José M. Herreras

Ocular Surface Alteration after Long-term Treatment with an Antiglaucomatous Drug

BACKGROUND This study was undertaken to see whether long-term locally applied ocular medications produced any alterations in the ocular surface, and, in particular, whether it caused damage to the mucus layer of the tear film. METHODS The authors studied the ocular surface of 40 control subjects (group 1), 21 patients (group 2) chronically treated with a commercial preparation of 0.5% timolol maleate, and 20 previously untreated glaucomatous patients (group 3) in need of treatment with the same drug. Parameters studied were Schirmers test, lacrimal meniscus height, break-up time, fluorescein and rose Bengal stains, conjunctival impression cytology, mucus staining, and the ferning test. RESULTS Patients in groups 2 and 3 showed a significant decrease (P less than 0.001) in the number of normal Schirmers and break-up time tests. All had positive vital stains. Results showed a significant decrease (P less than 0.001) in goblet-cell density, mucus granules, and reticular sheets, and an increase (P less than 0.001) in pathologic crystallization patterns. CONCLUSION These results demonstrate that chronic application of a commercial preparation of timolol maleate damaged the ocular surface, especially the mucus layer of the tear film.

Pars planitis: epidemiology, treatment, and association with multiple sclerosis

During retrospective and prospective studies, we attempted to determine the clinical characteristics, treatment, and visual outcome of patients with pars planitis and to evaluate the association between pars planitis and multiple sclerosis (MS). The retrospective study included 44 patients with pars planitis, who had been examined between October 1986 and January 1999. We analyzed age, sex, visual acuity (VA), median follow-up time, and medical and surgical treatments. The prospective study, which included 21 consecutive patients with pars planitis, was performed to determine the presence of MS. In the retrospective study, the mean patient age was 22.4 years (SD ± 11.5) and the median follow-up was 34.9 months (SD ± 27.2). Complications included macular edema (47.7%), vitreous opacities (38.6%), papillitis (38.6%), vasculitis (36.4%), and cataract (20.5%). Forty patients (90.9%) had a final bilateral VA better than 20/40. In the prospective study, magnetic resonance imaging (MRI) was performed. Demyelinating lesions were found in 10 (47.6%) of the 21 patients and relapsing-remitting clinically definite MS was diagnosed in seven (33.3%). With the exception of age, no significantly statistical differences were observed when the visual prognosis and the clinical and epidemiologic characteristics were compared between the two groups of patients with and without associated MS; a diagnosis of MS was more frequently made in patients over 25 years of age. With appropriate treatment, patients with pars planitis have a good visual prognosis. Because the presence of demyelinating lesions seems to be high among patients with pars planitis, MRI should be considered, especially in patients over 25 years of age.

Dry Eye Disease as an Inflammatory Disorder

Purpose: Dry eye disease (DED) is a prevalent inflammatory disorder of the lacrimal functional unit of multifactorial origin leading to chronic ocular surface disease, impaired quality of vision, and a wide range of complications, eventually causing a reduction in quality of life. It still is a frustrating disease because of the present scarcity of therapies that can reverse, or at least stop, its progression. Methods: A comprehensive literature survey of English-written scientific publications on the role of inflammation in DED. Results: New investigations have demonstrated that a chronic inflammatory response plays a key role in the pathogenesis of human DED. Additionally, correlations between inflammatory molecules and clinical data suggest that inflammation can be responsible for some of the clinical symptoms and signs. Conclusions: Research efforts to clarify its pathophysiology are leading to a better understanding of DED, demonstrating that inflammation, in addition to many other factors, plays a relevant role.

Anti-TNF-α therapy in patients with refractory uveitis due to Behçet’s disease: a 1-year follow-up study of 124 patients

OBJECTIVE The aim of this study was to assess the efficacy of anti-TNF-α therapy in refractory uveitis due to Behçets disease (BD). METHODS We performed a multicentre study of 124 patients with BD uveitis refractory to conventional treatment including high-dose corticosteroids and at least one standard immunosuppressive agent. Patients were treated for at least 12 months with infliximab (IFX) (3-5 mg/kg at 0, 2 and 6 weeks and then every 4-8 weeks) or adalimumab (ADA) (usually 40 mg every 2 weeks). The main outcome measures were degree of anterior and posterior chamber inflammation, visual acuity, macular thickness and immunosuppression load. RESULTS Sixty-eight men and 56 women (221 affected eyes) were studied. The mean age was 38.6 years (s.d. 10.4). HLA-B51 was positive in 66.1% of patients and uveitis was bilateral in 78.2%. IFX was the first biologic agent in 77 cases (62%) and ADA was first in 47 (38%). In most cases anti-TNF-α drugs were used in combination with conventional immunosuppressive drugs. At the onset of anti-TNF-α therapy, anterior chamber and vitreous inflammation was observed in 57% and 64.4% of patients, respectively. In both conditions the damage decreased significantly after 1 year. At baseline, 50 patients (80 eyes) had macular thickening [optical coherence tomography (OCT) >250 μm] and 35 (49 eyes) had cystoid macular oedema (OCT>300 μm) that improved from 420 μm (s.d. 119.5) at baseline to 271 μm (s.d. 45.6) at month 12 (P < 0.01). The best-corrected visual acuity and the suppression load also showed significant improvement. After 1 year of follow-up, 67.7% of patients were inactive. Biologic therapy was well tolerated in most cases. CONCLUSION Anti-TNF-α therapy is effective and relatively safe in refractory BD uveitis.

