Pterigión: caso clínico y revisión

We present the case of a 32-year old Cuban who first consulted at our clinic for recurrent pterygium, OS. At our service, we did a pterygium excision with orthotopic conjunctival autoplasty. 4 months later, there was a second recurrence for which we did an excision with buccal mucosal graft. After six months, a third recurrence occurred along with ptosis and restrictive diplopía, for which we decided to do an excision with eccentric lamellar keratoplasty and buccal mucosal graft. Pterygia are non-involutional corneal degenerations presenting as a wing-like fibrovascular growth of the bulbar conjunctiva¿s connective tissue onto the cornea. Recent studies showing over expression of matrix metalloproteinases due to stimulation by interleukins and tumor necrosis factors support the concept that the alteration of basal limbal epithelial cells is a key factor in the pathogenesis. Careful assessment and individualized patient treatment are mandatory to prevent vision threatening complicationsPresentamos un caso de un varón de 32 años que acudió a nuestra consulta afecto de pterigión recidivante, OI. Se practicó extirpación del pterigion con autoplastia conjuntival. 4 meses post-op, recidiva. Se practica una segunda cirugía en nuestro centro extirpando el pterigión recidivante con transplante de mucosa bucal. 6 meses después, nueva recidiva (tercera) con ptosis y diplopía restrictiva practicandole de nuevo una extirpación con transplante laminar excéntrico mas transplante de mucosa bucal. El pterigión es una degeneración corneal no involutivas que presenta una proliferación fibrovascular del tejido conjuntival que crece desde la conjuntiva bulbar hacia la cornea. Investigaciones recientes mostrando una sobreproducción de ciertas metaloproteinasas de la matriz debido a una estimulación inflamatoria de interleukinas y de factores necrosis tumoral soporta la noción de que la alteración de células epiteliales basales limbares posee un papel primordial en la patogenia. Se trata de un examen cauteloso y un tratamiento individualizado para prevenir las complicaciones de la extirpación del pterigión

Transcriptome Analysis of Cultured Limbal Epithelial Cells on an Intact Amniotic Membrane following Hypothermic Storage in Optisol-GS

The aim of the present study was to investigate the molecular mechanisms underlying activation of cell death pathways using genome-wide transcriptional analysis in human limbal epithelial cell (HLEC) cultures following conventional hypothermic storage in Optisol-GS. Three-week HLEC cultures were stored in Optisol-GS for 2, 4, and 7 days at 4 °C. Partek Genomics Suite software v.6.15.0422, (Partec Inc., St. Louis, MO, USA) was used to identify genes that showed significantly different (P < 0.05) levels of expression following hypothermic storage compared to non-stored cell sheets. There were few changes in gene expression after 2 days of storage, but several genes were differently regulated following 4 and 7 days of storage. The histone-coding genes HIST1H3A and HIST4H4 were among the most upregulated genes following 4 and 7 days of hypothermic storage. Bioinformatic analysis suggested that these two genes are involved in a functional network highly associated with cell death, necrosis, and transcription of RNA. HDAC1, encoding histone deacetylase 1, was the most downregulated gene after 7 days of storage. Together with other downregulated genes, it is suggested that HDAC1 is involved in a regulating network significantly associated with cellular function and maintenance, differentiation of cells, and DNA repair. Our data suggest that the upregulated expression of histone-coding genes together with downregulated genes affecting cell differentiation and DNA repair may be responsible for increased cell death following hypothermic storage of cultured HLEC. In summary, our results demonstrated that a higher number of genes changed with increasing storage time. Moreover, in general, larger differences in absolute gene expression values were observed with increasing storage time. Further understanding of these molecular mechanisms is important for optimization of storage technology for limbal epithelial sheet

Serum-Free and Xenobiotic-Free Preservation of Cultured Human Limbal Epithelial Cells

