Influence of climate on clinical diagnostic dry eye tests: pilot study

Purpose. To analyze dry eye disease (DED) tests and their consistency in similar nonsymptomatic population samples living in two geographic locations with different climates (Continental vs. Atlantic). Methods. This is a pilot study including 14 nonsymptomatic residents from Valladolid (Continental climate, Spain) and 14 sex-matched and similarly aged residents from Braga (Atlantic climate, Portugal); they were assessed during the same season (spring) of two consecutive years. Phenol red thread test, conjunctival hyperemia, fluorescein tear breakup time, corneal and conjunctival staining, and Schirmer test were evaluated on three different consecutive visits. Reliability was assessed using the intraclass correlation coefficient and weighted kappa (J) coefficient for quantitative and ordinal variables, respectively. Results. Fourteen subjects were recruited in each city with a mean (TSD) age of 63.0 (T1.7) and 59.1 (T0.9) years (p = 0.08) in Valladolid and Braga, respectively. Intraclass correlation coefficient and J values of the tests performed were below 0.69 and 0.61, respectively, for both samples, thus showing moderate to poor reliability. Subsequently, comparisons were made between the results corresponding to the middle and higher outdoor relative humidity (RH) visit in each location as there were no differences in mean temperature (p Q 0.75) despite RH values significantly differing (p e 0.005). Significant (p e 0.05) differences were observed between Valladolid and Braga samples on tear breakup time (middle RH visit, 2.76 T 0.60 vs. 5.26 T 0.64 seconds; higher RH visit, 2.61 T 0.32 vs. 5.78 T 0.88 seconds) and corneal (middle RH, 0.64 T 0.17 vs. 0.14 T 0.10; higher RH, 0.60 T 0.22 vs. 0.0 T 0.0) and conjunctival staining (middle RH, 0.61 T 0.17 vs. 0.14 T 0.08; higher RH, 0.57 T 0.15 vs. 0.18 T 0.09). Conclusions. This pilot study provides initial evidence to support that DED test outcomes assessing the ocular surface integrity and tear stability are climate dependent. Future large-sample studies should support these outcomes also in DED patients. This knowledge is fundamental for multicenter clinical trials. Lack of consistency in diagnostic clinical tests for DED was also corroborated. (Optom Vis Sci 2015;92:e284Ye289)

End-of-day dryness, corneal sensitivity and blink rate in contact lens wearers

Purpose: To study the relationship among the variables intensity ofthe end-of-day (EOD) dryness, corneal sensitivity and blink rate in soft contact lens (CL) wearers. Methods: Thirty-eight soft CL wearers (25 women and 13 men; mean age 27.1 ± 7.2 years) were enrolled. EOD dryness was assessed using a scale of 0–5 (0, none to 5, very intense). Mechanical and thermal (heat and cold) sensitivity were measured using a Belmonte’s gas esthesiometer. The blink rate was recorded using a video camera while subjects were wearing a hydrogel CL and watching a film for 90 min in a controlled environmental chamber. Results: A significant inverse correlation was found between EOD dryness and mechanical sensitivity (r: −0.39; p = 0.02); however, there were no significant correlations between EOD dryness and thermal sensitivity. A significant (r: 0.56; p < 0.001) correlation also was observed between EOD dryness and blink rate, but no correlations were found between blink rate and mechanical or thermal sensitivity. Conclusions: CL wearers with higher corneal sensitivity to mechanical stimulation reported more EOD dryness with habitual CL wear. Moreover, subjects reporting more EOD dryness had an increased blink rates during wear of a standard CL type. The increased blink rate could act to improve the ocular surface environment and relieve symptoms

Evaluation of inflammatory response induced by different types of daily contact lenses

