Search

In the experimental autoimmune encephalomyelitis (EAE) mouse model of optic neuritis, we recently demonstrated that diet supplementation with a balanced mixture of fatty acids (FAs), including omega 3 and omega 6, efficiently limited inflammatory events in the retina and prevented retinal ganglion cell (RGC) death, although mechanisms underlying the efficacy of FAs were to be elucidated. Whether FAs effectiveness was accompanied by efficient rescue of demyelinating events in the optic nerve was also unresolved. Finally, the possibility that RGC rescue might result in ameliorated visual performance remained to be investigated. Here, the EAE model of optic neuritis was used to investigate mechanisms underlying the anti-inflammatory effects of FAs, including their potential efficacy on macrophage polarization. In addition, we determined how FAs-induced rescue of RGC degeneration was related to optic nerve histopathology by performing ultrastructural morphometric analysis with transmission electron microscopy. Finally, RGC rescue was correlated with visual performance by recording photopic electroretinogram, an efficient methodology to unravel the role of RGCs in the generation of electroretinographic waves. We conclude that the ameliorative effects of FAs were dependent on a predominant anti-inflammatory action including a role on promoting the shift of macrophages from the inflammatory M1 phenotype towards the anti-inflammatory M2 phenotype. This would finally result in restored optic nerve histopathology and ameliorated visual performance. These findings can now offer new perspectives for implementing our knowledge on the effectiveness of diet supplementation in counteracting optic neuritis and suggest the importance of FAs as possible adjuvants in therapies against inflammatory diseases of the eye.

Dry eye is the most prominent pathology among those involving the ocular surface: a decrease of the aqueous (less frequent) or the lipid (more frequent) component of the tear film is the cause of the diminished stability of tears that is observed in this pathology. Dry eye shows a clear distribution linked to both sex (being more frequent among women) and age (increasing with aging). Therefore, specific treatments taking into account the etiology of the disease would be desired. The role of lactoferrin and its functional mimetic lactobionic acid are reported here as a possible remedy for age-related dry eye.

Current knowledge of the benefits of nutrition supplements for eye pathologies is based largely on the use of appropriate animal models, together with defined dietary supplementation. Here, C57BL6 mice were subretinally injected with polyethylene glycol (PEG)-400, an established model of retinal degeneration with a dry age-related macular degeneration (AMD)-like phenotype, an eye pathology that lacks treatment. In response to PEG-400, markers of the complement system, angiogenesis, inflammation, gliosis, and macrophage infiltration were upregulated in both retinas and retinal pigment epithelium (RPE)/choroids, whereas dietary supplementation with a mixture based on fatty acids counteracted their upregulation. Major effects include a reduction of inflammation, in both retinas and RPE/choroids, and an inhibition of macrophage infiltration in the choroid, yet not in the retina, suggesting a targeted action through the choroidal vasculature. Histological analysis revealed a thinning of the outer nuclear layer (ONL), together with dysregulation of the epithelium layer in response to PEG-400. In addition, immunohistofluorescence demonstrated Muller cell gliosis and macrophage infiltration into subretinal tissues supporting the molecular findings. Reduced ONL thickness, gliosis, and macrophage infiltration were counteracted by the diet supplement. The present data suggest that fatty acids may represent a useful form of diet supplementation to prevent or limit the progression of dry AMD.

Preservatives are used in multi-dose ophthalmic topical medications in order to prevent contamination by bacteria and fungi. However, prolonged use of preserved eye drops, as it may happen in dry eye or glaucoma, may damage cells of the ocular surface. Therefore, an important goal is to find preservatives with low toxicity which are mild to host cells, still able to prevent drug contamination so to maintain their sterility and efficacy. Hence, aim of this study has been to compare the relative toxicity on a rabbit corneal cell line of a new preservative, made by the association of N-hydroxy-methyl-glycinate (NIG) with disodium-ethylene diamine tetra-acetate (EDTA), with other known and widely used eye-drops preservatives.Rabbit corneal cells (SIRC) were tested either in 96-well plates or in suspension culture. Treatments with preservatives (used at known bacteriostatic concentrations) included: benzalkonium chloride (BAK), polyquaternium-1 (PQ-1), sodium perborate (SP: NaBOAlmost no cell toxicity was evident for NIG and SP at either concentration (0.001% or 0.002%), while a low toxicity was observed for PQ-1 (62% at the highest dose at 120 hours). BAK, as expected, showed the highest toxicity (60-80% at 30 minutes, and over 90% from eight hours onward). EDTA 0.1% alone or in combination with NIG 0.002%, showed no toxicity at 24 hours, and even resulted in cell growth promotion (46% and 38%, respectively), after 48 hours of treatment.These data show that the new preservative NIG/EDTA, at doses known to have effective antimicrobial properties, has a very low toxicity on corneal cells, and so it can be safely used in multi-dose eye drops.

