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Galvis V.,Centro Oftalmologico Virgilio Galvis | Galvis V.,Autonomous University of Bucaramanga | Sherwin T.,University of Auckland | Tello A.,Centro Oftalmologico Virgilio Galvis | And 4 more authors.
Eye (Basingstoke) | Year: 2015

Keratoconus has been classically defined as a progressive, non-inflammatory condition, which produces a thinning and steepening of the cornea. Its pathophysiological mechanisms have been investigated for a long time. Both genetic and environmental factors have been associated with the disease. Recent studies have shown a significant role of proteolytic enzymes, cytokines, and free radicals; therefore, although keratoconus does not meet all the classic criteria for an inflammatory disease, the lack of inflammation has been questioned. The majority of studies in the tears of patients with keratoconus have found increased levels of interleukin-6 (IL-6), tumor necrosis factor-α(TNF-α), and matrix metalloproteinase (MMP)-9. Eye rubbing, a proven risk factor for keratoconus, has been also shown recently to increase the tear levels of MMP-13, IL-6, and TNF-α. In the tear fluid of patients with ocular rosacea, IL-1α and MMP-9 have been reported to be significantly elevated, and cases of inferior corneal thinning, resembling keratoconus, have been reported. We performed a literature review of published biochemical changes in keratoconus that would support that this could be, at least in part, an inflammatory condition. © 2015 Macmillan Publishers Limited.

Soria J.,Bioftalmik Applied Research | Villarrubia A.,Institute Oftalmologia La Arruzafa | Merayo-Lloves J.,University of Oviedo | Elortza F.,CIC Biomagune | And 5 more authors.
Molecular Vision | Year: 2015

Purpose: The etiology of keratoconus (KC) and the factors governing its progression are not well understood. It has been suggested that this disease might be caused by biochemical alterations in the cornea; changes in the expression profiles of human aqueous humor (hAH) proteins have been observed in some diseases. To gain a new insight into the molecular mechanisms of KC pathology, we examined the hAH proteomes of those in the advanced stages of this disease. We used a high-throughput mass spectrometry approach to compare hAH protein expression in patients with KC and in control subjects. Methods: Aqueous humor samples were acquired from five keratoconus patients during keratoplasty surgery and from five myopic control subjects during phakic intraocular lens implantation. Quantitative mass spectrometry analysis using spectral counting was performed to determine the relative amounts of hAH proteins in the samples from KC patients and control individuals. Results: All KC patients included in the study presented severe keratoconus (K2 >52 D), and slit-lamp examination revealed microfolds in Descemet’s membrane, without clinical signs of hydrops. We found significant differences between the expression levels of 16 proteins in the two groups. In KC samples, seven proteins were overexpressed and nine were underexpressed in comparison with the control group. Gene ontology analysis revealed that these deregulated proteins are implicated in several biologic processes, such as the regulation of proteolysis, responses to hypoxia, and responses to hydrogen peroxide, among others. Conclusions: The protein expression profiles in hAH from KC patients and myopic control subjects differ significantly. This result suggests that some components of the hAH proteome are involved in this disease. Further in-depth analysis of the hAH proteome should provide a better understanding of the mechanisms governing the pathophysiology of KC. © 2015, Molecular Vision. All rights reserved.

Azkargorta M.,CIC Biomagune | Soria J.,Bioftalmik Applied Research | Acera A.,Bioftalmik Applied Research | Iloro I.,CIC Biomagune | Elortza F.,CIC Biomagune
Journal of Proteomics | Year: 2016

Tears are a complex biological mixture containing electrolytes, metabolites, lipids, mucins, some small organic molecules, and proteins. The tear film has various roles in the lubrication, protection from the external environment, and nutrition of the cornea; it is also involved in the modulation of the optical properties of the eye. Tear composition reflects the physiological condition of the underlying tissues. Therefore, the tear fluid is useful in the evaluation of health and disease states and it is a valuable source of biomarkers for objective analysis of ocular and systemic diseases.The relatively high protein concentration of this fluid and the ease of noninvasive sample collection make it suitable for diagnostic and prognostic purposes. Efforts in proteomics research have positively affected to the field of ophthalmology, and the knowledge on the tear proteome has expanded considerably in the last few years. Nevertheless, despite a large amount of available data and the many biomarkers proposed for several eye and systemic diseases, the extent of translation to well-characterized and clinically useful tools has been largely insufficient. As for most of other biofluids, the road from discovery to clinical application is still long and full of pitfalls.In this review, we discuss the proteomic approaches used in the characterization of tear protein and peptide content, recapitulating the main studies and the progress done. We also present a brief summary of the path from discovery to clinical application of tear protein markers, with some representative examples of translation from the bench to the bedside. Significance: In this review we cover the most relevant proteomic approaches used in the characterization of the tear proteome, and for the first time we also focus in advances performed in the nowadays emerging peptide content characterization. In this context, we recapitulate on the main studies and the progresses done in this field. We also present a concise overview of the course that may be happen from discovery to clinical application for tear protein markers. Finally we include some representative examples of translation from the bench to the bedside. © 2016 Elsevier B.V.

Gonzalez N.,Bioftalmik Applied Research | Iloro I.,Proteomics Platform | Soria J.,Bioftalmik Applied Research | Duran J.A.,Instituto Clinico Quirurgico Of Oftalmologia Icqo | And 4 more authors.
EuPA Open Proteomics | Year: 2014

The goal of this study was to investigate differences in tear peptidome/proteome profiles of aqueous-deficient dry eye, Meibomian gland dysfunction and control individuals. Tears from 93 individuals were collected by capillary and subjected to solid-phase extraction followed by MALDI-TOF for profiling analysis. Obtained spectra were aligned by variable penalty dynamic time warping (VPdtw) and the resulting data analyzed using multivariate statistics. Comparative analyses revealed good performance of VPdtw and a high discrimination of groups with a correct assignment of 89.3% using twelve informative peaks. SDS-PAGE followed by MALDI-TOF/TOF analysis allowed identification of lipocalin-1 as a biomarker candidate. © 2014 The Authors.

Azkargorta M.,CIC Biomagune | Soria J.,Bioftalmik Applied Research | Ojeda C.,Pontifical Catholic University of Valparaiso | Guzman F.,Pontifical Catholic University of Valparaiso | And 4 more authors.
Journal of Proteome Research | Year: 2015

Endogenous peptides are valuable targets in the analysis of biological processes. The tear film contains proteins and peptides released by the tear duct mucosal cells, including antimicrobial peptides involved in the protection against exogenous pathogens; however, the peptide content of the tear liquid remains poorly characterized. We analyzed naturally occurring peptides isolated from human basal tears. Mass spectrometry analysis of endogenous peptides presents a number of drawbacks, including size heterogeneity and nonpredictable fragmentation patterns, among others. Therefore, CID, ETD, and HCD methods were used for the characterization of the tear peptide content. The contribution of DMSO as an additive of the chromatographic solvents was also evaluated. We identified 157, 131, and 122 peptides using CID-, ETD-, and HCD-based methods, respectively. Altogether, 234 different peptides were identified, leading to the generation of the biggest data set of endogenous tear peptides to date. The antimicrobial activity prediction analysis performed in silico revealed different putative antimicrobial peptides. Two of the extracellular glycoprotein lacritin peptides were de novo synthesized, and their antimicrobial activity was confirmed in vitro. Our findings demonstrate the benefits of using different fragmentation methods for the analysis of endogenous peptides and provide a useful approach for the discovery of peptides with antimicrobial activity. © 2015 American Chemical Society.

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