Dissertação de mestrado em Genética Molecular

Location of corneal epithelial stem cells Reply

The longstanding concept that corneal epithelial stem cells reside mainly in the limbus is supported by the absence of major corneal epithelial differentiation markers, that is, K3 and K12 keratins, in limbal basal cells (these markers are expressed, however, in corneal basal cells, thus distinguishing the mode of keratin expression in corneal epithelium from that of all other stratified epithelia), the centripetal migration of corneal epithelial cells, the exclusive location of slow-cycling cells in the limbal basal layer, the superior in vitro proliferative potential of limbal epithelial cells, and the transplanted limbal cells' ability to reconstitute corneal epithelium in vivo (reviewed in refs 1-4). Moreover, previous data indicate that corneal and conjunctival epithelia represent two separate cell lineages (reviewed in refs 1-4). Majo et al. suggested, however, that corneal and conjunctival epithelia are equipotent, and that identical oligopotent stem cells are present throughout the corneal, limbal and conjunctival epithelia. We point out here that these suggestions are inconsistent with many known growth, differentiation and cell migration properties of the anterior ocular epithelia.

In vivo characterisation of a novel water-soluble Cyclosporine A prodrug for the treatment of dry eye disease.

Cyclosporine A (CsA) has been demonstrated to be effective for the treatment of a variety of ophthalmological conditions, including ocular surface disorders such as the dry eye disease (DED). Since CsA is characterised by its low water solubility, the development of a topical ophthalmic formulation represents an interesting pharmaceutical question. In the present study, two different strategies to address this challenge were studied and compared: (i) a water-soluble CsA prodrug formulated within an aqueous solution and (ii) a CsA oil-in-water emulsion (Restasis, Allergan Inc., Irvine, CA). First, the prodrug formulation was shown to have an excellent ocular tolerance as well as no influence on the basal tear production; maintaining the ocular surface properties remained unchanged. Then, in order to allow in vivo investigations, a specific analytical method based on ultra high pressure liquid chromatography coupled with triple quadrupole mass spectrometer (UHPLC-MS/MS) was developed and optimised to quantify CsA in ocular tissues and fluids. The CsA ocular kinetics in lachrymal fluid for both formulations were found to be similar between 15 min and 48 h. The CsA ocular distribution study evidenced the ability of the prodrug formulation to penetrate into the eye, achieving therapeutically active CsA levels in tissues of both the anterior and posterior segments. In addition, the detailed analysis of the in vivo data using a bicompartmental model pointed out a higher bioavailability and lower elimination rate for CsA when it is generated from the prodrug than after direct application as an emulsion. The interesting in vivo properties displayed by the prodrug solution make it a safe and suitable option for the treatment of DED.

LRIG1 inhibits STAT3-dependent inflammation to maintain corneal homeostasis.

Corneal integrity and transparency are indispensable for good vision. Cornea homeostasis is entirely dependent upon corneal stem cells, which are required for complex wound-healing processes that restore corneal integrity following epithelial damage. Here, we found that leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1) is highly expressed in the human holoclone-type corneal epithelial stem cell population and sporadically expressed in the basal cells of ocular-surface epithelium. In murine models, LRIG1 regulated corneal epithelial cell fate during wound repair. Deletion of Lrig1 resulted in impaired stem cell recruitment following injury and promoted a cell-fate switch from transparent epithelium to keratinized skin-like epidermis, which led to corneal blindness. In addition, we determined that LRIG1 is a negative regulator of the STAT3-dependent inflammatory pathway. Inhibition of STAT3 in corneas of Lrig1-/- mice rescued pathological phenotypes and prevented corneal opacity. Additionally, transgenic mice that expressed a constitutively active form of STAT3 in the corneal epithelium had abnormal features, including corneal plaques and neovascularization similar to that found in Lrig1-/- mice. Bone marrow chimera experiments indicated that LRIG1 also coordinates the function of bone marrow-derived inflammatory cells. Together, our data indicate that LRIG1 orchestrates corneal-tissue transparency and cell fate during repair, and identify LRIG1 as a key regulator of tissue homeostasis.