Purpose The activation of the urokinase-type plasminogen activator and its receptor system is associated with retinal diseases. Among peptide inhibitors of this system, UPARANT acts by preventing the onset of pathologic signs of neovascular ocular diseases. We investigated whether systemic UPARANT may act in a therapeutic regimen by suppressing the retinal damage that characterizes diabetic retinopathy using a rat model of streptozotocin-induced diabetes. Methods In healthy rats, plasma, eye, and retina concentrations of UPARANT were evaluated by mass spectrometry. In rat models of streptozotocin-induced diabetes, the appearance of diabetic retinopathy was assessed by electroretinogram (ERG). UPARANT was then administered at different dosages and daily regimens. ERG recording, Evans blue perfusion, and real-time PCR were used to evaluate UPARANT efficacy. UPARANT safety was also determined. Results UPARANT was found in plasma, eye, and retina soon after its administration and remained detectable after 24 hours. Between the 4th and the 5th week after diabetes onset, UPARANT at 8 mg/kg (daily for 5 days) was effective in recovering dysfunctional ERG. Three-day treatments at 8 mg/kg or a half dose for 5 days were ineffective. ERG recovery lasted approximately 2 weeks. ERG recovery was accompanied by restored blood-retinal barrier integrity and inhibition of inflammatory and angiogenic responses. UPARANT showed a safety profile. Conclusions These data suggest that targeting the urokinase-type plasminogen activator and its receptor system by systemic UPARANT is a potential therapeutic approach for the treatment of early diabetic retinopathy, thus providing a potential alternative approach to delay disease progression in humans.

The mechanisms underlying cutaneous melanogenesis have been widely studied; however, very little is known about uveal melanogenesis. Melanin is normally produced by uveal melanocytes and gives the color to the iris. A derangement from this normal production may occur, for instance, by iatrogenic events, such as glaucoma therapy with prostaglandins that may enhance cutaneous and iris pigmentation. In this study, we investigated the mechanisms that regulate uveal melanogenesis in human uveal melanoma cells (92.1) and murine cutaneous melanoma cells (B16-F1). In the first part of the study, we compared the effects of known cutaneous pigmenting agents on the B16-F1 and 92.1 cells, showing an opposite response of the two cell lines. Subsequently, using argan oil, a known depigmenting agent for murine cutaneous melanoma cells, on 92.1 cells, we found that in these cells, it also functioned as an inhibitor of melanogenesis and tyrosinase expression. From a molecular perspective, treatment of the 92.1 cells with argan oil decreased melanogenesis-associated transcription factor (MITF) gene expression by inducing MITF phosphorylation at Ser73, thus leading to MITF ubiquitination and disposal. It also led to the downregulation of the extracellular signal-regulated kinase (ERK)1/2 and Akt pathways, also known to be involved in cutaneous melanogenesis, although with an opposing function. Taken together, our data indicate that: ⅰ) some differences exist in the regulation of melanogenesis between cutaneous and uveal melanoma cells; and ⅱ) argan oil exerts a depigmenting effect on 92.1 cells through its action on the ERK1/2 and Akt pathways.

To investigate the effects of gabapentin, a structural analog of γ-amino butyric acid (GABA), on the inflammatory response of lipopolysaccharide (LPS)-stimulated rabbit corneal cells (SIRC) and on endotoxin-induced uveitis (EIU) in rabbits. We investigated the LPS-induced expression of several inflammatory mediators, such as TNF-α, IL-1β, cPLA2, COX-2, and PGE2 in the SIRC cells with or without gabapentin treatment. Gabapentin treatment significantly (p < 0.05) attenuated cytokines production, cPLA2 activation, COX-2 expression, and PGE2 levels in SIRC. EIU was induced by an intraocular injection of 0.1 μg of LPS in albino rabbit eye. After 7 and 24 h from LPS injection clinical signs of ocular inflammation were examined by slit lamp with or without topical treatment of 0.5% gabapentin. Tears, aqueous, cornea, conjunctiva, and iris-ciliary body were collected and inflammatory biomarkers assessed. Topical treatment with gabapentin significantly (p < 0.05) reduced clinical signs and biomarkers of inflammation compared with the LPS group both at 7 and 24 h. In conclusion, the results generated in the present study suggest that ophthalmic formulation based on gabapentin may be useful in the treatment of inflammatory conditions associated to ocular pain such as uveitis, and that clinical studies to evaluate this possibility may be warranted.

Glaucoma is a multifactorial blinding disease with a major inflammatory component ultimately leading to apoptotic retinal ganglion cell (RGC) death. Pharmacological treatments lowering intraocular pressure can help slow or prevent vision loss although the damage caused by glaucoma cannot be reversed. Recently, nutritional approaches have been evaluated for their efficacy in preventing degenerative events in the retina although mechanisms underlying their effectiveness remain to be elucidated. Here, we evaluated the efficacy of a diet supplement consisting of forskolin, homotaurine, spearmint extract, and vitamins of the B group in counteracting retinal dysfunction in a mouse model of optic nerve crush (ONC) used as an in vivo model of glaucoma. After demonstrating that ONC did not affect retinal vasculature by fluorescein angiography, we determined the effect of the diet supplement on the photopic negative response (PhNR) whose amplitude is strictly related to RGC integrity and is therefore drastically reduced in concomitance with RGC death. We found that the diet supplementation prevents the reduction of PhNR amplitude (p &lt, 0.001) and concomitantly counteracts RGC death, as in supplemented mice, RGC number assessed immunohistochemically is significantly higher than that in non-supplemented animals (p &lt, 0.01). Major determinants of the protective efficacy of the compound are due to a reduction of ONC-associated cytokine secretion leading to decreased levels of apoptotic markers that in supplemented mice are significantly lower than in non-supplemented animals (p &lt, 0.001), ultimately causing RGC survival and ameliorated visual dysfunction. Overall, our data suggest that the above association of compounds plays a neuroprotective role in this mouse model of glaucoma thus offering a new perspective in inflammation-associated neurodegenerative diseases of the inner retina.

