D.J. Galarreta

Ocular Mucin Gene Expression Levels as Biomarkers for the Diagnosis of Dry Eye Syndrome

PURPOSE To evaluate mRNA levels of the ocular mucins MUC1, MUC2, MUC4, MUC5AC, and MUC7 in conjunctival impression cytology samples from patients with moderate to severe dry eye syndrome (DES) compared with a population of healthy subjects; and to investigate the use of the levels of these mucin genes as biomarkers of DES and subsequently as a potential diagnostic test for DES. METHODS This prospective study commenced in the year 2000 and ended in the year 2009. Thirty-eight patients with DES and 43 age- and sex-matched healthy subjects completed the initial part of the study. Investigations were repeated at a later stage in 16 healthy subjects and 30 patients with DES, which were used as external validation data. Conjunctival impression cytology was performed in all subjects to test gene expression of ocular mucin genes MUC1, MUC2, MUC4, MUC5AC, and MUC7. Statistical analysis was performed to determine whether there was a difference in the levels of mucin gene expression between the two groups of subjects. Sensitivity and specificity of mucin gene expression for the diagnosis of DES was calculated. RESULTS Expressions of MUC1, MUC2, MUC4, and MUC5AC (P < 0.0001) were significantly lower in conjunctival epithelium of patients with DES compared with that in normal subjects. These results were replicated in the external control subject and patient groups. MUC1 expression levels demonstrated the greatest sensitivity (83.3%) and specificity (87.5%) among all genes tested. CONCLUSIONS The data strongly suggest that the expression levels of MUC1 may be used as a diagnostic test in DES for investigational and selective clinical trials.

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.

Expresión de los genes de mucinas MUC13, MUC15, MUC16 y MUC17 en el epitelio conjuntival normal humano in vivo

espanolObjetivo: La superficie ocular expresa al menos cinco de los 17 genes de mucinas descritos hasta ahora. El presente estudio fue disenado para determinar el perfil de expresion de los genes de mucinas en muestras obtenidas mediante citologia por impresion conjuntival (CIC) de sujetos sanos. Metodos: Se aplicaron dos filtros de polietersulfona en la conjuntiva superior de ambos ojos de 8 voluntarios sanos. Posteriormente, se llevo a cabo la reaccion en cadena de la polimerasa (PCR) usando el ARN total extraido y retrotranscrito de las muestras de CIC, para posteriormente estudiar la expresion de todos los genes humanos de mucinas conocidos. Despues de la amplificacion, los productos de la PCR fueron sometidos a electroforesis en un gel de agarosa al 1,5% y tenidos con bromuro de etidio para confirmar la obtencion de una unica banda cuando los ADNc son amplificados con los adecuados oligonucleotidos. Resultados: Se detectaron los transcritos de los genes de mucinas conjuntivales publicados anteriormente MUC1, MUC2, MUC4, MUC5AC y MUC7 en todas las muestras. Ademas, tambien se detectaron los transcritos de los genes de mucinas MUC13, MUC15, MUC16 y MUC17. Los productos amplificados mediante PCR convencional mostraron el tamano de amplificacion esperado. No se detectaron transcritos de los genes de mucinas MUC3A, MUC3B, MUC5B, MUC6, MUC8, MUC11 y MUC12. Conclusion: Por primera vez, se ha demostrado la expresion de cuatro ARNm de mucinas (MUC13, MUC15, MUC16 y MUC17) en el epitelio conjuntival humano de voluntarios sanos, hecho aun inedito. La funcion de estos genes habra de ser estudiada posteriormente. EnglishPurpose: 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.

Update on twice-daily bromfenac sodium sesquihydrate to treat postoperative ocular inflammation following cataract extraction

Ophthalmic bromfenac sodium sesquihydrate is a topically applied selective cyclooxygenase (COX)-2 inhibitor. It is similar to amfenac, except for a bromine atom at the C4 of the benzoyl ring position, which markedly affects its in vitro and in vivo potency, extends the duration of anti-inflammatory activity, and enhances its inhibitory effect on COX-2 absorption across the cornea and penetration into ocular tissues. The United States Food and Drug Administration approved bromfenac in 2005 for the treatment of postoperative inflammation and the reduction of ocular pain in patients who have undergone cataract surgery. Nonsteroidal anti-inflammatory drugs (NSAIDs), and among them bromfenac, could be even more effective than steroids at reestablishing the blood–aqueous barrier, as revealed by flare on slit-lamp examination and as quantitatively measured using ocular fluorophotometry. Similar to other NSAIDs, it has a role in inhibiting intraoperative miosis during cataract surgery. However, bromfenac also seems to be useful in other situations, such as refractive surgery, allergic conjunctivitis (not useful in dry eye), choroidal neovascularization, and even ocular oncology. No reports of systemic toxicity have been published and bromfenac has good topical tolerance with a low incidence of adverse effects.

