Stefano Barabino

Topical Omega-3 and Omega-6 Fatty Acids for Treatment of Dry Eye

OBJECTIVE To study the efficacy of topical application of alpha-linolenic acid (ALA) and linoleic acid (LA) for dry eye treatment. METHODS Formulations containing ALA, LA, combined ALA and LA, or vehicle alone, were applied to dry eyes induced in mice. Corneal fluorescein staining and the number and maturation of corneal CD11b(+) cells were determined by a masked observer in the different treatment groups. Real-time polymerase chain reaction was used to quantify expression of inflammatory cytokines in the cornea and conjunctiva. RESULTS Dry eye induction significantly increased corneal fluorescein staining; CD11b(+) cell number and major histocompatibility complex Class II expression; corneal IL-1alpha and tumor necrosis factor alpha (TNF-alpha) expression; and conjunctival IL-1alpha, TNF-alpha, interferon gamma, IL-2, IL-6, and IL-10 expression. Treatment with ALA significantly decreased corneal fluorescein staining compared with both vehicle and untreated controls. Additionally, ALA treatment was associated with a significant decrease in CD11b(+) cell number, expression of corneal IL-1alpha and TNF-alpha, and conjunctival TNF-alpha. CONCLUSIONS Topical ALA treatment led to a significant decrease in dry eye signs and inflammatory changes at both cellular and molecular levels. CLINICAL RELEVANCE Topical application of ALA omega-3 fatty acid may be a novel therapy to treat the clinical signs and inflammatory changes accompanying dry eye syndrome.

The International Workshop on Meibomian Gland Dysfunction: Report of the Subcommittee on Tear Film Lipids and Lipid-Protein Interactions in Health and Disease

Understanding the molecular composition (e.g., proteins and lipids) of the tear film (TF) and the contribution of the meibomian gland to the TF is critical in gaining knowledge about TF instabilities, dry eye syndromes, contact lens (CL) incompatibilities, and other eye diseases. Among its functions, the lipid layer of the TF slows evaporation of the aqueous component, preserves a clear optical surface, and forms a barrier to protect the eye from microbial agents and organic matter, such as dust and pollen.1 The TF contains a complex mixture of proteins, enzymes, lipids, mucins, and salts that allows the TF to perform its functions (Fig. 1). Researchers believe the outer lipid layer is 5 to 10 molecules thick and is composed primarily of wax and sterol esters, possibly intercalated with each other and with proteins rather than forming distinct repeating layers of molecules.2,3 Evidence from interferometric studies indicate that the TF lipid layer thickness ranges from 20 to 160 nm.4 If the size of a lipid molecule is approximately 2.2 nm (22 Å), then the calculated thickness for one layer would be 11 to 44 nm. The addition of polar and nonpolar layers would add to the lipid thickness, which indicates that the lipid component of the TF may be multiple layers thick or have other contributing sources to correspond with reported thickness measurements.5 Figure 1. A proposed model of the precorneal tear film showing the relationship and interaction of lipid-binding proteins and the outer lipid layer. While the signs and symptoms of TF instability are reasonably well characterized, we are only beginning to understand the specific molecular components of the TF and their relationship with disease and TF stability. The purpose of this review is to examine the meibomian glands contribution to TF lipids and lipid–protein interactions in health and disease.

Systemic linoleic and γ-linolenic acid therapy in dry eye syndrome with an inflammatory component

Purpose. To evaluate the efficacy and anti-inflammatory activity of systemic linoleic (LA) and &ggr;-linolenic acid (GLA), which decrease chronic inflammation in rheumatoid arthritis, on the ocular surface of patients with keratoconjunctivitis sicca. Methods. In a randomized clinical trial, 26 patients with aqueous-deficient keratoconjunctivitis sicca were consecutively selected from patients presenting to Department of Neurosciences, Ophthalmology and Genetics, University of Genoa. The diagnosis was based on dry eye symptom survey score, Schirmer-1 test values, positive vital staining with lissamine green, and fluorescein break-up time (FBUT). All patients had ocular surface inflammation based on HLA-DR expression, a major histocompatibility class II antigen, on epithelial bulbar conjunctiva samples. The subjects were randomly divided into two groups of 13 patients each. The study group received tablets containing LA (28.5 mg) and GLA (15 mg) twice daily for 45 days and used tears; the control group received a tear substitute and a placebo tablet for 45 days. Results. Statistically significant changes in symptoms (p < 0.005), lissamine green staining (p < 0.005), and ocular surface inflammation (p < 0.05) occurred in the study group compared with controls. HLA-DR expression varied from 58.5 ± 14.1% positive conjunctival cells to 41.3 ± 18.9% in the treated group and from 61.4 ± 21.9% to 58.0 ± 13.3% in the controls. No statistically significant difference between groups was found for FBUT and the Schirmer-1 test. Conclusions. Therapy with LA and GLA and tear substitutes reduces ocular surface inflammation and improves dry eye symptoms. Long-term studies are needed to confirm the role of this new therapy for keratoconjunctivitis sicca.