The Joslin Medalist Study characterized people affected with type 1 diabetes for 50 years or longer. More than 35% of these individuals exhibit no to mild diabetic retinopathy (DR), independent of glycemic control, suggesting the presence of endogenous protective factors against DR in a subpopulation of patients. Proteomic analysis of retina and vitreous identified retinol binding protein 3 (RBP3), a retinol transport protein secreted mainly by the photoreceptors, as elevated in Medalist patients protected from advanced DR. Mass spectrometry and protein expression analysis identified an inverse association between vitreous RBP3 concentration and DR severity. Intravitreal injection and photoreceptor-specific overexpression of RBP3 in rodents inhibited the detrimental effects of vascular endothelial growth factor (VEGF). Mechanistically, our results showed that recombinant RBP3 exerted the therapeutic effects by binding and inhibiting VEGF receptor tyrosine phosphorylation. In addition, by binding to glucose transporter 1 (GLUT1) and decreasing glucose uptake, RBP3 blocked the detrimental effects of hyperglycemia in inducing inflammatory cytokines in retinal endothelial and Müller cells. Elevated expression of photoreceptor-secreted RBP3 may have a role in protection against the progression of DR due to hyperglycemia by inhibiting glucose uptake via GLUT1 and decreasing the expression of inflammatory cytokines and VEGF.

To investigate the effects of gabapentin, a structural analog of γ-amino butyric acid (GABA), on the inflammatory response of lipopolysaccharide (LPS)-stimulated rabbit corneal cells (SIRC) and on endotoxin-induced uveitis (EIU) in rabbits. We investigated the LPS-induced expression of several inflammatory mediators, such as TNF-α, IL-1β, cPLA2, COX-2, and PGE2 in the SIRC cells with or without gabapentin treatment. Gabapentin treatment significantly (p < 0.05) attenuated cytokines production, cPLA2 activation, COX-2 expression, and PGE2 levels in SIRC. EIU was induced by an intraocular injection of 0.1 μg of LPS in albino rabbit eye. After 7 and 24 h from LPS injection clinical signs of ocular inflammation were examined by slit lamp with or without topical treatment of 0.5% gabapentin. Tears, aqueous, cornea, conjunctiva, and iris-ciliary body were collected and inflammatory biomarkers assessed. Topical treatment with gabapentin significantly (p < 0.05) reduced clinical signs and biomarkers of inflammation compared with the LPS group both at 7 and 24 h. In conclusion, the results generated in the present study suggest that ophthalmic formulation based on gabapentin may be useful in the treatment of inflammatory conditions associated to ocular pain such as uveitis, and that clinical studies to evaluate this possibility may be warranted.

PURPOSE: To report short-term results of pulsed ciliary muscle electrostimulation to improve near vision, likely through restoring accommodation in patients with emmetropic presbyopia. METHODS: In a prospective non-randomized trial, 27 patients from 40 to 51 years old were treated and 13 age- and refraction-matched individuals served as untreated controls. All patients had emmetropia and needed near sphere add between +0.75 and +1.50 diopters. The protocol included four sessions (one every 2 weeks within a 2-month period) of bilateral pulsed (2 sec on; 6 sec off) micro-electrostimulation with 26 mA for 8 minutes, using a commercially available medical device. The uncorrected distance visual acuity (UDVA) (logMAR) for each eye, uncorrected near (40 cm) visual acuity in each eye (UNVA) and with both eyes (UNVA OU) (logMAR), and reading speed (number of words read per minute at 40 cm) were measured preoperatively and 2 weeks after each session. Overall satisfaction (0 to 4 scale) was assessed 2 weeks after the last session. RESULTS: UDVA did not change and no adverse events were noted in either group. Bilateral and monocular UNVA and reading speed were stable in the control group, whereas they continuously improved in the treated group (Friedman, P < .00001). Post-hoc significant differences were found for monocular and binocular UNVA after the second treatment and after the first treatment considering words read per minute ( P < .001). One patient (3.7%) was not satisfied and 18 patients (66.7%) were very satisfied (score of 4). Average satisfaction score was 3 (satisfied). CONCLUSIONS: Ciliary muscle contraction to restore accommodation was safe and improved the short-term accommodative ability of patients with early emmetropic presbyopia. [ J Refract Surg. 2017;33(9):578–583.]

PDR is characterized by neovascularization and a persistent grade of inflammation. N-formyl peptide receptors (FPRs) belong to a transmembrane G protein-coupled receptor superfamily able to modulate angiogenic and inflammatory responses. However, their involvement in PDR remains to be elucidated. Here, we examined FPR expression in human umbilical vein endothelial cells (HUVECs). We further assessed the capacity of vitreous fluid obtained from PDR patients after pars plana vitrectomy to induce pro-angiogenic/pro-inflammatory responses in endothelium and the contribution of FPR inhibitors to abrogate these responses.

