, MMP-9, and SDF-1 genes. Changes in the expression of each gene in different treatments were calculated. Finally, the graphs were analyzed by SPSS (Version 18) software. Results The highest expression of CXCR4 and CXCR7 was in the Li-treated group. Additionally, the highest expression levels of MMP-9 and CD44 genes were observed in the VA-treated group. In contrast, the expression level of SDF-1a, MMP2, and Ki67 genes in all three treatment groups reduced compared to the control group. Conclusion Increasing the LSCs migration genes (CXCR4 and MMP9) was more evident than cell proliferation genes (Ki67). In sum, Li and VA can affect the process of proliferation and migration of LSCs in vitro.

We present a new case of Pai syndrome and review the current literature on this disease. Our PubMed search was conducted using the term “Pai syndrome.” We included original case reports and case se...

To evaluate the gene transfer of the interleukin (IL)-10 cytokine as a treatment modality for prolonging limbal allograft survival in a rat model.Adenoviral (AV) and lentiviral (LV) vectors were produced for ex vivo gene transfer into limbal graft tissue prior to orthotopic transplantation. Experimental groups comprised unmodified isografts, unmodified allografts, allografts transfected with a reporter gene, and allografts transfected with IL-10. The functional effects of the transgenes were determined by clinical assessment and by following donor cell survival in the recipient animal. Group comparisons were made using survival analysis and tested with the log-rank test. Differences in mean rejection times between groups were tested using the Wilcoxon rank-sum test.Isografts survived during the entire observation period of 56 days. Allografts underwent clinical rejection at a mean of 6.7 days (standard deviation 2.0) postoperatively, irrespective of the presence of transgenes (p 0.001 for difference in rejection times). For both the AV and LV vector systems, Kaplan-Meier analysis showed a statistically significant difference with respect to time-to-graft failure when comparing allografts transfected with IL-10 with allografts transfected with reporter gene alone (p = 0.011 and p 0.001, respectively). In the isografts, donor cells could be detected during the complete observation period. In all the allograft groups, however, donor cell detection declined after 1 week and was lost after 4 weeks.Under the conditions tested in the present model, both the AV and the LV vector systems were able to transfect limbal graft tissue ex vivo with biologically active IL-10, leading to delayed rejection compared to the controls.

This review surveys available literature for corneal and limbal dimensions. With modern scleral lenses, these measures have become central to determining the overall size of a lens for satisfactory fitting. In general, published values are not based on a definition of what is being measured. In addition, the most widely accepted average corneal diameter measurement, 11.7 × 10.6 mm, emanates from one source published more than 50 years ago. This value was not based on a measurement but appears to be the cumulative impression of measurements from seven studies conducted between 67 and 127 years ago. Furthermore, in most instances, if at all stated, quoted measures are based on horizontal visible iris diameter, providing limited acknowledgement of limbal width and its inclusion as part of the corneal diameter. The corneo-scleral sulcus from one side to the other has been measured, giving a larger diameter, but may include at least part of the limbus. More objective measurements are possible with modern ophthalmic instrumentation but the lack of structural definition and low magnification resolution with these techniques raises concerns with the accuracy of the results. Measurement of the horizontal visible iris diameter does not include the limbal width, which means that the horizontal visible iris diameter is an underestimate of the true corneo-limbal diameter. This review concludes that the width of the limbus has been neither structurally defined nor accurately measured and that there is a need for the development of new protocols for determining the dimensions of the average cornea and limbus. It is predicted that more accurate measures will indicate that to vault across cornea with limbus and provide excellent comfort, the average cornea will need a lens to have a diameter of 16.0 mm or larger.

Salzmann's nodular degeneration (SND) typically occurs in patients who are female, 50-60 years old, and have a history of corneal inflammation and irritation. Multiple case reports have documented associations between SND and trachoma, viral infections, trauma, contact lens wear, corneal surgeries and corneal exposure. The authors describe a patient with bilateral SND confirmed by anterior segment optical coherence tomography (OCT) imaging in the context of thyroid eye disease (TED) and history of LASIK. Treatment involved propylthiouracil (PTU), artificial tear use, loteprednol etabonate ophthalmic gel, eyelid taping and selenium supplementation and prospective superficial keratectomy with diamond burr polish.

