Purpose.: To identify early biomarkers of retinal Müller cell activation in diabetic eyes with or without clinically detectable signs of diabetic retinopathy (DR). Methods.: This study was a cross-sectional comparative case series. The aqueous humor (AH) of 34 eyes was collected in 12 healthy controls, 11 diabetic patients without DR, and 11 diabetic patients with nonproliferative DR. Full ophthalmic examination and spectral-domain optical coherence tomography were performed in all eyes. Glial fibrillary acidic protein (GFAP), aquaporin 1 (AQP1), and aquaporin 4 (AQP4) were quantified in AH samples as biomarkers of Müller cell activity by ELISA. Statistical analysis was performed with ANOVA followed by Tukey-Kramer post hoc test. Results.: There was no significant difference in the age among the three groups. Mean concentration of GFAP, AQP1, and AQP4 significantly increased in diabetic eyes versus controls (P < 0.05, for each comparison). Glial fibrillary acidic protein and AQP1 showed an approximate 2-fold increase, whereas AQP4 showed an approximate 25-fold increase in diabetics with DR versus controls. In diabetics without DR, AQP4 showed an approximate 6-fold increase versus controls. Conclusions.: Glial fibrillary acidic protein, AQP1, and AQP4—biomarkers of Müller cell activity—are significantly increased in human eyes with diabetes, confirming that Müller cells are precociously affected by diabetes mellitus.

Purpose: We characterize age-associated alterations in the expression of inflammatory mediators and tissue remodeling factors in human tears. Methods: A total of 75 consecutive volunteers (32 male/44 female; 19–93 years) underwent clinical assessment of ocular surface status, ocular surface disease index (OSDI) grading and tear sampling. The volunteers were categorized into three groups: young (18–40 years), middle-aged (41–60 years), and old (>60 years). Total protein profiles and chip-based protein array evaluations were conducted to investigate the expression of 60 potential candidates, including pro-/anti-inflammatory mediators and tissue remodeling factors. Appropriate validations were performed using conventional assays. Multiple comparisons for regression between potential candidates and age were performed, as well as statistical analyses among the three age groups. Nonpooled samples were used for quantifications. Results: Pearson analysis of chip-arrays identified 9 of 60 potential candidates. Specifically, IL-8, IL-6, and regulated on activation, normal T cell expressed and secreted (RANTES; P < 0.0083) protein as well as matrix metalloproteinase (MMP)-1, IL-3, and TNF-α (P < 0.05) correlated positively with aging. MIP-3β showed an opposite tendency. Western blot and ELISA analysis corroborated the array data. OSDI grading did not correlate with aging. Conclusions: Dynamic changes to tear protein profiles occur with aging. Our study identifies the expression of IL-8, IL-6, RANTES, MMP-1, and MIP-3β as increasing with age. These select inflammatory and matrix remodeling factors may be relevant to the development of novel diagnostic tools and therapeutics in the context of age-related ocular surface disease.

Introduction Primary open angle glaucoma (POAG) is a progressive optic neuropathy characterized by impaired aqueous outflow and extensive remodeling in the trabecular meshwork (TM). The aim of this study was to characterize and compare the expression patterns of selected proteins belonging to the tissue remodeling, inflammation and growth factor pathways in ex vivo glaucomatous and post-mortem TMs using protein-array analysis. Methods TM specimens were collected from 63 white subjects, including 40 patients with glaucoma and 23 controls. Forty POAG TMs were collected at the time of surgery and 23 post-mortem specimens were from non-glaucomatous donor sclerocorneal tissues. Protein profiles were evaluated using a chip-based array consisting of 60 literature-selected antibodies. Results A different expression of some factors was observed in POAG TMs with respect to post-mortem specimens, either in abundance (interleukin [IL]10, IL6, IL5, IL7, IL12, IL3, macrophage inflammatory protein [MIP]1δ/α, vascular endothelial growth factor [VEGF], transforming growth factor beta 1 [TGFβ1], soluble tumor necrosis factor receptor I [sTNFRI]) or in scarcity (IL16, IL18, intercellular adhesion molecule 3 [ICAM3], matrix metalloproteinase-7 [MMP7], tissue inhibitor of metalloproteinase 1 [TIMP1]). MMP2, MMP7, TGFβ1, and VEGF expressions were confirmed by Western blot, zymography, and polymerase chain reaction. No difference in protein profile expression was detected between glaucomatous subtypes. Conclusion The analysis of this small TM population highlighted some proteins linked to POAG, some previously reported and others of new detection (IL7, MIPs, sTNFαRI). A larger POAG population is required to select promising disease-associated biomarker candidates. Funding This study was partially supported by the Fondazione Roma, the Italian Ministry of Health and the “National 5xMille 2010 tax donation to IRCCS-G.B. Bietti Foundation”.

Objective. Vernal keratoconjunctivitis (VKC) is a chronic recurrent bilateral inflammation of the conjunctiva associated with atopy. Several inflammatory and tissue remodeling factors contribute to VKC disease. The aim is to provide a chip-based protein analysis in tears from patients suffering from quiescent or active VKC.Methods. This study cohort included 16 consecutive patients with VKC and 10 controls. Participants were subjected to clinical assessment of ocular surface and tear sampling. Total protein quantification, total protein sketch, and protein array (sixty protein candidates) were evaluated.Results. An overall increased Fluorescent Intensity expression was observed in VKC arrays. Particularly, IL1β, IL15, IL21, Eotaxin2, TACE, MIP1α, MIP3α, NCAM1, ICAM2,βNGF, NT4, BDNF,βFGF, SCF, MMP1, and MMP2 were increased in quiescent VKC. Of those candidates, only IL1β, IL15, IL21,βNGF, SCF, MMP2, Eotaxin2, TACE, MIP1α, MIP3α, NCAM1, and ICAM2 were increased in both active and quiescent VKC. Finally, NT4,βFGF, and MMP1 were highly increased in active VKC.Conclusion. A distinct “protein tear-print” characterizes VKC activity, confirming some previously reported factors and highlighting some new candidates common to quiescent and active states. Those candidates expressed in quiescent VKC might be considered as predictive indicators of VKC reactivation and/or exacerbation out-of-season.

Purpose: To investigate Nerve Growth Factor (NGF) and nitric oxide synthase (iNOS) levels in tears obtained from Video Display Terminal (VDT) workers and correlate their expression with ocular signs and symptoms. Methods: A total of 120 VDT workers (62M/58F; 31–63 years old) and 40 age/sex matched no-VDT volunteers (19M/21F; 30–60 years old) were enrolled in the study. Participants completed the OSDI questionnaire and were subjected to clinical assessment of ocular surface status, including ocular symptoms and tear film parameters. NGF and iNOS levels were quantified in tear samples and their expressions correlated with OSDI, ocular symptoms and tear film parameters. Results: 59.17% of the studied population was symptomatic based on OSDI scores. Women were more commonly affected. The most frequent symptom was asthenopia and except for dryness, no differences were found between genders regarding other symptoms. A statistically significant decrease in NGF levels was found between normal and moderate (p < 0.05) and between mild and moderate (p < 0.05) OSDI grading. iNOS expression was increased in moderate OSDI grading compared to normals (p < 0.05). A negative correlation was found between NGF and respectively OSDI results, dryness and blurry vision (p < 0.05). No correlations were found among NGF, iNOS and ocular surface parameters (Schirmer, BUT, ocular surface staining). Conclusion: Our data suggest that NGF and iNOS levels contribute to VDT ocular discomfort. Further studies are required to better understand the relationship between NGF and iNOS in VDT ocular surface.