Claudia Yahalom

Expression and activation of toll-like receptor 3 and toll-like receptor 4 on human corneal epithelial and conjunctival fibroblasts

BackgroundToll-like receptors (TLRs) are recognized as important contributors to the initiation and modulation of the inflammatory response in the eye. This study investigated the precise expression patterns and functionality of TLRs in human corneal epithelial cells (HCE) and in conjunctival fibroblasts (HCF).MethodsThe cell surface expression of TLRs 2-4, TLR7 and TLR9 in HCE and HCF was examined by flow cytometry with or without stimulation with lipopolysaccharide (LPS) or polyinosinic:polycytidylic acid (poly I:C). The mRNA expression of the TLRs was determined by real-time PCR. The protein content levels of interleukin (IL)-6, IL-8, IL-1β and tumor necrosis factor-α (TNF-α) were measured in HCE and HCF using multiplex fluorescent bead immunoassay (FBI).ResultsThe surface expression of TLR3 and TLR4 was detected on both HCE and HCF. Following incubation with LPS, the percentage of HCE cells staining for TLR4 decreased from 10.18% to 0.62% (Pu2009<u20090.001). Incubation with poly I:C lowered the percentage of HCE cells positive for TLR3 from 10.44% to 2.84% (Pu2009<u20090.001). The mRNA expression of TLRs2, 4, 7 and 9 was detected in HCE only. Activation of HCE with LPS complex elicited protein secretion up to 4.51u2009±u20090.85-fold higher levels of IL-6 (Pu2009<u20090.05), 2.5u2009±u20090.36-fold IL-8 (Pu2009>u20090.05), 4.35u2009±u20091.12-fold IL-1β (Pu2009>u20090.05) and 29.35u2009±u20092.3-fold TNFα (Pu2009<u20090.05) compared to cells incubated in medium.ConclusionsHCF and HCE both express TLRs that respond to specific ligands by increasing cytokine expression. Following activation, the surface expression of TLR3 and TLR4 on HCE is decreased, thus creating a negative feedback loop, mitigating the effect of TLR activation.

Combined Occurrence of Autosomal Dominant Aniridia and Autosomal Recessive Albinism in Several Members of a Family

Abstract Purpose: To characterize clinical and genetic aspects of a family with a unique combination of two hereditary blinding eye diseases. Methods: Comprehensive eye examination of proband and family members. Molecular analyses of the TYR and PAX6 genes. Results: A young couple, both legally blind, requested genetic counselling regarding their ocular condition. The female was previously diagnosed with oculocutaneous albinism (OCA1A) and her spouse was diagnosed with Peters anomaly. A comprehensive clinical examination revealed that the female had OCA1A combined with signs of another ocular disease, showing some similarity to aniridia. A complete ocular examination of her family members revealed that her brother also suffered from the same combined phenotype, her father had typical OCA1A signs, and her mother and sister had aniridia-like phenotype, without clinical diagnosis until the time of presentation. Molecular analysis identified two compound heterozygous TYR mutations known to cause OCAIA and cosegregate with oculocutaneous albinism. In addition, we identified a novel heterozygous PAX6 mutation confirming the atypical aniridia phenotype. Conclusions: We report here a unique and rare clinical phenotype that is explained by the segregation of two severe inherited eye diseases. The clinical and genetic analysis in this family allowed them to receive accurate genetic counseling.

Nitric oxide secretion in human conjunctival fibroblasts is inhibited by alpha linolenic acid.