Epidermolysis bullosa (EB) is an inherited connective tissue disease that causes skin blisters due to a lack of anchoring between epidermis and dermis, resulting in friction and skin fragility. Usu...

Experimental animals have been used extensively in the goal of developing sight-saving therapies for humans. One example is the development of transplantation of cultured limbal epithelial stem cells (LESC) to restore vision following ocular surface injury or disease. With clinical trials of cultured LESC therapy underway in humans and a potential companion animal population suffering from similar diseases, it is perhaps time to give something back. Comparatively to humans, what is known about the healthy limbus and corneal surface physiology of companion animals is still very little. Blinding corneal diseases in animals such as symblepharon in cats with Feline Herpes Virus-1 infections require a basic understanding of the functional companion animal limbus and corneal stem cells. Our understanding of many other vision threatening conditions such as scarring of the cornea post-inflammation with lymphocytic-plasmacytic infiltrate in dogs (aka chronic superficial keratitis) or pigment proliferation with Pigmentary Keratitis of Pugs would benefit from a better understanding of the animal cornea in health and disease. This is also vital when new therapeutic approaches are considered. This review will explore the current challenges and future research directions that will be required to increase our understanding of corneal diseases in animals and consider the potential development and delivery of cultured stem cell therapy to veterinary ocular surface patients.

The purpose of this study is to assess the stability of the growth factors (GF) in autologous serum eyedrops under different storage conditions.The concentration of epidermal growth factor (EGF), transforming growth factor-β (TGF-β1), platelet-derived growth factor AB (PDGF-AB), and albumin was measured in fresh and defrosted samples of autologous serum under different storage conditions. The fresh and defrosted samples were cooled at 4 °C, and they were studied immediately after preparation, or after defrosting, and after 1, 2, 3, and 4 weeks. The concentration of GF was also assessed after 1, 3, 6, and 9 months at -20 °C. We also investigated how the different storage conditions influence the biological effects of autologous serum on conjunctival and corneal cell cultures.The concentration of EGF, TGF-β1, PDGF-AB, and albumin remained stable over the 4 weeks at 4 °C, both in fresh and in defrosted samples. Likewise, no statistically significant differences were found between the GF concentration in fresh samples and after 1, 3, 6, and 9 months of freezing at -20 °C. Moreover, no differences were found on the cell proliferation and differentiation between cultured cells with fresh or defrosted samples after 4 weeks at 4 °C or after 1, 3, 6, or 9 months at -20 °C.Long-term storage of autologous serum eyedrops at -20 °C does not affect the concentration of GF, simplifies clinical logistics, and reduces the frequency of blood extractions from the patients.

To compare outcomes of live related limbal allograft (Lr-CLAL) versus cadaveric keratolimbal allograft (KLAL) in limbal stem cell deficiency (LSCD) secondary to ocular burns.Twenty patients with stage IIb LSCD were randomized so that cases underwent either Lr-CLAL or KLAL. Fibrovascular pannus was removed and superficial keratectomy done on the recipient bed. Limbal lenticule of 2-3 clock hours' length was harvested from the donor, which was placed over the host bed and sutured followed by bandage contact lens application. Parameters assessed were uncorrected visual acuity (UCVA), best corrected visual acuity (BCVA), conjunctivalization, corneal neovascularization, epithelial defects, corneal clarity, Schirmer's test, tear film breakup time (tBUT), and ultrasonic pachymetry.At 6 months follow-up, the Lr-CLAL group had a higher gain in vision (p = 0.029), decrease in conjunctivalization (p = 0.009), and increase in Schirmer's values (p = 0.009).Lr-CLAL seems to have better result in terms of vision gain and ocular surface restoration.

