Levels of the oxidative stress biomarker malondialdehyde in tears of patients with central serous chorioretinopathy relate to disease activity.

Purpose: Central serous chorioretinopathy (CSCR) has been associated with oxidative stress-related risk factors. The objective of this study was to optimize an analytical method for evaluating the oxidative stress biomarker malondialdehyde (MDA) in human tears and determine its level in the tears of patients with CSCR. Methods: In this pilot study, tear samples were obtained from 34 healthy donors and 31 treatment-naïve CSCR male patients (eight with acute CSCR and 23 with chronic CSCR). Two analytical methods based on high-performance liquid chromatography followed by fluorescence detection were evaluated, with either 2-thiobarbituric derivative (TBA) or 2-aminoacridone (2-AA). Activity of CSCR was defined by the serous retinal detachment (SRD) height, which was measured by two independent observers on spectral-domain optical coherence tomography. Results: The 2-AA method showed higher sensitivity and precision compared to the TBA method. When the 2-AA method was applied to tears from healthy donors, the levels of MDA were statistically significantly higher in men compared to women (mean ± standard deviation, SD: 9,914 nM ± 6,126 versus 4,635 nM ± 1,173, p = 0.006). No difference was found in tear MDA levels between male patients with CSCR and age-matched control men (p = 0.17). However, MDA levels were statistically significantly higher in acute compared to chronic CSCR cases (mean ± SD: 12,295 nM ± 8,495 versus 6,790 ± 3,969 nM, p = 0.03). Additionally, there was a correlation between MDA levels and RPE leakage, quantified by the height of the serous retinal detachment (p = 0.02, r = 0.40). Conclusions: Levels of MDA in tears, measured with an optimized analytical method, correlate with RPE leakage in CSCR.

Investigation of the global protein content from healthy human tears.

Considering absence of invasiveness and side effects, tears emerge as a particularly attractive fluid for biomarker discovery and therefore for daily clinical use. However, to date this fluid remains poorly studied in healthy condition. Here, we present an updated in-depth characterisation of the human healthy tear protein composition using proteomics approach. Both eyes of 8 healthy controls (4 men and 4 women, average age: 38 ±â€¯18) were collected using the Schirmer's strip method. After liquid digestion and off-gel electrophoresis fractionation, three independent proteomics analyses were performed on a LTQ-Orbitrap Velos Pro. Globally, 1351 proteins were identified with 2 unique peptides and 1% FDR. Gene ontology analyses showed up that 39% of the tear proteins were enzymes, with high numbers of dehydrogenases, phosphatases, kinases and ligases. Immunoglobulins, serpins and 14-3-3 domains proteins also emerged as the main tear protein families. The glycolysis and the coagulation and complement cascades, which were already shown in tears as involved in ocular and systemic diseases, were highlighted performing pathway analyses. Our study therefore complements the existing data on healthy tears proteome. Nevertheless, extensive studies for deeply and definitively characterise this promising fluid are required in the near future in order to be able to routinely use this fluid in clinics. A better understanding of its protein content will probably open new avenues in the biomarker discovery and clinical practice in the near future.

Differential profiling of lacrimal cytokines in patients suffering from thyroid-associated orbitopathy.

The aim was to investigate the levels of cytokines and soluble IL-6R in the tears of patients with thyroid-associated orbitopathy (TAO) disease. Schirmer's test was adopted to collect tears from TAO patients (N = 20, 17 women, mean age (±SD): 46.0 years (±13.4)) and healthy subjects (N = 18, 10 women, 45.4 years (±18.7)). Lacrimal cytokines and soluble IL-6R (sIL-6R) were measured using a 10-plex panel (Meso Scale Discovery Company) and Invitrogen Human sIL-6R Elisa kit, respectively. Tear levels of IL-10, IL-12p70, IL-13, IL-6 and TNF-α appeared significantly higher in TAO patients than in healthy subjects. Interestingly, IL-10, IL-12p70 and IL-8 levels increased in tears whatever the form of TAO whereas IL-13, IL-6 and TNF-α levels were significantly elevated in inflammatory TAO patients, meaning with a clinical score activity (CAS) ≥ 3, compared to controls. Furthermore, only 3 cytokines were strongly positively correlated with CAS (IL-13 Spearman coeff. r: 0.703, p = 0.0005; IL-6 r: 0.553, p = 0.011; IL-8 r: 0.618, p = 0.004, respectively). Finally, tobacco use disturbed the levels of several cytokines, especially in patient suffering of TAO. The differential profile of lacrimal cytokines could be useful for the diagnosis of TAO patients. Nevertheless, the tobacco use of these patients should be taken into account in the interpretation of the cytokine levels.