A history of the renewal of the corneal epithelium : a 3D animation

Background: To compare the different schemes that have been proposed during the last thirteen years to explain the renewal of the corneal epithelium. Material and Methods:We analyzed all the data present in the literature to explain the renewal of the corneal epithelium in mammals. According to the schemes proposed in the literature we developed a 3D animation to facilitate the understanding of the different concepts. Results:Three different schemes have been proposed to explain the renewal of the corneal epithelium in mammals during the last thirteen years. 1950-1981: the corneal epithelium was thought being renewed by mitosis of cells located in the basal layer. At this time scientist were not talking about stem cells. 1981-1986 was the period of the "XYZ hypothesis" or the transdifferentiation paradigm. At this time the conjunctival epithelium renewed the corneal epithelium in a centripetal migration. 1986-2008: the limbal stem cell paradigm, there were no stem cells in the corneal epithelium, all the corneal stem cells were located in the limbus and renewed the central cornea after a migration of 6 to 7 mm of transient amplifying cells toward the centre of the cornea. 2008, epithelial stem cells were found in the central cornea in mammals (Nature, Majo et al. November 2008). Discussion:We thought that the renewal of the corneal epithelium was completely defined. According to the last results we published in Nature, the current paradigm will be revisited. The experiments we made were on animals and the final demonstration on human has still to be done. If we find the same results in human, a new paradigm will be define and will change the way we consider ocular surface therapy and reconstruction.

A novel cyclosporin a aqueous formulation for dry eye treatment: in vitro and in vivo evaluation.

PURPOSE: The aim of the present study was the in vitro and in vivo evaluation of a novel aqueous formulation based on polymeric micelles for the topical delivery of cyclosporine A for dry eye treatment. METHODS: In vitro experiments were carried out on primary rabbit corneal cells, which were characterized by immunocytochemistry using fluorescein-labeled lectin I/isolectin B4 for the endothelial cells and mouse monoclonal antibody to cytokeratin 3+12 for the epithelial ones. Living cells were incubated for 1 hour or 24 hours with a fluorescently labeled micelle formulation and analyzed by fluorescence microscopy. In vivo evaluations were done by Schirmer test, osmolarity measurement, CyA kinetics in tears, and CyA ocular distribution after topical instillation. A 0.05% CyA micelle formulation was compared to a marketed emulsion (Restasis). RESULTS: The in vitro experiments showed the internalization of micelles in the living cells. The Schirmer test and osmolarity measurements demonstrated that micelles did not alter the ocular surface properties. The evaluation of the tear fluid gave similar CyA kinetics values: AUC = 2339 ± 1032 min*μg/mL and 2321 ± 881.63; Cmax = 478 ± 111 μg/mL and 451 ± 74; half-life = 36 ± 9 min and 28 ± 9 for the micelle formulation and Restasis, respectively. The ocular distribution investigation revealed that the novel formulation delivered 1540 ± 400 ng CyA/g tissue to the cornea. CONCLUSIONS: The micelle formulation delivered active CyA into the cornea without evident negative influence on the ocular surface properties. This formulation could be applied for immune-related ocular surface diseases.

Oeil et environnement

Résumé L'oeil est exposé à de multiples facteurs environnementaux qui influent sur le développement visuel, le confort et la santé visuelle. Une exposition excessive à la lumière solaire peut causer des pathologies de la surface oculaire et de la rétine, mais une exposition insuffisante à la lumière du jour pourrait être incriminée dans l'apparition ou la progression de la myopie. Les lumières artificielles, LED en particulier, comportent un risque accru de phototoxicité rétinienne. L'augmentation importante de la prévalence des allergies oculaires pourrait être liée à la multiplication des polluants environnementaux, comme la fumée de tabac, les dérivés de combustion de carburants ou les phtalates. Enfin, certains compléments alimentaires peuvent jouer un rôle protecteur dans certains types d'atteintes oculaire, rétinienne en particulier. Abstract The eyes are exposed to multiple environmental factors, which affect visual development, comfort, and visual health. While overexposure to sunlight can cause ocular surface and retinal pathologies, insufficient exposure to daylight could significantly contribute to myopia progression. New artificial lights, namely LED, have a higher risk of retinal phototoxicity, and could alter ocular circadian rhythm. The significant increase of prevalence of ocular allergies could be caused by the proliferation of environmental polluting substances, like tobacco smoke, fuel combustion by-products, or phtalates, which are found in many types of plastics. Finally, some dietary supplements could play a protective role in certain types of ocular pathologies, namely retinal pathologies.