H syndrome is an autosomal recessive histiocytosis with multisystemic involvement caused by mutations in the SLC29A3 gene. The term H syndrome was coined to denote the major clinical findings which include hyperpigmentation, hypertrichosis, hearing loss, hepatosplenomegaly, hypogonadism, hyperglycemia/diabetes mellitus and hallux valgus/flexion contractures. Almost 100 individuals affected with this disorder have been reported, however, a thorough evaluation of the ophthalmologic features of H syndrome has not yet been performed.Ophthalmic examination of a 50-year-old male with H syndrome. Mutation analysis of SLC29A3 was also performed in this patient.Ophthalmic findings included; shallow orbits with exorbitism, bilateral pterygium, limbal thickening, corneal arcus and cortical cataract. We also review ophthalmologic findings in previously reported H syndrome patients.The presence of dilated lateral scleral vessels, corneal arcus and shallow orbits should raise the suspicion of H syndrome, especially when seen in young age.

Glaucoma may develop or worsen after ocular surface transplantation and often requires surgical management for adequate intraocular pressure control. Traditional glaucoma filtering procedures in patients with prior ocular surface transplant may be problematic for several reasons, which include mechanical disruption of the pre-existing graft, epithelial and stem cell toxicity induced by antifibrotic agents, and increased risk of future corneal transplantation failure. We describe the implantation of a glaucoma drainage implant via a limbal-based conjunctival incision with tube placement in the ciliary sulcus in three eyes of two patients with prior ocular surface transplantation. At a follow-up interval of 3-7 months, all three eyes have excellent postoperative control of intraocular pressure, stable vision, and healthy ocular surface grafts.

Corneal epithelium is maintained by limbal epithelial stem cells (LESCs), the loss of which can be catastrophic for corneal transparency. Effective therapies include the transplantation of cultivated LESCs, requiring optimization of in vitro cultivation protocols. Unfortunately, optimization studies are hampered by the limited number of ocular tissue donors. We investigated the feasibility of obtaining more than one limbal primary culture (LPC) from the same 1-2 mm(2) limbal explant (LE).LEs were plated and maintained until outgrowth surrounded each, being removed at this point. LPCs were allowed to reach confluence (LPC0). The same removed LE was plated again, following the same procedure, obtaining LPC1. This procedure was repeated as often as possible up to six times. LPCs from each passage were analysed by real time reverse transcription-polymerase chain reaction and immunofluorescence-microscopy.LPCs from LPC0 to LPC2 presented a heterogeneous cell population, with cells positive for LESC markers K14, K15, ABCG2 and p63, differentiated corneal epithelial cell-specific markers K3 and K12, and for the fibroblast marker S100A4. These cells had an epithelial-like morphology. In LPC3-LPC4, elongated cell morphology appeared, and the presence of LESC markers decreased, while the presence of differentiated corneal epithelial-cell and fibroblast markers increased.One LE can be successfully cultivated up to three consecutive times while maintaining the LESC phenotype in the LPC cells. This protocol provides several homologous LPCs for basic research. Additionally, by using a cell-carrier, the resulting LPCs could serve reservoirs for potential autologous expanded LESC transplantations and/or for making correlations between laboratory and clinical outcomes.

To evaluate how corneal deturgescence after Descemet membrane endothelial keratoplasty (DMEK) influences the posterior corneal surface area and the endothelial cell density (ECD) measurements.A mathematical model was formulated to estimate the increase in posterior corneal surface area associated with postoperative corneal deturgescence and its effect on ECD measurements. Important input parameter for the model was the change in pachymetry from 1 h to 6 months after surgery. To this end, the clinical records of 25 patients (25 eyes) who underwent DMEK were reviewed retrospectively and the central corneal thickness (CCT) measurements taken after 1 h, 1 month, 3 months and 6 months were noted. ECD measurements before surgery and at 1, 3 and 6 months after surgery were also recorded and decrease in pachymetry and ECD loss were calculated.The average decrease in CCT due to corneal deturgescence was 267 µm (±39 µm), which corresponds to an 8.6% increase in total posterior corneal surface area, as calculated using our mathematical model. The stretching of the endothelial cell layer associated with this increase of posterior corneal surface area may result in an apparent endothelial cell "loss". This might account for approximately 25% of the observed average ECD decrease of 34% (±17%).The observed decrease in ECD within the first 6 months after DMEK may overestimate the actual loss of endothelial cells by about 8% due to increased posterior corneal surface area associated with postoperative corneal deturgescence.