Thyroid-Associated Orbitopathy and Biomarkers: Where We Are and What We Can Hope for the Future.

BACKGROUND: Thyroid-associated orbitopathy (TAO) is the most common autoimmune disease of the orbit. It occurs more often in patients presenting with hyperthyroidism, characteristic of Graves' disease, but may be associated with hypothyroidism or euthyroidism. The diagnosis of TAO is based on clinical orbital features, radiological criteria, and the potential association with thyroid disease. To date, there is no specific marker of the orbital disease, making the early diagnosis difficult, especially if the orbital involvement precedes the thyroid dysfunction. SUMMARY: The goal of this review is to present the disease and combine the available data in the literature concerning investigation of TAO biomarkers. CONCLUSIONS: Despite the progress done in the understanding of TAO disease, some important pieces are still missing. Typically, for the future, major efforts have to be done in the discovery of new biomarkers, validation of the suspected candidates on multicenter cohorts with standardized methodologies, and establishment of their clinical performances on the specific clinical application fields in order to improve not only the management of the TAO patients but also the therapeutic options and follow-up.

Thyroid-associated orbitopathy and tears: A proteomics study.

To date, Thyroid-Associated Orbitopathy (TAO), an autoimmune inflammatory disease affecting the eye, remains poorly characterised and its diagnosis challenging. The aim of this study was to investigate the tears of the TAO patients in order to identify potential biomarkers. Two independent quantitative Tandem Mass Tag™ 6-plex experiments were done. After in-solution digestion and isoelectric fractionation, the 12 fractions were analysed with a LTQ Orbitrap Velos coupled to a liquid chromatography. Raw files were searched against Swiss-Prot-AC database using Proteome Discoverer software, with a false discovery rate of 1% at peptide and protein levels. The differential proteins were then verified using orthogonal approaches in independent patients. Globally, 712 tear proteins were quantified with 2 unique peptides. Interestingly, cystatin c (TAO/controls ratio: 1.53), alpha-1 antichymotrypsin (ratio: 1.70) and retinal dehydrogenase (ratio: 0.68), displaying differential levels in the tears of TAO patients using proteomics experiments emerged as highly promising biomarkers after verification. In conclusion, this proteomics study supports the idea that tears reflect biological modifications occurring in a disease context and can therefore be a promising fluid for biomarker discovery. Moreover, our study identified three candidates that could in the future open new avenues in the diagnosis of TAO disease. SIGNIFICANCE: Thyroid associated orbitopathy (TAO) is the most common disease affecting the orbit. Moreover, the later, severe stages of the disease can be sight threating [1]. On the other hand, the early sign and symptoms can be mistaken with other ocular pathologies [2]. Here we explore the modification of the tear content of the TAO patients using proteomics strategies and we proposed three new biomarker candidates, which could allow the early diagnosis of the disease and prompt action to prevent more severe stages. Moreover, our findings could also help to better understand the pathophysiology of the disease.

Total IgE and eotaxin (CCL11) contents in tears of patients suffering from seasonal allergic conjunctivitis.