Spontaneously diabetic Torii (SDT) rats are an inbred strain of rats with a non-obese type 2 diabetes mellitus that were isolated from an outbred colony of Sprague–Dawley (SD) rats. They develop diabetes mellitus at about 20 weeks of age and, about 24 weeks later, are characterized by alterations of electroretinogram (ERG) and blood retinal barrier (BRB) dysfunction followed by later proliferative neovascularization. SDT rats are a useful model to investigate possible therapeutical approaches that may counteract the pathological signs of diabetic retinopathy (DR). The urokinase-type plasminogen activator (uPA)/uPA receptor (uPAR) system is up-regulated in rat models of DR and treatments downregulating this system may have some effects. We tested the possibility that systemic administration of UPARANT, a highly resistant peptide inhibitor of the uPA/uPAR system, might prevent visual dysfunction and BRB leakage in SDT rats.

AIMS/HYPOTHESIS: Angiogenesis and inflammation characterise proliferative diabetic retinopathy (PDR), a major complication of diabetes mellitus. However, the impact of inflammation on the pathogenesis of PDR neovascularisation has not been elucidated. Here, we assessed the capacity of PDR vitreous fluid to induce pro-angiogenic/proinflammatory responses in endothelium and the contribution of the inflammation-related pattern recognition N-formyl peptide receptors (FPRs) in mediating these responses. METHODS: Pooled and individual pars plana vitrectomy-derived PDR vitreous fluid ('PDR vitreous') samples were assessed in endothelial cell proliferation, motility, sprouting and morphogenesis assays, and for the capacity to induce proinflammatory transcription factor activation, reactive oxygen species production, intercellular junction disruption and leucocyte-adhesion molecule upregulation in these cells. In vivo, the pro-angiogenic/proinflammatory activity of PDR vitreous was tested in murine Matrigel plug and chick embryo chorioallantoic membrane (CAM) assays. Finally, the FPR inhibitors Boc-Phe-Leu-Phe-Leu-Phe (Boc-FLFLF) and Ac-L-Arg-Aib-L-Arg-L-Cα(Me)Phe-NH2 tetrapeptide (UPARANT) were evaluated for their capacity to affect the biological responses elicited by PDR vitreous. RESULTS: PDR vitreous activates a pro-angiogenic/proinflammatory phenotype in endothelial cells. Accordingly, PDR vitreous triggers a potent angiogenic/inflammatory response in vivo. Notably, the different capacity of individual PDR vitreous samples to induce neovessel formation in the CAM correlates with their ability to recruit infiltrating CD45+ cells. Finally, the FPR inhibitor Boc-FLFLF and the novel FPR antagonist UPARANT inhibit neovessel formation and inflammatory responses triggered by PDR vitreous in the CAM assay. CONCLUSIONS/INTERPRETATION: This study provides evidence that inflammation mediates the angiogenic activity of PDR vitreous and paves the way for the development of FPR-targeting anti-inflammatory/anti-angiogenic approaches for PDR therapy.

This study assessed the effect of a systemic oral treatment with antioxidants (AOs) in preretinopathic diabetes (PRD) patients, through the evaluation of oxidative stress in plasma and changes in the full-field electroretinogram (ERG).Thirty-two PRD subjects with good metabolic control were recruited. Patients were randomized in two groups, one of which received oral AO treatment with α-lipoic acid at 400 mg/day in association with genistein and vitamins, whereas the other group received a placebo. Free radicals and the AO barrier were evaluated in plasma with the Free Radical Analytical System 4 instrument (HD srl, Parma, Italy), and the same day the electrophysiological response was measured by ERG. These analyses were performed at enrollment and after 30 days of treatment.Statistically significant increases of plasma AO levels and ERG oscillatory potential values were observed in the group treated with AO, but not in the control group.Results of this preliminary study suggest that an oral treatment with AOs in PRD subjects may have a protective effect on retinal cells, as detected by ERG analysis, through the strengthening of the plasma AO barrier.

PURPOSE A mouse model of age-related macular degeneration (AMD) was used to investigate the anti-angiogenic and anti-inflammatory role of UPARANT in laser-induced choroidal neovascularization (CNV). METHODS Choroidal neovascularization was induced by laser photocoagulation, and UPARANT was intravitreally injected. Some experiments were also performed after either intravitreal injection of anti-VEGF drugs or systemic administration of UPARANT. Immunohistochemistry using CD31 antibodies was used to evaluate the area of CNV. Evans blue dye extravasation was quantitatively assessed. Transcripts of markers of outer blood retinal barrier were measured by quantitative RT-PCR, also used to evaluate angiogenesis and inflammation markers. Western blot was used to determine levels of transcription factors encoding genes involved in angiogenesis and inflammation. Levels of urokinase-type plasminogen activator (uPA), its receptor (uPAR), and formyl peptide receptors (FPRs) were determined at the transcript and the protein level. RESULTS Intravitreal UPARANT reduced the CNV area and the leakage from the choroid. The uPA/uPAR/FPR system was upregulated in CNV, but was not influenced by UPARANT. UPARANT recovered laser-induced upregulation of transcription factors encoding angiogenic and inflammatory markers. Accordingly, angiogenic and inflammatory factors were also reduced. UPARANT as compared to anti-VEGF drugs displayed similar effects on CNV area. CONCLUSIONS UPARANT mitigates laser-induced CNV by inhibiting angiogenesis and inflammation through an action on transcription factors encoding angiogenesis and inflammatory genes. The finding that UPARANT is effective against CNV may help to establish uPAR and its membrane partners as putative targets in the treatment of AMD.

