V. Saez

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.

Conjunctival mucin mRNA expression in contact lens wear.

Purpose. To investigate the influence of the water content in non-ionic hydrogel contact lenses (HCL) on the mRNA levels of human conjunctival mucin genes (MUCs). Methods. Sixteen healthy subjects with no history of contact lenses wear were selected and randomized into two equal groups. Group 1 subjects wore low water content (38%, Soflens 38) non-ionic HCLs. Group 2 wore high water content (66%, Soflens 66) non-ionic HCLs. Conjunctival impression cytology was applied to the superior bulbar conjunctiva of both eyes before, 6 months, and 1 year after HCL fitting, and 15 days after discontinuation of wearing. Total RNA was isolated, retrotranscribed, and amplified by conventional polymerase chain reaction (PCR) and by quantitative real time PCR to study the mRNA levels of MUCs and to analyze variations during the study period. Time- and HCL-dependent variations in mRNA expression were analyzed using Student’s test. Results. From the known MUCs, transcripts from MUC1, MUC2, MUC4, MUC5AC, MUC7, MUC13, MUC15, MUC16, and MUC17 genes were detected in all subjects before HCL fitting. Except for MUC2, the expression of some MUC genes significantly increased whereas others significantly decreased at either the 6- and 12-month period. Statistically significant differences between both HCL groups (p < 0.001) were found in the MUC4, MUC13, and MUC15 mRNA expression after 1 year of wear and after the 15 days without HCL wear. However, these differences were not clearly related to the water content of the lenses. Conclusions. Low and high water content non-ionic HCLs induced different changes in the mRNA levels of several MUCs, but the water content was not related to the changes. Recovery to basal levels of conjunctival MUC mRNA expression after wearing HCL lenses for a year takes longer than 15 days for some MUCs.

Human epithelium from conjunctival impression cytology expresses MUC7 mucin gene

Purpose To prove that noninvasive methods of obtaining conjunctival tissue, such as conjunctival impression cytology (CIC), could be valid alternatives that are simpler, faster, and more convenient for patients than biopsy to analyze mRNA levels of mucin genes. Methods Using the semiquantitative reverse-transcriptase polymerase chain reaction, we studied the presence of the mucin genes described on the ocular surface thus far and attempted to detect the presence of MUC7 in CIC samples from 10 healthy donors. Results Conjunctival cells recovered by CIC expressed all the genes studied. There were no statistically significant differences between male and female subjects, and there was a significant correlation between the two eyes of the same donor only in the expression of MUC7. Conclusion CIC is a valid, noninvasive technique to detect the mRNAs of ocular genes in healthy individuals. MUC1, MUC2, MUC4, MUC5AC, and MUC7 mucin genes could be all detected in each CIC sample. This technique may be a useful tool to study the expression of some genes in ocular diseases.

α2-Adrenergic Receptors Are Present in Normal Human Conjunctiva

Purpose: Despite the passage of medications, including antiglaucoma drugs, through the ocular surface, and despite the increasing relevance of neurogenic inflammation in the ocular surface, the presence of some neuroreceptors in the conjunctiva has not been ascertained. This study describes the presence of α2-adrenergic receptors in normal human conjunctiva. Methods: Immunofluorescence microscopy, electrophoresis, and Western blot analyses were done in human conjunctival biopsies and rat control tissues. Antibodies against α2-adrenergic receptor subtypes α2A, α2B, and α2C were used. Results: Immunoreactivity for α2A- and α2B-, but not α2C-adrenergic receptors was evenly distributed in epithelial cells of human conjunctiva cryosections. Immunoreactive bands were detected for the three α2-adrenergic receptor subtypes: a major band of 48–50 kDa and fellow bands of 65–67 kDa. Conclusions: Normal human conjunctival epithelial cells express α2A-, α2B-, and α2C-adrenergic receptors. Further studies to determine the functional implications of these receptors in ocular surface homeostasis are warranted.