The health effects of sea buckthorn berries and oil

Sea buckthorn (Hippophaë) berries are ingredients of the Chinese traditional medicine. In addition to China, they are nowadays cultivated for food in several European countries, Russia, Canada, the USA, and Japan. Sea buckthorn berries are a rich source of flavonoids, mainly flavonol glycosides and proanthocyanidins. Depending on the genetic background, growth conditions, and ripeness of the berries, vitamin C concentrations up to over 1 g/100 ml juice, have been reported. Sea buckthorn berries contain inositols and methyl inositols, components of messenger molecules in humans. Sea buckthorn seed oil is rich in essential aplha-linolenic and linoleic acids, whereas the most abundant fatty acids in the berry oil are palmitoleic, palmitic and oleic acids. Other potentially beneficial lipophilic compounds of sea buckthorn seeds and berries include carotenoids, phytosterols, tocopherols and tocotrienols. The effects of sea buckthorn fractions on inflammation, platelet aggregation, oxidation injuries, the liver, skin and mucosa, among others, have been reported. The aim of the thesis work was to investigate the health effects of sea buckthorn berries and oil in humans. The physiological effects of sea buckthorn berries, berry components, and oil have mostly been studied in vitro and in animal models, leaving a demand for more clinical trials. In the first randomized, placebo-controlled trial of this thesis healthy adults consumed 28 g/day of sea buckthorn berries for three months. The main objective was to investigate the effects on the common cold. In addition, effects on other infections, inflammation and circulating lipid markers associated with cardiovascular disease risk were studied. In the second randomized, placebocontrolled trial participants reporting dry eye symptoms consumed 2 g/day of sea buckthorn oil from the seeds and berries for three months. The effects on symptoms and clinical signs of dry eye were monitored. In addition, the effects on circulating markers of inflammation and liver functions were analyzed. Sea buckthorn berries did not affect the common cold or other infections in healthy adults. However, a decrease in serum C-reactive protein was detected, indicating effects on inflammation. Fasting concentrations of serum flavonols, typical to sea buckthorn berry, increased without affecting the circulating total, HDL, LDL cholesterol, or triacylglycerol concentrations. Tear film hyperosmolarity and activation of inflammation at the ocular surface are among the core mechanisms of dry eye. Combined sea buckthorn berry and seed oil attenuated the rise in tear film osmolarity taking place during the cold season. It also positively affected some of the dry eye symptoms. Based on the tear film fatty acid analysis, the effects were not mediated through direct incorporation of sea buckthorn oil fatty acids to tear film lipids. It is likely that the fatty acids, carotenoids, tocopherols and tocotrienols of sea buckthorn oil affected the inflammation of the ocular surface, lacrimal and/or meibomian glands. The effects on the differentiation of meibomian gland cells are also possible. Sea buckthorn oil did not affect the serum concentrations of inflammation markers or liver enzymes investigated. In conclusion, this thesis work suggests positive effects of sea buckthorn berries and oil on inflammation and dry eye, respectively, in humans.