A multicentre, double-masked, randomized, controlled trial assessing the effect of oral supplementation of omega-3 and omega-6 fatty acids on a conjunctival inflammatory marker in dry eye patients

Purpose:  To determine whether oral supplementation with omega‐3 and omega‐6 fatty acids can reduce conjunctival epithelium expression of the inflammatory marker human leucocyte antigen‐DR (HLA‐DR) in patients with dry eye syndrome (DES).

Distribution of conjunctival HLA-DR expression and the pathogenesis of damage in early dry eyes

Purpose: To examine the expression of HLA-DR, a marker of inflammation, in the early stages of dry eye disease and to locate the appearance of this marker on specific areas of the bulbar conjunctiva. Methods: Dry eye patients were identified and their condition classified as mild (n = 16) or moderate (n = 16) based on Schirmer testing, vital staining, tear break-up time, and symptom questionnaire scores. Brush cytology was used to collect epithelial cells from the nasal, temporal, and superior conjunctivae of patients and age-matched controls. HLA-DR positive cells were detected by immunohistochemical staining and quantified. Results: Patients with moderate dry eye had the highest rate of conjunctival HLA-DR-positive cells, with significantly higher rates than controls regardless of which region of the conjunctiva was sampled (P < 0.01). The mild dry eye group had similar rates of HLA-DR-positive cells in the superior conjunctival region compared with controls. However, in the nasal and temporal regions, they displayed a significantly higher rate of HLA-DR-positive cells than controls (P < 0.01) and the nasal region showed a significant difference (P < 0.01) when compared with the temporal one. Some of these mild dry eyes had no vital staining. Conclusions: The HLA-DR expression pattern in mild and moderate dry eyes appears to reflect disease progression. Overexpression of HLA-DR in mild dry eyes showing no vital staining suggests that inflammation may be a primary cause of ocular surface damage. These data support the use of immunomodulatory drugs in the treatment of dry eye disease.

Role of amniotic membrane transplantation for conjunctival reconstruction in ocular-cicatricial pemphigoid

PURPOSE To evaluate the role and the effectiveness over time of amniotic membrane transplantation (AMT) as a first-step procedure to treat conjunctival reconstruction in late-stage ocular-cicatricial pemphigoid (OCP). DESIGN Prospective interventional noncomparative case series. PARTICIPANTS Nine eyes (9 patients) with advanced OCP. METHODS Preoperatively, the ocular surface conditions were evaluated by immunohistochemistry of conjunctival biopsy and impression cytology specimens. The amniotic membrane was obtained during cesarean section from women who were 39 weeks pregnant and seronegative for human immunodeficiency virus, hepatitis B and C, and syphilis; it was processed, histologically tested, and stored at -80 degrees C. After scar tissue was removed, the preserved amniotic membrane was placed over the cornea, the bulbar, and tarsal conjunctiva, and was secured with 8-0 Vicryl sutures to the conjunctival edges and the deep fornices with double-armed 6-0 silk sutures. In 2 cases a double layer of amniotic membrane was transplanted. All patients received immunosuppressive systemic therapy and preservative-free tear substitutes and steroids topically for at least 6 months. During follow-up (average, 48 weeks; range, 28-96 weeks), a new standardized method was used to evaluate the fornix depth, and impression cytology testing was performed and conjunctival inflammation recorded and used as parameters for monitoring disease activity. MAIN OUTCOME MEASURES Symblepharon, increased inferior fornix depth, presence of conjunctival goblet cells, and the degree of conjunctival inflammation. RESULTS The conjunctival surface was free from symblepharon in all subjects for the first 16 weeks. At the week 28 examination, a small area of symblepharon was present in four eyes (44.4%). The depth of the fornix was significantly (P < 0.0001, analysis of variance) improved at weeks 4, 16, and 28. The normal conjunctival epithelium with goblet cells was restored in 6 of 9 eyes (66.7%) at the week 4 examination and in 4 eyes (44.4%) at the week 28 examination. Conjunctival inflammation was clinically but not statistically reduced. The visual acuity improved in 5 subjects. CONCLUSIONS AMT can be a first-step procedure for ocular surface reconstruction in OCP, but its effectiveness deteriorates slightly over time.