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.

Characterization of epithelial primary cultures from human conjunctiva

Primary cultures of human epithelial cells from normal conjunctiva were developed and characterized to determine whether they retained epithelial characteristics. Conjunctival explants were obtained from the upper fornix of healthy donors and cultured in supplemented DMEM/F-12 medium for 5 days. The epithelial outgrowth was maintained for an additional 10 days. Primary cultures were then processed for light microscopy, transmission and scanning electron microscopy (TEM, SEM), and immunocytochemistry. They exhibited typical features of conjunctival epithelium on light microscopy (polygonal morphology, intimate cohesion, production of mucins), TEM (abundant desmosomes, keratin bundles, granules, microvilli), SEM (polygonal shape, microvilli, intimate cohesion), and immunocytochemistry (positivity for the receptor of epidermal growth factor, desmosomal proteins, and cytokeratins). In conclusion, primary cultures developed from normal human conjunctiva maintained the epithelial characteristics in vitro. Because the conjunctiva is a major component of the anterior ocular surface, we propose this in vitro system as suitable for physiopathologic and toxicologic studies.

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.

CARBOMER- VERSUS CELLULOSE-BASED ARTIFICIAL-TEAR FORMULATIONS : MORPHOLOGIC AND TOXICOLOGIC EFFECTS ON A CORNEAL CELL LINE

PURPOSE Frequent instillation of artificial tears is the primary disadvantage of the treatment for dry-eye syndrome. Recently gel formulations have been proposed as an alternative to classic cellulose formulations. The higher viscosity of these gels presumably prolongs tear-retention time in the eye and results in fewer daily applications. However, no toxicologic studies with gel formulations have been performed. Our aim was to study the toxic effects of these formulations on corneal cells. METHODS SIRC cells from rabbit cornea were exposed for 30 min, and 1, 3, and 6 h to five commercially available artificial tears, three of them carbomer gel formulations (Lacrivisc, Lacrivisc unit-doses, and Viscotears) and two carboxymethylcellulose (CMC) formulations (Celluvisc and Cellufresh). A cytotoxicity assay and a scanning electron microscopy (SEM) study were used to analyze the putative toxic effects of the formulations. The preservatives of the gel formulations also were tested. RESULTS Carbomer gel formulations, both with and without preservatives, caused more in vitro toxic effects in the corneal cells than did CMC formulations and caused severe damage even after 30 min of exposure. SEM revealed dramatic cell-surface alterations. Preservatives added to Lacrivisc and Viscotears also had toxic effects on cells, whose effects were not significantly different from those of the commercial preparations. CONCLUSION These results demonstrate that in the in vitro study, CMC artificial tears are less toxic than carbomer gel formulations. Questions about the benefits of using high-viscosity gels in the treatment of dry-eye syndrome still remain.

Anti-TNF-α therapy in refractory uveitis associated with sarcoidosis: Multicenter study of 17 patients

OBJECTIVES To assess anti-TNF-α therapy response in uveitis associated with sarcoidosis refractory to conventional immunosuppressive therapy. METHODS Open-label, multicenter, retrospective study on patients with sarcoid uveitis who underwent anti-TNF-α therapy because of inadequate response to conventional therapy including corticosteroids and at least 1 systemic synthetic immunosuppressive drug. The main outcome measurements were degree of anterior and posterior chamber inflammation, visual acuity, macular thickness, and immunosuppression load. RESULTS A total of 17 patients (8 men; 29 affected eyes; mean ± standard deviation age 38.4 ± 16.8; range: 13-76 years) were studied. The patients had bilateral hilar lymphadenopathy (58.8%), lung parenchyma involvement (47.1%), peripheral lymph nodes (41.2%), and involvement of other organs (52.9%). Angiotensin-converting enzyme was elevated in 58.8%. The most frequent ocular pattern was bilateral chronic relapsing panuveitis. The first biologic agent used was adalimumab in 10 (58.8%) and infliximab in 7 (41.2%) cases. Infliximab 5mg/kg intravenously every 4-8 weeks and adalimumab 40mg subcutaneously every 2 weeks were the most common administration patterns. In most cases anti-TNF-α therapy was given in combination with immunosuppressive drugs. The mean duration of follow-up was 33.9 ± 17.1 months. Significant improvement was observed following anti-TNF-α therapy. Baseline results versus results at 2 years from the onset of biologic therapy were the following: the median of cells in the ocular anterior chamber (interquartile range-IQR) 0.5 (0-2) versus 0 (0-0) (p = 0.003), vitritis 0 (0-1.25) versus 0 (0-0) (p = 0.008), macular thickness (391.1 ± 58.8 versus 247 ± 40.5µm) (p = 0.028), and visual acuity 0.60 ± 0.33 versus 0.74 ± 0.27; p = 0.009. The median daily (interquartile range) dose of prednisone was also reduced from 10 (0-30)mg at the onset of the anti-TNF-α therapy to 0 (0-0)mg at 2 years (p = 0.02). Significant reduction was also achieved in the immunosuppressive load. CONCLUSION Anti-TNF-α therapy is effective in sarcoid uveitis patients refractory to conventional immunosuppressive therapy. Infliximab and adalimumab allowed a substantial reduction in prednisone dose despite having failed standard therapy.

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.