This study was designed to measure vitamin D metabolites in the aqueous and vitreous humor and in tear fluid, and to determine if dietary vitamin D3 supplementation affects these levels. We also determined if the corneal epithelium can synthesize vitamin D following UV-B exposure.Rabbits were fed a control or vitamin D3 supplemented diet. Pilocarpine-stimulated tear fluid was collected and aqueous and vitreous humor were drawn from enucleated eyes. Plasma vitamin D was also measured. To test for epithelial vitamin D synthesis, a human corneal limbal epithelial cell line was irradiated with two doses of UV-B (10 and 20 mJ/cm(2)/day for 3 days) and vitamin D was measured in control or 7-dehydrocholesterol treated culture medium. Measurements were made using mass spectroscopy.25(OH)-vitamin D3 and 24,25(OH)(2)-vitamin D3 increased significantly following D3 supplementation in all samples except vitreous humor. Tear fluid and aqueous humor had small but detectable 1,25(OH)(2)-vitamin D3 levels. Vitamin D2 metabolites were observed in all samples. Vitamin D3 levels were below the detection limit for all samples. Minimal vitamin D3 metabolites were observed in control and UV-B-irradiated epithelial culture medium except following 7-dehydrocholesterol treatment, which resulted in a UV-B-dose dependent increase in vitamin D3, 25(OH)-vitamin D3 and 24,25(OH)(2)-vitamin D3.There are measurable concentrations of vitamin D metabolites in tear fluid and aqueous and vitreous humor, and oral vitamin D supplementation affects vitamin D metabolite concentrations in the anterior segment of the eye. In addition, the UV exposure results lead us to conclude that corneal epithelial cells are likely capable of synthesizing vitamin D3 metabolites in the presence of 7-dehydrocholesterol following UV-B exposure.

Cholera toxin and isoproterenol (β-adrenergic receptor agonist) are largely used to enhance cell proliferation. The aim of the study was to assess the effects of cholera toxin and isoproterenol on growth and differentiation of cells cultured from human superficial limbal explants.Limbal epithelial cells were cultured from superficial limbal explantsin basal medium either supplemented with cholera toxin or isoproterenol for 3 weeks. Growth kinetics and morphometry were studied by light and confocal microscopy. Progenitor and differentiated epithelial cell markers were studied by immunocytochemistry, flow cytometry, Colony Formation Assay, and reverse transcription and polymerase chain reaction.Cell proliferation was significantly higher with 0.5 µg/ml (p = 0.049), 1 µg/ml (p = 0.005), and 2 µg/ml (p = 0.008) isoproterenol whereas, cholera toxin and 4 µg/ml isoproterenol did not significantly increase cell proliferation. Multilayered epithelial cell sheets were obtained in all culture conditions. Addition of isoproterenol resulted in smaller cell size (p0.05) 14 days after cells were cultured, whereas cholera toxin had no effects. Strong expression of cytokeratins 3 and 4/5/6/8/10/13/18 and lower expression of cytokeratin 19, vimentin, and Delta N p63α were observed after 3 weeks of culture with no significant differences in the percentage of positive cells according to culture medium. Colony-forming efficiencies were observed after 2 weeks in all culture condition but not after 3 weeks.Isoproterenol was more efficient than cholera toxin for enhancing cell proliferation and resulted in smaller cell size. It appears to be useful and safe for growing human limbal epithelial progenitors from limbal explants with no feeders before transplantation to patients with limbal deficiency.

Ocular injuries following exposure to the chemical agent sulfur mustard (SM) are characterized by acute corneal erosions and inflammation of the anterior segment that may be followed by delayed Partial Limbal Stem Cell Deficiency (LSCD), expressed clinically by corneal neovascularization and epithelial defects. LSCD may derive from direct destruction of limbal stem cells or indirectly from altered limbal stromal niche. The aim of this study was to investigate the mechanism underlying LSCD in SM injuries, focusing on the effects of the chemical on limbal epithelium.Rabbit eyes were exposed to SM vapor and were observed by slit lamp examinations and pachymetry. Eyes were taken for histological and molecular biology evaluations at different time points (4 h-4 weeks), to include acute and delayed injuries. Epithelial stem cells were identified by ABCG2, p63 and by in vivo BrdU labeling for slow cycling cells.Limbal stem cells were not damaged during the acute phase following SM exposure, in contrast to the severe injury of the central corneal epithelium. On the contrary, limbal epithelium became activated, responding to corneal insult with a wound healing process, as shown by histology and by transient elevation of the stem cells markers. Simultaneously, inflammation was taking place in the limbal stroma lasting for weeks. A gradual loss of stem cells was observed later-on (2-4 weeks), associated with typical symptoms of LSCD.LSCD associated with SM ocular toxicity was not derived from a direct cytotoxic effect on the epithelial stem cells, but apparently from pathological events at the limbal stroma, that produced an abnormal microenvironment for the stem cells, triggering their gradual death. The results, and in particular the absence of a primary damage to the epithelial stem cells, indicate the presence of a therapeutic window for intervention to avoid the development of the delayed LSCD.