Treatment of retinal neovascular diseases by anti-VEGF based therapies has two major flaws: one third of patients respond poorly, and chronic depletion of VEGF may have neurotoxic effects. We investigated in experimental rodent models of neovascular diseases the anti-angiogenic effects of a new synthetic molecule (UPARANT) designed to inhibit endothelial cells response to a wide variety of angiogenic triggers.

Purpose To investigate the molecular mechanisms of the antiangiogenic activity of UPARANT, an antagonist of the urokinase-type plasminogen activator receptor (uPAR), on primary human retinal endothelial cells (HREC) as a model of in vitro angiogenesis. Methods The antiangiogenic activity of UPARANT was evaluated on endothelial cell migration, invasion, and tube formation. Human REC were further analyzed for viability, transendothelial electrical resistance (TEER), and tight junction (TJ) expression at the protein and mRNA levels. Vascular endothelial growth factor-related signaling molecules were also analyzed by Western and northern blots. Results UPARANT inhibited in a dose-dependent fashion HREC motility, invasion, and tube formation stimulated by VEGF-A, in a range of doses (1-100 nM) that had no effect on cell viability and proliferation. UPARANT also prevented the loss of permeability induced by VEGF-A, restoring normal TEER values and TJ protein expression. At the molecular level, UPARANT inhibited VEGFR-2 and STAT3 phosphorylation, thus decreasing VEGF and hypoxia-inducible factor 1-alpha expression, finally resulting in decreased activation of MEK/ERK, JNK, p38, and AKT signaling proteins. Conclusions These findings indicate that UPARANT exerts its antiangiogenic effects through the inhibition of the downstream signaling activated by angiogenic factors such as VEGF-A.

Autophagy is an important homeostatic cellular process encompassing a number of consecutive steps indispensable for degrading and recycling cytoplasmic materials. Basically autophagy is an adaptive response that under stressful conditions guarantees the physiological turnover of senescent and impaired organelles and, thus, controls cell fate by various cross-talk signals. Diabetic retinopathy (DR) is a serious microvascular complication of diabetes and accounts for 5% of all blindness. Although, various metabolic disorders have been linked with the onset of DR, due to the complex character of this multi-factorial disease, a connection between any particular defect and DR becomes speculative. Diabetes increases inflammation, advanced glycation end products (AGEs) and oxidative stress in the retina and its capillary cells. Particularly, a great number of evidences suggest a mutual connection between oxidative stress and other major metabolic abnormalities implicated in the development of DR. In addition, the intricate networks between autophagy and apoptosis establish the degree of cellular apoptosis and the progression of DR. Growing data underline the crucial role of reactive oxygen species (ROS) in the activation of autophagy. Depending on their delicate balance both redox signaling and autophagy, being detrimental or beneficial, retain opposing effects. The molecular mechanisms of autophagy are very complex and involve many signaling pathways cooperating at various steps. This review summarizes recent advances of the possible molecular mechanisms in autophagic process that are involved in pathophysiology of DR. In-depth analysis on the molecular mechanisms leading to autophagy in the retinal pigment epithelial (RPE) will be helpful to plan new therapies aimed at preventing or improving the progression of DR.

Aim of the present study is to extend our previous observations on a model of primary epithelial cell culture obtained from bovine conjunctiva, and analyse the maintenance of the conjunctival phenotype, relative to cytokeratin (CK) expression, through extended periods of cultivation under different conditions. Conjunctival epithelial cells were grown in transwell filters, and cultured either under liquid covered (LC), or air-interface (AI) conditions. The physiological state of the cells was monitored daily by measurement of the trans-epithelial electrical resistance (TEER). Analysis of cytokeratin expression was then carried out at different time points (up until 14 days), and compared to the original profile of the conjunctival tissue in order to assess deviations from the primitive phenotype. Immunodetection studies, carried out by both western immunoblot and immunofluorescence analyses, revealed constant expression of the pan-epithelial marker AE3 (recognizing basic type cytokeratins), confirming the epithelial nature of the culture. Other cytokeratins characteristic of non-keratinized stratified epithelia (CK4 and CK13) were absent in corneal tissue, while in conjunctival epithelial cells were more expressed under AI than under LC culture conditions. Expression of CK12, a specific marker of corneal tissue, revealed by the antibody AE5, was never observed in conjunctival epithelial cells. These results indicate that the conjunctival phenotype is conserved during extended periods of culturing, making this system a reliable substitute of conjunctival tissue for pharmaceutical analyses.