Evidence for aerobic metabolism in retinal rod outer segment disks.

The disks of the vertebrate retinal rod Outer Segment (OS), devoid of mitochondria, are the site of visual transduction, a very energy demanding process. In a previous proteomic study we reported the expression of the respiratory chain complexes I-IV and the oxidative phosphorylation Complex V (F(1)F(0)-ATP synthase) in disks. In the present study, the functional localization of these proteins in disks was investigated by biochemical analyses, oxymetry, membrane potential measurements, and confocal laser scanning microscopy. Disk preparations, isolated by Ficoll flotation, were characterized for purity. An oxygen consumption, stimulated by NADH and Succinate and reverted by rotenone, antimycin A and KCN was measured in disks, either in coupled or uncoupled conditions. Rhodamine-123 fluorescence quenching kinetics showed the existence of a proton potential difference across the disk membranes. Citrate synthase activity was assayed and found enriched in disks with respect to ROS. ATP synthesis by disks (0.7 micromol ATP/min/mg), sensitive to the common mitochondrial ATP synthase inhibitors, would largely account for the rod ATP need in the light. Overall, data indicate that an oxidative phosphorylation occurs in rod OS, which do not contain mitochondria, thank to the presence of ectopically located mitochondrial proteins. These findings may provide important new insight into energy production in outer segments via aerobic metabolism and additional information about protein components in OS disk membranes.

The TFOS International Workshop on Contact Lens Discomfort: Report of the contact lens interactions with the ocular surface and adnexa subcommittee

Efron, N., Jones, L., Bron, A. J., Knop, E., Arita, R., Barabino, S., … Markoulli, M. (2013). The TFOS International Workshop on Contact Lens Discomfort: Report of the Contact Lens Interactions With the Ocular Surface and Adnexa Subcommittee. Investigative Opthalmology & Visual Science, 54(11), TFOS98. https://doi.org/10.1167/iovs.13-13187

Dry eye syndromes.

Over the past 20 years it has become clear that dry eye syndrome (DES) or keratoconjunctivitis sicca (KCS) is a complex multifactorial disease characterized by an immune and inflammatory process that affects the lacrimal glands and ocular surface. In this paradigm, inflammation is seen as both the cause and consequence of conjunctival and corneal cell damage. In this chapter, we identify the unique characteristics of the lacrimal gland, the role of epithelial cells, regulatory T cells, and cytokines in maintaining ocular surface homeostasis and tear secretion function. We analyze the factors inducing loss of the lacrimal gland homeostasis and its consequences, and in so doing hope to provide a picture of the role of the immune system in the pathophysiology of KCS and useful information to help understand the complexity of DES.

Immune response in the conjunctival epithelium of patients with dry eye

The aim of our project was to test the hypothesis that patients with dry eye have a significant degree of inflammation and lymphocyte infiltration in conjunctival epithelium by using flow cytometry analysis of cells stored in cell culture medium. Impression cytology specimens were collected in 15 normal subjects and 15 dry eye patients. Samples collected from the right eye were placed in Phosphate Buffered Saline containing 0.05% paraformaldehyde (PFA), and samples from the left eye in cell culture medium containing 10% foetal calf serum (FCS). Phenotypic analysis was performed on cells derived from 21 dry eye patients and 16 healthy controls. The cells collected in FCS were stained for the expression of CK19, CD3, CD4, CD8, CD56, CD19, CD20, CD14 and HLA-DR, and analyzed by flow cytometry. FCS samples contained a statistically increased number of cells when compared to PFA samples. No statistically significant differences were present in the number of CD45+CK19- cells, CD3+ and CD4+T cells, B and NK cells in dry eye patients compared to healthy controls. In the dry eye group there was a significant difference in the CD4/CD8 ratio respect to what observed in normal subjects, and an increased number of CD14+ cells. HLA-DR expression was increased only in CK19+ conjunctival epithelial cells of dry eye patients. This study indicates that immune cells isolated from the superficial layer of the conjunctiva may play a pivotal role in the pathogenesis of dry eye, and that a new method of preservation of impression cytology samples can enhance flow cytometry analysis of epithelial and immune cells of the conjunctiva.