Reconstruction of the conjunctiva is an essential part of ocular surface regeneration, especially if an extensive area or the whole ocular surface is affected, such as in patients with ocular cicatricial pemphigoid, Stevens-Johnson syndrome, toxic epidermal necrolysis, or chemical/thermal burns. In these situations, corneal reconstruction almost inevitably fails unless the conjunctival surface is first repaired and a deep fornix is restored. The growing field of tissue engineering and advances in stem cell research offer promising new alternatives for these challenges. This article reviews the present approaches for reconstruction of the conjunctival surface, considering the established strategies and new potential methodologies.

To investigate adhesion complex formation in cultivated human limbal epithelium after transplantation into the limbal deficient model.Cultivated epithelium on amniotic membrane was transplanted into limbal deficient rabbits. The transplanted rabbits and the controls were sacrificed at 1, 2, 3, and 4 weeks. The adhesion complex was examined by electron microscopy and immunohistochemistry.Morphologically identifiable hemidesmosomes appeared at 1 week, and matured adhesion complex was found at 3 weeks. Collagen VII was partly stained after transplantation. The mean numbers of hemidesmosomes/2.25 microm were 2.3 +/- 0.9, 2.5 +/- 0.5, 5.2 +/- 1.0, and 4.0 +/- 0.9 at 1, 2, 3, and 4 weeks, and all they were smaller than those in the control, respectively (p0.05). It reached 137.4% of the density of hemidesmosomes in human cornea at 3 weeks. The average depths of anchoring fibril were 0.10 +/- 0.03, 0.27 +/- 0.06, 0.45 +/- 0.06, and 0.46 +/- 0.12 microm at 1, 2, 3, and 4 weeks, reaching 75.0% of that in the human cornea after 3 weeks, although they were shallower than that of the control, respectively (p0.05).Assembly of adhesion complex in cultivated epithelium transplanted in limbal deficient rabbit might recover to the level of that in the human after 3 weeks, although it was delayed compared with that in normal wound healing of the rabbit.

Diseases that affect the limbal stem cells are multifactorial and present with different stages of severity. The most important features to be considered in evaluating these patients include the degree of limbal stem cell loss, the extent of conjunctival disease, and the presence and etiology of ocular surface inflammation. Other important factors are tear film and eyelid abnormalities, keratinization of the ocular surface, laterality of the disease process, health and age of the patient. Careful consideration of all of these factors help tremendously in tailoring the most suitable method of treatment for each patient. The management of severe ocular surface disease has benefited from numerous advances in recent years. At one time, available techniques for visual rehabilitation consisted of superficial keratectomy, use of artificial tears, tarsorraphy as well as lamellar and penetrating keratoplasty. A lamellar or penetrating keratoplasty procedure resulted in a stable surface only for as long as the donor epithelium was present and once the epithelium sloughed off, the ocular surface failed due to conjunctivalization. The last few decades enjoyed the development and, especially, progress of new ocular surface reconstruction techniques such as amniotic membrane transplantation, limbal stem cell transplant procedures, transplantation of cultivated oral mucosal or limbal stem cell sheets. This review will briefly focus on the indications and methodology of each procedure and the currently available clinical data on the results of these procedures.