Epigallocatechin-gallate (EGCg), the major catechin present in green tea extracts, has been shown to have several antibacterial activities, limiting bacterial growth and invasion and acting in synergy with β-lactam antibiotics. In this article, we report that EGCg at doses half and below its calculated MIC of 100 μg/ml, is able to reverse tetracycline resistance in staphylococcal isolates expressing the specific efflux pump Tet(K) and appears to improve the MICs of tetracycline for susceptible staphylococcal isolates as well. The visible effect of EGCg is an increased accumulation of tetracycline inside bacterial cells. This effect is likely due to the inhibition of pump activity, and it is evident not only for Tet(K) pumps but also for efflux pumps of a different class [Tet(B)]. In summary, our data indicate that the observed dramatic enhancement by EGCg of tetracycline activity for resistant staphylococcal isolates is caused by impairment of tetracycline efflux pump activity and increased intracellular retention of the drug, suggesting a possible use of EGCg as an adjuvant in antibacterial therapy.

The objective of this paper is to assess the gelatinase production by some ocular pathogenic bacterial strains, and evaluate the ability of (-)epigallocatechin-3-gallate (EGCg) to inhibit this gelatinase activity and thus limit bacterial invasion. The effect of EGCg on bacterial gelatinase activity was tested by classic zymography methods, while its effect on bacterial invasion was evaluated through the ability of growing bacteria to liquefy and thus penetrate a semisolid gelatine substrate. It was found that EGCg inhibits bacterial gelatinases with an IC(50) of about 0.2 mM, and limits invasion of gelatinase-positive bacteria at concentrations above 2 mM. These results show for the first time that EGCg, as well as having direct antibacterial activity, can also inhibit bacterial gelatinases, thus limiting their invasion on gelatine. Possible use of EGCg is thus suggested as an adjuvant in antibacterial chemotherapy.

Purpose Pharmacologic control of neovascularization is a promising approach for the treatment of retinal angiogenesis. UPARANT, an inhibitor of the urokinase-type plasminogen activator receptor (uPAR), inhibits VEGF-driven angiogenesis in vitro and in vivo. This study investigates for the first time the effectiveness of UPARANT in counteracting pathologic neovascularization in the retina. Methods Murine retinal fragments and a mouse model of oxygen-induced retinopathy (OIR) were used. In mice with OIR, UPARANT-treated retinas were analyzed for avascular area and neovascular tuft formation. Levels of transcription and proangiogenic factors were determined. UPARANT effects on the blood-retinal barrier (BRB), visual function, retinal cytoarchitecture, and inflammatory markers were also assessed. Human umbilical vein endothelial cells (HUVECs) and chick embryo chorioallantoic membrane (CAM) in which angiogenesis was induced by the vitreous fluid from patients with proliferative diabetic retinopathy (PDR) were also used. Results UPARANT reduced VEGF-induced angiogenesis in retinal fragments. In mice with OIR, UPARANT decreased neovascular response, VEGF, and VEGF receptor-2 activity. Transcription factors regulating VEGF expression were also reduced. UPARANT restored BRB integrity, recovered visual loss, and reduced levels of inflammatory markers. Restored electroretinogram does not involve any rescue in the retinal cytoarchitecture. Finally, UPARANT blocked PDR vitreous fluid-induced angiogenesis in HUVEC and CAM assays. Conclusions The finding that UPARANT is effective against neovascularization may help to establish uPAR as a target in the treatment of proliferative retinopathies. The potential application of UPARANT in retinal diseases is further supported by UPARANT capacity to counteract the angiogenic activity of PDR vitreous fluid.

Objective: To study the neovascularization in regenerating and proliferating corneal cells following a standard alkali injury in rabbit eye. Methods: Three and six weeks after the creation of an alkali burn in the center of the right cornea of six albino rabbits, the animals were killed and histological sections from the cornea of both eyes were stained, photographed and studied for a possible formation of a neovascularization. The photographs were examined using the Quantimet image analyzer (Leica) and statistical analysis of the data was performed. Results: Sections of the injured cornea showed the formation of neovessels in the epithelial and superficial stromal layers. The neovascularization is present after 3 weeks of the corneal injury. After 6 weeks from the corneal alkali burn, neovessels are increased. Conclusions: There is a growing body of evidence suggesting that vascular abnormalities may play a crucial role in several ocular diseases. To improve our knowledge of the vascular involvement in these conditions, there is a need for a non-invasive imaging modality capable of assessing microcirculation within ocular tissue beds both in vitro and in vivo. This study shows that ultra-high sensitive optical microangiography, associated with other experimental techniques, is an adequate technique to visualize the eye surface microcirculations and to quantify microvascular vessel density under both normal and physio-pathological conditions.

Purpose Agomelatine is an agonist of melatonin that is used in the treatment of major depressive disorders. It has also shown an ability to decrease IOP in experiment animals and in normal human subjects. This pilot study addresses for the first time agomelatine effects on the IOP of patients affected by POAG. Methods Ten patients affected by hypertensive POAG treated by multiple hypotensive topical drugs and under further treatment with agomelatine (25 mg day−1 per os) for psychiatric problems, were enrolled. IOP tonometric values were measured at enrolment and after 15 and 30 days of agomelatine supplementation. Results Agomelatine given orally showed a significant hypotonising effect, stably decreasing IOP by roughly 30% of the enrolment value after 15 and 30 days of treatment. Conclusions The hypotonising effect of oral systemic agomelatine at 25 mg day−1 was able to further decrease IOP in both eyes of all enrolled POAG patients in which multiple drug treatment with anti-glaucoma eye drops had no further effect.