BACKGROUND: To prospectively investigate patients with seasonal allergic conjunctivitis (SAC) during the pollen season and test associations between tears total IgE, eotaxin concentrations, and SAC severity. METHODS: Enrolled patients presented ocular symptoms and clinical signs of SAC at the time of presentation. Ocular itching, hyperaemia, chemosis, eyelid swelling, and tearing were scored, and the sum of these scores was defined as the clinical score. Conjunctival papillae were separately graded. We measured eotaxin concentration in tears by an enzyme-linked immunosorbent assay (ELISA) and total tear IgE by Lacrytest strip. RESULTS: Among thirty patients (30 eyes), 11 showed neither tear IgE nor tear eotaxin, while 15 out of 19 patients with positive IgE values presented a positive amount of eotaxin in their tears (Fisher's test: p < 0.001). The mean eotaxin concentration was 641 ± 154 (SEM) pg/ml. In patients with no amount of tear IgE, we observed a lower conjunctival papilla grade than in patients whose tears contained some amount of IgE (trend test: p = 0.032). In the 15 patients whose tear eotaxin concentration was null, tear IgE concentration was 5.3 ± 3.5 arbitrary units; in the other 15 patients whose eotaxin was positive, IgE reached 21 ± 4.3 arbitrary U (Mann-Whitney: p < 0.001). We measured 127 ± 47 pg/ml eotaxin in patients with no history of SAC but newly diagnosed as suffering from SAC, and 852 ± 218 pg/ml eotaxin in patients with a known SAC (p = 0.008). In contrast, tear IgE concentrations of both groups did not differ statistically significantly (p = 0.947). CONCLUSIONS: If IgE and eotaxin secreted in tears are major contributors in SAC pathogenesis, they however act at different steps of the process.

A new drug delivery system inhibits uveitis in an animal model after cataract surgery.

Cataract surgery is a common ocular surgical procedure consisting in the implantation of an artificial intraocular lens (IOL) to replace the ageing, dystrophic or damaged natural one. The management of postoperative ocular inflammation is a major challenge especially in the context of pre-existing uveitis. The association of the implanted IOL with a drug delivery system (DDS) allows the prolonged intraocular release of anti-inflammatory agents after surgery. Thus IOL-DDS represents an "all in one" strategy that simultaneously addresses both cataract and inflammation issues. Polymeric DDS loaded with two model anti-inflammatory drugs (triamcinolone acetonide (TA) and cyclosporine A (CsA)) were manufactured in a novel way and tested regarding their efficiency for the management of intraocular inflammation during the 3 months following surgery. The study involved an experimentally induced uveitis in rabbits. Experimental results showed that medicated DDS efficiently reduced ocular inflammation (decrease of protein concentration in aqueous humour, inflammatory cells in aqueous humour and clinical score). Additionally, more than 60% of the loading dose remained in the DDS at the end of the experiment, suggesting that the system could potentially cover longer inflammatory episodes. Thus, IOL-DDS were demonstrated to inhibit intraocular inflammation for at least 3 months after cataract surgery, representing a potential novel approach to cataract surgery in eyes with pre-existing uveitis.

Long-term close follow-up of chorioretinal lesions in presumed ocular tuberculosis.

PURPOSE: To evaluate the long-term outcome (up to 7 years) of presumed ocular tuberculosis (TB) when the therapeutic decision was based on WHO guidelines. METHODS: Twelve out of 654 new uveitic patients (1998-2004) presented with choroiditis and positive tuberculosis skin test (TST) (skin lesion diameter >15 mm). Therapy was administered according to WHO recommendations after ophthalmic and systemic investigation. The area size of ocular lesions at presentation and after therapy, measured on fluorescein and indocyanine green angiographies, was considered the primary outcome. Relapse of choroiditis was considered a secondary outcome. The T-SPOT TB test was performed when it became available. RESULTS: Visual acuity significantly improved after therapy (p=0.0357). The mean total surface of fluorescein lesions at entry was 44.8 ± 20.9 (arbitrary units) and decreased to 32.5 ± 16.9 after therapy (p=0.0165). The mean total surface of indocyanine green lesions at entry was 24.5 ± 13.3 and decreased to 10.8 ± 5.4 after therapy (p=0.0631). The T-SPOT TB revealed 2 false TST-positive results. The mean follow-up was 4.5 ± 1.5 years. Two relapses out of 10 confirmed ocular TB was observed after complete lesion healing, 2.5 years and 4.5 years after therapy, respectively. CONCLUSIONS: A decrease of ocular lesion mean size and a mean improvement of VA were observed after antituberculous therapy. Our long-term follow-up of chorioretinal lesions demonstrated relapse of ocular tuberculosis in 10% of patients with confirmed ocular TB, despite complete initial retinal scarring.