To investigate the capability of cultivated allogeneic epithelial stem cells to restore a functional ocular surface in a limbal deficient cornea; to verify the long term survival of epithelial allograft; and to examine the host immune response to heterologous cell transplant in a rabbit model.Limbal deficiency was established by performing limbectomy on rabbits (n = 100). Corneal epithelial stem cells were obtained from the limbus and replicated in vitro without a supporting layer. The cell (3 x 10(5)) suspension was then transplanted via topical application as eye drops. Animals were divided into allograft, autograft, and control groups. Females were used as recipients and males as donors for the allograft. Corneas were collected at 7, 14, 21, 40 days as well as 2, 3, 7 and 8 months after cell transplantation. Experimental corneas were evaluated by histology, immunofluorescence, immunohistochemistry and Y chromosome analysis.A well-differentiated corneal epithelium was recognized at 14 to 40 days after cell transfer overlying an infiltrated corneal stroma. Corneal re-epitheliazation was confirmed in 31 of 36 allograft corneas. No significant immune rejection was noted. Stromal abnormality caused by previous limbal deficiency was mostly resolved three months after the regeneration of corneal epithelium.Transplanted corneal epithelial stem cells were able to differentiate into normal corneal epithelium in vivo without the use of membrane scaffolding. This non-autologous donor cell-derived corneal epithelium survived up to 8 months without immunosuppression and was able to reverse the stromal scarring. Thus, cultivated epithelial stem cells have great potential as an alternative to multiple-surgical procedures in the treatment of limbal deficiency states.

Purpose: To report a case of a child with Pfeiffer syndrome, unique ocular anterior segment findings and a mutation in FGFR2 (Trp290Cys).Methods: Case Report.Results: We describe a patient with Pfeiffer syndrome with a unique constellation of ocular anterior segment anomalies including microcornea, limbal scleralization, corectopia and glaucoma. Genomic DNA extraction was heterozygous for a G to T mutation at nucleotide 870 of the fibroblast growth factor receptor 2 gene (FGFR2) which changes tryptophan (TGG) to cysteine (TGT) at amino acid position 290 (Trp290Cys).Conclusion: This case supports the association between Pfeiffer syndrome and severe ocular anterior segment anomalies, including glaucoma, and underscores the possible role that FGFR2 has in development of the anterior segment of the eye.

Purpose: To report a case of pyogenic granuloma associated with ocular acne rosacea.Design: Case report.Methods: Interventional case report.Results: The patient developed an unusual lesion of the conjunctiva and limbus, histologically confirmed to be pyogenic granuloma. Excision of the lesion resulted in symblepharon formation, adhesions between lid margins, tarsal conjunctiva, and cornea, and reductions in visual acuity. The patient underwent surgical separation of the corneal lid adhesions with a mucosal autograft for the tarsal conjunctiva and an amniotic membrane graft for the cornea with good results.Conclusions: Ocular rosacea is a common condition but such excessive and unusual fibrotic healing responses following excision of pyogenic granulomas have not been previously reported. This case demonstrated significant morbidity and decreased visual acuity. However, early recognition and effective surgical management can lead to good visual outcomes.

Superior limbic keratoconjunctivitis (SLK) is a disease of unknown etiology which showed various degrees of keratinization. The cytokeratins (CKs) are known to be the hallmark of epithelial differentiations and each CK has a characteristic distribution dependent on the type and the differentiation status of epithelium. The authors have studied the expression of cytokeratins (CKs) as well as proliferating cell nuclear antigen (PCNA) in the conjunctiva of SLK.Three superior limbic conjunctivae of patients with SLK and two normal conjunctivae were examined immunohistochemically using monoclonal antibodies against CKs and PCNA.In SLK conjunctivae, increased expression of a basal cell marker (CK14) was observed throughout the epithelium. Sporadic positive staining with CK10, which is a specific marker for keratinization, was correlated to the severity of the disease. The disappearance of CK13 staining, the marker for nonkeratinized stratified epithelia, at the basal cell layer of SLK was noted. Moreover, increased expression of PCNA in SLK was observed.The altered expression of CKs in SLK suggests an abnormality of differentiation in the conjunctival epithelium of the disease. Upregulated proliferation of conjunctival epithelial cells may be another feature of the disease.