BACKGROUND Up to 50% of patients with uveal melanoma develop metastases but none of the existing treatments of the primary tumor has been able to reduce the metastatic rate. Probably, micrometatases have already developed before treatment of the uveal melanoma and dormant micrometastases can persist for years before they start growing. This long time-span provides the possibility to treat micrometastases. METHODS In order to develop an animal model for metastatic uveal melanoma, B16 melanoma cells were injected into the posterior ocular compartment of C57BL6 mice. These cells grew and metastasised to the lungs and liver. Immunological factors for the metastatic process and possible neoadjuvant treatments were investigated. RESULTS Natural killer cells (NK) are of significance in the rejection of metastases and HLA-I expression of uveal melanomas correlates with the melanoma cell type. Interferon-alpha-2b increases the activity of NK cells and reduces the metastatic rate in the animal model. CONCLUSION Treatment with interferon-alpha-2b results in decreased metastases from intraocular melanoma in a murine model.

Metastasis to the liver is a frequent event in clinical oncology, the molecular mechanisms of which are not fully understood. We have recently reported a consistent overexpression of c-met in B16 melanoma cells selected in vivo for enhanced liver metastatic ability. In this study we address the question as to whether constitutive activation of c-met is a necessary and sufficient condition for enhanced liver colonization B16 melanoma cells. Different levels of c-met expression and/or activation in B16 cells were achieved subcloning, or by c-DNA transfection with either HGF/SF or the oncogenic form of c-met (tpr-met). Metastatic ability of the different populations was then evaluated in vivo by the lung colonization (experimental metastasis) assay. Results indicate that c-met (but not tpr-met) activation in B16 melanoma cells may increase their liver colonizing potential, probably by enhancing motility and invasion in response paracrine interactions with its ligand. C-met expres sion per se, however, is not able to change the organ specificity of the cells. C-met activation appears instead to be required at later stages of liver colonization by B16 melanoma cells, in order to enhance their site-specific metastatic ability. © Rapid Science 1998

B16 melanoma cells selected in mice for liver-specific metastasis (B16-LS9) overexpress a constitutively active form of the hepatocyte growth factor/scatter factor receptor (HGF/SFr), the product of the c-met proto-oncogene. HGF/SF can affect both invasion and growth of receptive cells. In fact, we show that overexpression of c-met in B16-LS9 cells results in a higher inducibility of two different proteolytic activities (uPA and gelatinase), in correlation with a stronger invasive and motility response to HGF/SF treatment. However, HGF/SF treatment inhibits growth of B16 cells, which might appear in contradiction with the observation that c-met overexpression and constitutive activation seems to be required for efficient liver colonization. However, this apparent discrepancy is resolved by the finding that liver-derived, but not lung-derived factor(s), can efficiently rescue B16-LS9 cells from the growth inhibitory effects of HGF/SF, while not changing their motility response. Therefore, overexpression of c-met in B16-LS9 cells might give a specific advantage in liver colonization, because specifically at this site B16-LS9 cells can take full advantage of the positive effects exerted by HGF/SF stimulation on motility and invasion, while the negative effects on growth are counteracted by other paracrine factor(s).

A third of people affected by primary open angle glaucoma (POAG) have normal or even low intraocular pressure (IOP), such as in the case of normotensive glaucoma and low pressure glaucoma. Therefore, high IOP, while remaining the most important risk factor for POAG development, is not the only one. POAG has been recognized to belong to the class of neurodegenerative diseases, such as Alzheimer's (AD) and Parkinson's, and several recent studies have suggested possible links between Alzheimer's and glaucoma both from an epidemiological and a pathogenetic point of view. In fact, several pathogenetic factors are common between the two diseases: the diffuse axonal degeneration of ganglion cells, the presence of specific markers (such as amyloid-β) in the aqueous humor, the activation of caspases and the abnormal processing of amyloid precursor protein in retinal ganglion cells, the reduction of the intracranial pressure of the cerebrospinal fluid, the mutations of the gene that encodes for optineurine. Conflicting data have been reported concerning apolipoprotein E. Finally, numerous publications affirm the utility of molecules traditionally used in the treatment of neurodegenerative diseases (e.g. forskolin, L-carnosine, homotaurine, folic acid, acetylcholinesterase inhibitors, antioxidant vitamins, glatiramer acetate, etc.) also in the treatment of glaucoma. In the present research we analyze the common pathogenetic pathways of Alzheimer's disease and Glaucoma, we discuss the two diseases with respect to their differences and similarities and we speculate about common therapies, based on the similar pathogenetic findings.