Trans-scleral diffusion of triamcinolone acetonide.

PURPOSE: To assess ex vivo human scleral permeability to triamcinolone acetonide (TA). METHODS: The experiments were carried out using scleral samples and a Franz-type vertical diffusion cell. A suspension containing TA was prepared and placed in the donor chamber. The concentration of TA in the receptor chamber was measured by high-performance liquid chromatography (HPLC) assay and expressed as a percentage relative to TA concentration dissolved in the donor chamber. Control experiments using a commercial TA suspension were performed. RESULTS: TA (+/-SEM) dissolved in the donor suspension was 10.69 +/- 1.28 microg/ml. The diffusion rate of TA varied from 30% after 1 day to 72% after 4 days, after which equilibrium was reached. The human scleral permeability coefficient (P(s) +/- SEM) was 1.47+/- 0.17 x 10(- 5) cm/s. CONCLUSIONS: TA crossed human sclera. The mean amount of drug retained in the sclera increased with time, 4 days being necessary to equilibrate the unidirectional flux. The TA permeability coefficient was comparable to that of other corticosteroids.

Increased eotaxin in tears of patients wearing contact lenses.

PURPOSE: Giant papillary conjunctivitis in patients wearing contact lenses occurs after intolerance and/or allergy to contact lenses. Eotaxin is a CC chemokine with a potent and specific chemotactic effect for eosinophils, which are involved in allergies. The purpose of this study is to measure the eotaxin levels in tears of patients wearing contact lenses and in normal subjects. Eotaxin levels were also correlated with the grade of giant papillary conjunctivitis. METHODS: Around 10 microL of tears were collected with glass capillaries in 16 patients wearing contact lenses and in 10 normal volunteers. Giant papillary conjunctivitis was graded from 0 to 4 by reference to standard slit-lamp photographs of the superior tarsal conjunctiva. Eotaxin concentration in tears was measured by ELISA using mouse anti-human eotaxin monoclonal antibodies. For the statistical analysis of the results, the paired Wilcoxon/Kruskal-Wallis test was used. RESULTS: The mean concentration of eotaxin was 2698+/-233 (SEM) pg/mL in patients wearing contact lenses and 1498+/-139 pg/mL in normal subjects. The difference was statistically significant (P=0.0004). The mean score of papilla grade was 1.75+/-0.19 in patients wearing contact lenses and 0.2+/-0.13 in normal subjects (P<0.0001). Papilla grade could be correlated to the eotaxin level in tears (R2=0.6562 and P<0.0001). CONCLUSION: An increase of eotaxin levels in tears was measured in patients wearing contact lenses. Eotaxin levels correlated with the severity of giant papillary conjunctivitis. These data suggest that eotaxin could play a role in papilla formation.

Eotaxin-1 (CCL11) up-regulation in tears during seasonal allergic conjunctivitis.

PURPOSE: To compare in-season eotaxin-1 levels in tears of patients suffering from seasonal allergic conjunctivitis (SAC) with (1) tears of normal subjects and (2) tears of SAC patients out of season. METHODS: Tears of 11 SAC patients and six control volunteers were collected during the pollen season. Tears of five SAC patients showing a strong sensitivity to grass pollen (skin-prick tests and specific serum IgE) were collected both in season and out of season. ELISA measured eotaxin-1 level. RESULTS: Eotaxin-1 concentration in tears of SAC patients [2,100+/-503 (SEM) pg/ml] and normal subjects (1,193+/-176 pg/ml) were significantly different (P=0.0049). Regarding allergic patients, the clinical score (sum of five allergic criteria) was significantly different in season and out of season (P=0.0043) as was also the case with eotaxin-1 concentration (P=0.024). CONCLUSIONS: The eotaxin-1 concentration in tears of patients showing hay fever could confirm a diagnosis of seasonal ocular allergy.