Altres ajuts: The research was funded by the South-Eastern Norway Regional Health Authority, grant number 2010092, project name "Treating blindness using novel tissue storage technology". The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.To develop a one-week storage method, without serum and xenobiotics, that would maintain cell viability, morphology, and phenotype of cultured human limbal epithelial sheets. Human limbal explants were cultured on intact human amniotic membranes for two weeks. The sheets were stored in a hermetically sealed container at 23°C in either a serum-free medium with selected animal serum-derived compounds (Quantum 286) or a xenobiotic-free medium (Minimal Essential Medium) for 4 and 7 days. Stored and non-stored cultures were analyzed for cell viability, amniotic membrane and epithelial sheet thickness, and a panel of immunohistochemical markers for immature cells (ΔNp63α, p63, Bmi-1, C/EBP∂, ABCG2 and K19), differentiated cells (K3 and Cx43), proliferation (PCNA), and apoptosis (Caspase-3). The cell viability of the cultures was 98 ± 1% and remained high after storage. Mean central thickness of non-stored limbal epithelial sheets was 23 ± 3 μm, and no substantial loss of cells was observed after storage. The non-stored epithelial sheets expressed a predominantly immature phenotype with ΔNp63α positivity of more than 3% in 9 of 13 cultures. After storage, the expression of ABCG2 and C/EBP∂ was reduced for the 7 day Quantum 286-storage group; (P = 0.04), and Bmi-1 was reduced after 4 day Quantum 286-storage; (P = 0.02). No other markers varied significantly. The expression of differentiation markers was unrelated to the thickness of the epithelia and amniotic membrane, apart from ABCG2, which correlated negatively with thickness of limbal epithelia (R = -0.69, P = 0.01) and ΔNp63α, which correlated negatively with amniotic membrane thickness (R = -0.59, P = 0.03). Limbal epithelial cells cultured from explants on amniotic membrane can be stored at 23°C in both serum-free and xenobiotic-free media, with sustained cell viability, ultrastructure, and ΔNp63α-positivity after both 4 and 7 days

Data from: Serum-free and xenobiotic-free preservation of cultured human limbal epithelial cells

Aim/Purpose of the Study: To develop a one-week storage method, without serum and xenobiotics, that would maintain cell viability, morphology, and phenotype of cultured human limbal epithelial sheets. Materials and Methods: Human limbal explants were cultured on intact human amniotic membranes for two weeks. The sheets were stored in a hermetically sealed container at 23°C in either a serum-free medium with selected animal serum-derived compounds (Quantum 286) or a xenobiotic-free medium (Minimal Essential Medium) for 4 and 7 days. Stored and non-stored cultures were analyzed for cell viability, amniotic membrane and epithelial sheet thickness, and a panel of immunohistochemical markers for immature cells (ΔNp63α, p63, Bmi-1, C/EBP∂, ABCG2 and K19), differentiated cells (K3 and Cx43), proliferation (PCNA), and apoptosis (Caspase-3). Results: The cell viability of the cultures was 98 ± 1% and remained high after storage. Mean central thickness of non-stored limbal epithelial sheets was 23 ± 3 μm, and no substantial loss of cells was observed after storage. The non-stored epithelial sheets expressed a predominantly immature phenotype with ΔNp63α positivity of more than 3% in 9 of 13 cultures. After storage, the expression of ABCG2 and C/EBP∂ was reduced for the 7 day Quantum 286-storage group; (P = 0.04), and Bmi-1 was reduced after 4 day Quantum 286-storage; (P = 0.02). No other markers varied significantly. The expression of differentiation markers was unrelated to the thickness of the epithelia and amniotic membrane, apart from ABCG2, which correlated negatively with thickness of limbal epithelia (R = -0.69, P = 0.01) and ΔNp63α, which correlated negatively with amniotic membrane thickness (R = -0.59, P = 0.03). Conclusion: Limbal epithelial cells cultured from explants on amniotic membrane can be stored at 23°C in both serum-free and xenobiotic-free media, with sustained cell viability, ultrastructure, and ΔNp63α-positivity after both 4 and 7 days

Effects of explant size on epithelial outgrowth, thickness, stratification, ultrastructure and phenotype of cultured limbal epithelial cells