To compare the supporting mechanism between the serum-free and the fibroblast-cocultured single-cell clonal culture systems.Clonal growth, measured by colony forming efficiency (CFE) and size, was compared between rabbit corneal and limbal epithelial cells in a previously-established serum-free MCDB medium supplemented with growth factors, and in a coculture system with a feeder layer of mitomycin C-treated mouse 3T3 fibroblasts grown in the MCDB or DMEM medium plus 20% fetal bovine serum (FBS).Limbal epithelial cells in the serum-free MCDB medium had a significantly lower CFE than corneal epithelial cells (p0.001), suggesting that this system promoted more clonal growth of corneal progenitor cells. In contrast, with cocultured 3T3 fibroblasts limbal CFE was significantly increased (p0.001), while corneal CFE was not changed, indicating that the 3T3 system promoted more clonal growth of limbal progenitor cells. Addition of 20% FBS in the MCDB medium cocultured with 3T3 fibroblasts significantly promoted both limbal and corneal CFEs (p0.001). For both cultures, switching the serum-containing MCDB medium to the serum-containing DMEM medium produced clonal growth only with cocultured fibroblasts. This epithelial growth-promoting activity was not present on the cell surface or in the extracellular matrix, but present in pre-centrifuged and prefiltered 3T3 fibroblast-conditioned media. Both growth-promoting and anti-apoptotic activities were present in fibroblast-derived serum-free conditioned media. In the presence of this anti-apoptotic activity, serum addition promoted clonal growth, and the expression of cornea-type K3 keratin in limbal colonies was negative using AE-5 monoclonal antibody.Further purification and characterization of this fibroblast-derived anti-apoptotic survival factor will facilitate understanding of the mechanism by which epithelial stem cells are regulated via epithelial-mesenchymal interactions.

In the present study we have analyzed the architecture of the episcleral microvasculature in the owl and cynomolgus monkey using scanning electron microscopy of resin casts. Due to the topical pretreatment with nitroprusside the complete vasculature including segments of the aqueous humor outflow channels could be visualized. We found that 1-3 mm posterior to the limbus corneae the episcleral vessels form numerous arteriovenous anastomoses (AVA) in the size of small arterioles and venules. These AVA are also located at the collector channels near Schlemm's canal and at the episcleral venous plexus which drains the collector channels. It is assumed that the AVA might influence not only the circulation in the episcleral venous plexus but also in the aqueous humor outflow routes.

The goal of this study was to determine the cell origin of human pterygia. In order to determine the origin of these cells, longitudinal cryostat sections through five primary and two recurrent pterygia were studied immunohistochemically by finding limbal basal stem cell staining patterns as defined by monoclonal antibodies AE1 (staining positive) and AE5 (staining negative). In addition, sections were stained with antivimentin antibody. Altered limbal basal cells invading normal cornea along the basement membrane were identified in seven human pterygia with these specific monoclonal antibodies. A group of limbal basal cells (vimentin and AE1 positive) was always present between the dissolved edge of Bowman's layer and vascularized conjunctiva which contained goblet cells. Scattered patches of cells staining positive with both vimentin and AE5 (in addition to their AE1 staining) were also found in conjunctival epithelium growing on corneal basement membrane adjacent to the migrating limbal cells, indicating local infiltration by the altered limbal basal cells. This same pattern was also found in recurrent pterygia. Based on this data we propose that the pathogenesis of pterygia is due to a normal stationary parental limbal epithelial basal cell becoming altered and giving rise to a zone of motile daughter cells, the pterygium cells, which leave the limbal region and migrate as a group centripetally along the corneal basement membrane dissolving Bowman's layer. Since these altered limbal basal cells are found at the microscopic advancing edge over Bowman's layer with no fibroblast mass under them, the pterygium cell apparently precedes the rapid growth of the fibroblasts from the stroma.(ABSTRACT TRUNCATED AT 250 WORDS)

An amelanotic, circum-corneal nevus in a 2-year-old child is described. The nevus presented at birth as a red spot in the nasal conjunctiva that subsequently enlarged to completely encircle the cornea. The tumour was partially removed three times, but at the age of 6 years, the nevus still covers the entire limbal region. The case illustrates that circum-corneal redness in a child may be caused by a nevus and that a conjunctival limbal nevus in a child tend to recur after incomplete excision.