Simone Eperon

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 μg/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 (Ps ± SEM) was 1.47± 0.17× 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 μL 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.

Inflammation in cataract surgery

Cataract surgery induces rupture of the blood–ocular barrier. Topical corticosteroids or NSAIDs have been demonstrated to be effective in the management of postoperative inflammation after cataract surgery. Endophthalmitis, toxic anterior segment syndrome or retained intraocular lens material may be responsible for increased postoperative inflammation. The choice of a less traumatic phacoemulsification technique, small incision, adequate infusion solution and implantation in the bag, may reduce postoperative inflammation. In severe uveitis, adequate control of inflammation should be achieved for at least 3 months and surgery should be performed when intraocular inflammation is brought under control, using systemic corticosteroids or immunosuppressive therapy as needed. In the presence of decreased visual acuity postoperatively, cystoid macular edema should be ruled out clinically, by either optical coherence tomography or fluorescein angiography. The recent development of biopolymers as drug-delivery systems has shown their efficacy in the management of intraocular inflammation after cataract surgery.

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.

Experimental uveitis can be maintained in rabbits for a period of six weeks after a safe sensitization method.

Abstract Purpose: New treatments against long-lasting uveitis need to be tested. Our aim was to develop a six-week model of uveitis in rabbits. Methods: Rabbits were presensitized with an s.c. injection of Mycobacterium tuberculosis H37RA emulsified with TiterMax® Gold adjuvant. Uveitis was induced at day 28 and 50, by intravitreal challenges of antigen suspension. Ocular inflammation was assessed till euthanasia at day 71 after s.c. injection of M. tuberculosis H37RA by: (a) the number of inflammatory cells in aqueous humor (AH); (b) the protein concentration in AH; (c) the clinical score (mean of conjunctival hyperaemia, conjunctival chemosis, oedema and secretion); (d) the microscopical score (mean presence of fibrin and synechiae, aqueous cell density and aqueous flare grade, as scored by slit lamp). Results: At the sites of presensitization injection, rabbits presented flat nodules which progressively vanished. The first challenge induced a significant increase in the four parameters (p < 0.05 the Wilcoxon/Kruskal–Wallis test). The AH contained 764 ± 82 cells/µl and 32 ± 0.77 mg protein/ml. During the following days, inflammatory parameters decreased slightly. The second intravitreal challenge increased inflammation (3564 ± 228 cells/µl AH and 31 ± 1 mg protein/ml), which remained at a high level for a longer period of time. Conclusion: We developed a model of long-term uveitis, which could be maintained in rabbits for at least six weeks. Such a model could be used to test the efficacy of either new drugs or various drug delivery systems intended to deliver active agents during a few months.

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.

Investigation of the global protein content from healthy human tears

ABSTRACT 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 ± 18) were collected using the Schirmers 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. Graphical abstract Summary of the experimental workflow followed in this study. GPF: gas‐phase fractionation mode; LTQ: linear trap quadrupole; IPG: immobilized pH gradient. DAVID: Database for Annotation, Visualization and Integrated Discovery; STRING: Search Tool for the Retrieval of Interacting Genes/Proteins; KEGG: Kyoto Encyclopedia of Genes and Genomes. Figure. No Caption available. HighlightsTear fluid contains a high variety of proteins.A lot of enzymes, more precisely hydrolases and transferases, were identified.Pathways involved in different diseases can be greatly mapped in tears.Combination of different studies is essential to establish a reliable tear proteome.