Ex vivo expansion of limbal stem cells is affected by substrate properties

Limbal epithelial stem cells play a key role in the maintenance and regulation of the corneal surface. Damage or destruction of these cells results in vascularisation and corneal opacity. Subsequent limbal stem cell transplantation requires an ex vivo expansion step and preserving cells in an undifferentiated state remains vital. In this report we seek to control the phenotype of limbal epithelial stem cells by the novel application of compressed collagen substrates. We have characterised the mechanical and surface properties of conventional collagen gels using shear rheology and scanning electron microscopy. In doing so, we provide evidence to show that compressive load can improve the stiffness of collagen substrates. In addition Western blotting and immunohistochemistry display increased cytokeratin 3 (CK3) protein expression relating to limbal epithelial cell differentiation on stiff collagen substrates. Such gels with an elastic modulus of 2900 Pa supported a significantly higher number of cells than less stiff collagen gels (3 Pa). These findings have substantial influence in the development of ocular surface constructs or experimental models particularly in the fields of stem cell research, tissue engineering and regenerative medicine.

Towards the use of hydrogels in the treatment of limbal stem cell deficiency

Corneal blindness caused by limbal stem cell deficiency (LSCD) is a prevailing disorder worldwide. Clinical outcomes for LSCD therapy using amniotic membrane (AM) are unpredictable. Hydrogels can eliminate limitations of standard therapy for LSCD, because they present all the advantages of AM (i.e. biocompatibility, inertness and a biodegradable structure) but unlike AM, they are structurally uniform and can be easily manipulated to alter mechanical and physical properties. Hydrogels can be delivered with minimum trauma to the ocular surface and do not require extensive serological screening before clinical application. The hydrogel structure is also amenable to modifications which direct stem cell fate. In this focussed review we highlight hydrogels as biomaterial substrates which may replace and/or complement AM in the treatment of LSCD.

In vivo study of the biocompatibility of a novel compressed collagen hydrogel scaffold for artificial corneas

The experiments were designed to evaluate the biocompatibility of a plastically compressed collagen scaffold (PCCS). The ultrastructure of the PCCS was observed via scanning electron microscopy. Twenty New Zealand white rabbits were randomly divided into experimental and control groups that received corneal pocket transplantation with PCCS and an amniotic membrane, respectively. And the contralateral eye of the implanted rabbit served as the normal group. On the 1st, 7th, 14th, 21st, 30th, 60th, 90th, and 120th postoperative day, the eyes were observed via a slit lamp. On the 120th postoperative day, the rabbit eyes were enucleated to examine the tissue compatibility of the implanted stroma. The PCCS was white and translucent. The scanning electron microscopy results showed that fibers within the PCCS were densely packed and evenly arranged. No edema, inflammation, or neovascularization was observed on ocular surface under a slit lamp and few lymphocytes were observed in the stroma of rabbit cornea after histological study. In conclusion, the PCCS has extremely high biocompatibility and is a promising corneal scaffold for an artificial cornea. (c) 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Differential effects of alpha-helical and beta-hairpin antimicrobial peptides against Acanthamoeba castellanii

In this work we evaluated the ability of different types of antimicrobial peptides to promote permeabilization and growth inhibition of Acanthamoeba castellanii trophozoites, which cause eye keratitis. We used cationic alpha-helical peptides P5 and a beta-hairpin amphipathic molecule (gomesin), of the spider Acanthoscurria gomesiana haemocytes. A. castellanii permeabilization was obtained after 1 h incubation with micromolar concentrations of both types of peptides. While permeabilization induced by gomesin increased with longer incubations, P5 permeabilization did not increase with time and occurred at doses that are more toxic for SIRC cells, P5, however, at doses below the critical dose used to kill rabbit corneal cells was quite effective in promoting growth inhibition. Similarly, P5 was more effective when serine protease inhibitor was added simultaneously to the permeabilization assay. High performance chromatography followed by mass spectrometry analysis confirmed that, in contrast to gomesin, P5 is hydrolysed by A. castellanii culture supernatants. We conclude that the use of antimicrobial peptides to treat A. castellanii infections requires the search of more specific peptides that are resistant to proteolysis.

Conjunctival effects of canine distemper virus-induced keratoconjunctivitis sicca

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)