Corneal inflammation from pine processionary caterpillar hairs.

Purpose: Hairs from the caterpillar of the pine processionary moth, Thaumetopoea pityocampa, located in the pine forests of southern Europe and elsewhere, can become embedded in the cornea and conjunctiva. Disorders produced by the urticating hairs can be classified into a broad spectrum of severity from mild keratoconjunctivitis to anaphylactic shock. This report presents 3 cases that illustrate the range of corneal responses to embedded hairs of the processionary moth caterpillar. Methods: Case series and review of the literature. Results: A 51-year-old man (case 1), a 46-year-old woman (case 2), and a 67-year-old man (case 3) presented different manifestations as a result of contact with pine processionary caterpillar hairs. Case 1 had acute keratitis with decreased vision. He was treated with a corneal depot steroid 4 times daily for 3 months. After 4 months, he was asymptomatic. Case 2 had acute keratitis without vision symptoms. She was released without treatment, and 1 month later was asymptomatic. Case 3 experienced dry gritty sensations, probably because of blepharitis, but also presented asymptomatic caterpillar hairs embedded in the corneal stroma. He was instructed regarding lid hygiene for his blepharitis, and after 3 months the embedded caterpillar hairs had disappeared. Conclusions: The most common ocular presentation of embedded hairs from the pine processionary moth caterpillar is keratitis. The urticating hairs can cause corneal infiltrates that disappear progressively over time. Steroid eye drops accelerate the resorption of these infiltrates.

Antioxidant enzyme mRNA expression in conjunctival epithelium of healthy human subjects.

OBJECTIVE To determine the profile, relative quantitation, and correlation of gene expression of the antioxidant enzymes copper-zinc superoxide dismutase (CuZn-SOD), Mn-superoxide dismutase (Mn-SOD), extracellular superoxide dismutase (EC-SOD), catalase (CAT), glutathione synthetase (GSS), and glutathione reductase (GSR) in conjunctival samples from healthy subjects. DESIGN Descriptive study. PARTICIPANTS Sixteen healthy donors (32 eyes) were included in this study. METHODS Conventional and real-time polymerase chain reactions (PCR) were performed using total RNA isolated from conjunctival impression cytology taken from bulbar superior conjunctiva from both eyes. RESULTS Products amplified by conventional PCR had only 1 band with the expected size for each target gene. Melt-curve analysis of real-time PCR products also identified a single amplified product for each gene. Log-transformed antioxidant enzyme mRNA expression levels, relative to glyceraldehyde-3-phosphate dehydrogenase expression (mean ± standard error of the mean [SEM]), were CuZn-SOD, 1.52 ± 0.13; Mn-SOD, 1.72 ± 0.08; EC-SOD, 0.35 ± 0.08; GSS, 2.43 ± 0.20; GSR, 2.52 ± 0.16; and CAT, 0.90 ± 0.08. The mRNA levels for CuZn-SOD were strongly correlated with GSS, GSR, Mn-SOD, and EC-SOD. Similarly, the levels of mRNA of GSS and GSR were strongly correlated with each other and with Mn-SOD and EC-SOD. CONCLUSIONS Normal human conjunctiva expresses the antioxidant enzymes genes CuZn-SOD, Mn-SOD, EC-SOD, CAT, GSS, and GSR. The relative quantitation of these genes expressed in conjunctivas of normal eyes will allow further comparisons in pathological circumstances. Knowledge of correlated gene expression will provide a better understanding of the antioxidant balance in the ocular surface.

Inter-examiner agreement of the AS-OCT Visante corneal thickness

Purpose To determine the inter-examiner agreement of the manual values of central and peripheral corneal thickness as measured by optical coherence tomography (AS-OCT Visante) and to assess the agreement between AS-OCT Visante pachymetry, Orbscan II and ultrasound pachymetry (USP).