Purpose Transplantation of limbal stem cells is a promising therapy for limbal stem cell deficiency. Limbal cells can be harvested from either a healthy part of the patient’s eye or the eye of a donor. Small explants are less likely to inflict injury to the donor site. We investigated the effects of limbal explant size on multiple characteristics known to be important for transplant function. Methods Human limbal epithelial cells were expanded from large versus small explants (3 versus 1 mm of the corneal circumference) for 3 weeks and characterized by light microscopy, immunohistochemistry, and transmission electron microscopy. Epithelial thickness, stratification, outgrowth, ultrastructure and phenotype were assessed. Results Epithelial thickness and stratification were similar between the groups. Outgrowth size correlated positively with explant size (r = 0.37; P = 0.01), whereas fold growth correlated negatively with explant size (r = –0.55; P < 0.0001). Percentage of cells expressing the limbal epithelial cell marker K19 was higher in cells derived from large explants (99.1±1.2%) compared to cells derived from small explants (93.2±13.6%, P = 0.024). The percentage of cells expressing ABCG2, integrin β1, p63, and p63α that are markers suggestive of an immature phenotype; Keratin 3, Connexin 43, and E-Cadherin that are markers of differentiation; and Ki67 and PCNA that indicate cell proliferation were equal in both groups. Desmosome and hemidesmosome densities were equal between the groups. Conclusion For donor- and culture conditions used in the present study, large explants are preferable to small in terms of outgrowth area. As regards limbal epithelial cell thickness, stratification, mechanical strength, and the attainment of a predominantly immature phenotype, both large and small explants are sufficient

Effects of explant size on epithelial outgrowth, thickness, stratification, ultrastructure and phenotype of cultured limbal epithelial cells

Purpose Transplantation of limbal stem cells is a promising therapy for limbal stem cell deficiency. Limbal cells can be harvested from either a healthy part of the patient’s eye or the eye of a donor. Small explants are less likely to inflict injury to the donor site. We investigated the effects of limbal explant size on multiple characteristics known to be important for transplant function. Methods Human limbal epithelial cells were expanded from large versus small explants (3 versus 1 mm of the corneal circumference) for 3 weeks and characterized by light microscopy, immunohistochemistry, and transmission electron microscopy. Epithelial thickness, stratification, outgrowth, ultrastructure and phenotype were assessed. Results Epithelial thickness and stratification were similar between the groups. Outgrowth size correlated positively with explant size (r = 0.37; P = 0.01), whereas fold growth correlated negatively with explant size (r = –0.55; P < 0.0001). Percentage of cells expressing the limbal epithelial cell marker K19 was higher in cells derived from large explants (99.1±1.2%) compared to cells derived from small explants (93.2±13.6%, P = 0.024). The percentage of cells expressing ABCG2, integrin β1, p63, and p63α that are markers suggestive of an immature phenotype; Keratin 3, Connexin 43, and E-Cadherin that are markers of differentiation; and Ki67 and PCNA that indicate cell proliferation were equal in both groups. Desmosome and hemidesmosome densities were equal between the groups. Conclusion For donor- and culture conditions used in the present study, large explants are preferable to small in terms of outgrowth area. As regards limbal epithelial cell thickness, stratification, mechanical strength, and the attainment of a predominantly immature phenotype, both large and small explants are sufficient

Antibodies used in the study.

<p>Antibodies used in the study.</p

Staining Pattern of Immunohistochemical Markers Used in the Study.

<p>Staining Pattern of Immunohistochemical Markers from representative areas of Non-Stored Cultured Limbal Epithelial Cell Sheets. The pictures show nuclear markers A) ΔNP63α, B) pan-p63, C) Bmi-1, and D) C/EBP∂; membrane markers E) ABCG2, and F) Cx-43; keratin markers G) K19, and H) K3, proliferating cell marker I) PCNA, and finally, apoptosis marker J) Caspase-3. Original magnification 400x. Arrow in C) marks positively stained cell.</p

Mean Central Thickness of Non-Stored and Stored Cultured Limbal Epithelial Sheets.

<p>Scatterplot illustrating average central thickness (<i>μ</i>m) of cultured human limbal epithelial cell sheets stored for 4 and 7 days in Quantum 286 or MEM, compared to a non-stored control group. Horizontal bars represent mean thickness per group.</p