Levels of mucin gene expression in normal human conjunctival epithelium in vivo.

PURPOSE: Conjunctival impression cytology (CIC) samples were used to determine the mean and normal range of mRNA levels of human MUC1, MUC2, MUC4, MUC5AC, and MUC7 mucin genes. METHODS: Real time PCR was performed to determine normal mRNA levels in CIC samples of 24 male and 19 female healthy donors. Correlation coefficients between gene expression levels were obtained. RESULTS: All five mucin genes were expressed in the CIC samples. MUC1 and MUC4 were present at the highest level and MUC2 was at the lowest. There were no gender differences. Significant positive correlations existed between MUC2 and MUC4 and between MUC2 and MUC7 levels. CONCLUSIONS: Normal levels and ranges of mRNAs for MUC1, MUC2, MUC4, MUC5AC and MUC7 conjunctival mucin genes have been established for the first time. These data may serve as the normal threshold values for future comparisons in different experimental and pathological conditions involving the ocular surface.

[In vitro toxicity of non-preserved artificial-tear formulations]. / Lágrimas artificiales sin conservantes: estudio comparativo in vitro.

PURPOSE: To analyse the putative toxic effect of three commercially available non-preserved artificial tear formulations on in vitro human conjunctival cells. MATERIAL AND METHOD: A conjunctival human epithelial cell line was exposed to Cellufresh, Oculotect and Acuolens formulations during 1, 3 and 24 hours. Cytotoxicity was measured by calculating the percentage of cell viability examination and scanning electron microscopy (SEM). Controls underwent exposure to supplement free DMEM-F12 (negative control) and exposure to 0.005% benzalkonium chloride solution (positive control). RESULTS: Cell viability after 1 or 3 hours incubation with Cellufresh and Oculotect was similar to that obtained for negative controls. With Acuolens incubation however, cell viability showed significant reduction after 3 and 24 hours compared to control. SEM showed that Cellufresh and Oculotect exposed cells presented similar behavior to control cells. All three cell lines presented evidence of cellular surface alteration after incubation for 1 or 3 hours compared to controls, Acuolens showing the highest rate of alterations in exposed cells and an additional increment in cell loss was observed. CONCLUSION: In the present study, non preserved artificial tears formulations showed a different degree in their in vitro toxicity, Acuolens being more toxic than Cellufresh or Oculotect.

[Normal human conjunctival epithelium expresses MUC13, MUC15, MUC16 and MUC17 mucin genes]. / Expresión de los genes de mucinas MUC13, MUC15, MUC16 y MUC17 en el epitelio conjuntival normal humano in vivo.

PURPOSE: The ocular surface epithelia express at least five mucin genes of the total of 17 human mucin genes that have been identified so far. This study was designed to determine the expression profile of mucin genes in conjunctival impression cytology (CIC) samples from healthy subjects. METHODS: Two polyethersulfone filters were applied to the superior conjunctiva of both eyes from eight healthy donors. Polymerase chain reaction (PCR) was performed using isolated and retrotranscripted total RNA obtained from the CIC samples to study the expression of all known human mucin genes. Following amplification, PCR products were electrophoresed on 1.5% agarose gel and stained with ethidium bromide to confirm that only a single band was obtained when amplifying all cDNAs with the convenient primers. RESULTS: Transcripts of the previously reported conjunctival mucin genes MUC1, MUC2, MUC4, MUC5AC, and MUC7 were detected in all samples. In addition, transcripts of MUC13, MUC15, MUC16 and MUC17 mucin genes also were detected. Amplified products by conventional PCR showed the expected amplicon size. Transcripts of MUC3A, MUC3B, MUC5B, MUC6, MUC8, MUC11, and MUC12 mucin genes were not detected. CONCLUSION: The expression of four additional mucin mRNA (MUC13, MUC15, MUC16, and MUC17) has been proved in human conjunctival epithelium from healthy donors for the first time. The function of these genes remains to be further elucidated.

Homeostatic control of conjunctival mucosal goblet cells by NKT-derived IL-13.

Although the effects of the interleukin 13 (IL-13) on goblet cell (GC) hyperplasia have been studied in the gut and respiratory tracts, its effect on regulating conjunctival GC has not been explored. The purpose of this study was to determine the major IL-13-producing cell type and the role of IL-13 in GC homeostasis in normal murine conjunctiva. Using isolating techniques, we identified natural killer (NK)/natural killer T (NKT) cells as the main producers of IL-13. We also observed that IL-13 knockout (KO) and signal transducer and activator of transcription 6 knockout (STAT6KO) mice had a lower number of periodic acid Schiff (PAS)+GCs. We observed that desiccating stress (DS) decreases NK population, GCs, and IL-13, whereas it increases interferon-γ (IFN-γ) mRNA in conjunctiva. Cyclosporine A treatment during DS maintained the number of NK/NKT cells in the conjunctiva, increased IL-13 mRNA in NK+ cells, and decreased IFN-γ and IL-17A mRNA transcripts in NK+ and NK- populations. C57BL/6 mice chronically depleted of NK/NKT cells, as well as NKT cell-deficient RAG1KO and CD1dKO mice, had fewer filled GCs than their wild-type counterparts. NK depletion in CD1dKO mice had no further effect on the number of PAS+ cells. Taken together, these findings indicate that NKT cells are major sources of IL-13 in the conjunctival mucosa that regulates GC homeostasis.