PurposeIt is known that both human conjunctival fibroblasts (HCF) and corneal epithelial (HCE) cells contribute to the inflammatory process in the ocular surface by releasing inflammatory cytokines. In addition, nitric oxide (NO) has an important role in inflammatory responses in the ocular surface. In the present study, we aimed to characterize the capacity of these cells to release nitric oxide in response to cytokines and Lipopolysaccharide (LPS), and show that Alpha-linoleic acid (ALA) inhibits these responses.MethodsHCF, HCE cells, peripheral blood mononuclear cells (PBMCs) and co-culture of HCF and PBMC were treated with different combinations of inflammatory inducers, including interleukin)IL- (6, tumor necrosis factors (TNF)-α, interferon (IFN)- γ and IL-1β and LPS. Nitrite levels were measured in cell supernatants with and without ALA by the Griess reaction test at 24, 48 and 72xa0h respectively. Expression of nitric oxide synthase 2 (NOS-2) was evaluated by real-time PCR.ResultsAll cytokine combinations had an inducible effect on nitrite secretion in HCF, PBMC and co-cultured PBMC and HCF, but not in HCE cells. Treatment with a combination of IL-6, LPS, TNF-α, IFN- γ and IL-1β induced the highest nitrite secretion (2.91 fold, Pu2009<u20090.01) as compared to cells incubated in medium alone. nitrite secretion was reduced by 38.9xa0% (Pu2009<u20090.05) after treatment with ALA alone. Co-culturing PBMC with HCF with and without ALA treatment demonstrated similar results in nitrite level as,compared to PBMC alone. In addition, ALA significantly decreased NOS-2 expression in HCF by 48.9xa0% (Pu2009<u20090. 001) after 72xa0h.ConclusionsThe decrease in nitrite release and inhibition of NOS-2 expression indicate that ALA may have an anti-inflammatory effect both on HCF and on peripheral immune cells. This indicates that ALA may serve as a potent anti-inflammatory agent in ocular surface inflammation.

Preimplantation genetic diagnosis as a strategy to prevent having a child born with an heritable eye disease

ABSTRACT Background: In developed countries, genetically inherited eye diseases are responsible for a high percentage of childhood visual impairment. We aim to report our experience using preimplantation genetic diagnostics (PGD) in order to avoid transmitting a genetic form of eye disease associated with childhood visual impairment and ocular cancer. Material and Methods: Retrospective case series of women who underwent in vitro fertilization (IVF) and PGD due to a familial history of inherited eye disease and/or ocular cancer, in order to avoid having a child affected with the known familial disease. Each family underwent genetic testing in order to identify the underlying disease-causing mutation. IVF and PGD treatment were performed; unaffected embryos were implanted in their respective mothers. Results: Thirty-five unrelated mothers underwent PGD, and the following hereditary conditions were identified in their families: albinism (10 families); retinitis pigmentosa (7 families); retinoblastoma (4 families); blue cone monochromatism, achromatopsia, and aniridia (2 families each); and Hermansky-Pudlak syndrome, Leber congenital amaurosis, Norrie disease, papillorenal syndrome, primary congenital cataract, congenital glaucoma, Usher syndrome type 1F, and microphthalmia with coloboma (1 family each). Following a total of 88 PGD cycles, 18 healthy (i.e., unaffected) children were born. Conclusions: Our findings underscore the importance an ophthalmologist plays in informing patients regarding the options now available for using prenatal and preimplantation genetic diagnosis to avoid having a child with a potentially devastating genetic form of eye disease or ocular cancer. This strategy is highly relevant, particularly given the limited options currently available for treating these conditions.

Mild aniridia phenotype: an under-recognized diagnosis of a severe inherited ocular disease

PurposeAniridia is a rare panocular disorder caused by mutations in the PAX6 gene and characterized mainly by iris hypoplasia. Here, we present six families with a history of low vision/blindness with a previously undiagnosed mild aniridia phenotype with minimal iris changes.MethodsRetrospective case series of patients diagnosed with a subtle aniridia phenotype characterized by minimal iris abnormalities, foveal hypoplasia, and an identified mutation in PAX6. Data collection from patient’s charts included ocular examination findings, visual acuity, refraction, and clinical pictures when available. Genetic analysis was performed by isolation of genomic DNA from peripheral blood. The main outcome was the identification of patients with mild aniridia harboring a PAX6 mutation.ResultsIn all six families, the phenotype included minimal corectopia and foveal hypoplasia; nystagmus was present in 10 out of 11 patients. A PAX6 mutation was identified in all six families; three of these mutations were identified previously, and three are novel mutations. All the mutations are located within the conventional 128-residue paired domain of PAX6.ConclusionsA mild form of aniridia should be considered in the differential diagnosis of patients with low vision associated with mild iris abnormalities, nystagmus, and foveal hypoplasia. To ensure an accurate diagnosis of aniridia, minimal pupillary changes and/or incipient keratopathy should be examined. The broad phenotypic heterogeneity among aniridia leads to the fact that eye care clinicians must have a high index of suspicion for the disease when seeing undiagnosed low vision patients, because proper diagnosis can improve management as well as facilitate genetic testing and counselling.