AIM: To establish a threshold value of intraocular pressure (IOP) increase after the closed-eyelid test (CET) that correlates with the highest probability of developing overt primary open-angle glaucoma (OAG) in an Italian native population from 1980 to 2010. METHODS: Retrospective analysis of data obtained from 161 patients with ocular hypertension who performed the CET in 1980, and were subsequently followed to see whether they developed OAG. CET was performed always in the morning Eyelids were closed by bandaging for 1 h in a quiet environment, with the patient seated and not sleeping. IOP was measured again 8 to 10s after opening the eyelids. RESULTS: Accurate statistical analysis of the obtained values indicated that 77% of the subjects showing an IOP increase after 1 hour of eyelid closure in a sitting position developed OAG in the following 30 years and that IOP increase values above 4 mmHg led to a subsequent diagnosis of glaucoma in more than 80% of the patients. CONCLUSION: Eyelid closure for 60 minutes results in a net elevation of IOP the extent of which depends on the balance between the increase of aqueous humour secretion and its outflow. Therefore, the CET may discriminate individuals with a normal outflow from individuals with a less functional outflow, which are evidently those at a higher risk of developing glaucoma.

The impact of glycosaminoglycans on intraocular pressure in glaucoma patients and in healthy young or aging subjects is explored.Thirty small autoptic samples were harvested from the tissue localized around the iridocorneal angle of the eye, taking care not to cause aesthetic damage. The samples came from three groups (young, old, and subjects with glaucoma). All samples were divided in two fragments and both were used for morphological and biochemical analyses. Quantitative data were obtained from image analysis to correlate with biochemical values. All results were statistically analyzed.The findings show the following changes of iridocorneal angle are caused by glycosaminoglycans both in aging and in glacoumatous patients: (1) deposition of fibrous granular material and increased electron density of the structures close to the iridocorneal angle; and (2) strong decrease of hyaluronic acid content and increase of sulfated glycosaminoglycans.Similar to what happens in other tissues in the body, glycosaminoglycans of the human iridocorneal angle undergo physiological and pathological changes. The trabecular meshwork is the structure responsible for the regulation of the aqueous humor outflow that is often altered in primary open-angle glaucoma patients.

In most studies concerning organ-specific metastasis, different or selected lines are compared with one another. Here we report results with the same cell line (the F9 murine teratocarcinoma) which can be directed towards liver or lung, depending on whether or not it is treated with retinoic acid and cyclic AMP. Thus, organ-specific colonization by tail-vein-injected murine F9 teratocarcinoma cells shows a particular pattern: unselected, undifferentiated F9 cells preferentially colonize the liver of the syngeneic animal. The lungs, the first capillary bed encountered by cells thus injected, are only very rarely colonized. By contrast, the lungs become the main target organ of F9 cells induced to differentiate by treatment with retinoic acid and cyclic AMP. We have recently shown that liver colonization by undifferentiated F9 cells correlated with the adhesiveness of the cells (higher to fibronectin and liver-derived extracellular matrix than to laminin and lung-derived extracellular matrix) as well as with their growth response to organ-derived extracts (no response with lung extracts and good response with liver extracts). The data reported below indicate that induction of differentiation causes at most a decreased adhesiveness of F9 cells to all the substrata tested (laminin, fibronectin, type-IV collagen, organ-derived extracellular matrix), suggesting that the shift in organ colonization observed with differentiated F9 cells is not due to an enhancement of the specific adhesion to the lung matrix. On the other hand, differentiated cells, but not undifferentiated ones, were able to respond to growth stimulation mediated by lung-derived extracts, thereby implying a relevant role for organ-specific growth stimulation in lung colonization by differentiated F9 cells. © 1992 Wiley-Liss, Inc.

Metastatic spread of tumor cells is one of the most common causes of death in cancer patients. Therefore, elucidation of the molecular mechanisms that underlie the formation of metastatic colonies has been one of the major objectives of cancer research during the last two decades. In this review we will mainly discuss the mechanisms that cause a malignant cell to grow at a given site rather than at other possible sites, taking into account experimental and clinical evidence published on the subject. As a whole this evidence tends to confirm the hypothesis that organ-specific colonization by malignant cells often follows very specific and close interactions between the cancer cell and the target organ, either in terms of specific cellular adhesion or growth promotion. In this paper we would like to underscore the fact that cellular adhesion, either specific or unspecific, is a necessary but, by itself, insufficient condition for the development of metastases. It is the ability of the tumor cells to grow at the site where they arrested that ultimately determines whether a metastatic colony develops or fails to develop at that site.

Unselected F9 murine embryonal carcinoma cells preferentially colonize the liver upon injection into tail veins of syngeneic mice, while the lungs are only very rarely colonized. Here we show that F9 cells attach better to fibronectin than to laminin in an adhesion assay, like other liver-colonizing cell lines. Moreover, assays of adhesion to extracellular matrix (ECM) prepared from rat organs (liver, lung and kidney) demonstrate that, in the absence of serum, F9 cells adhere better to liver- than to kidney- or lung-derived ECM. Even in the presence of FCS, the adhesion to lung ECM remains very low. This very low adhesiveness of F9 cells to lung-derived ECM correlates well with the finding that, in an organ distribution assay, tail-vein-injected F9 cells are very rapidly released from the lungs, when compared to the retention times of the lung-specific murine melanoma cell line B16-F10. Yet another property appears to contribute to organ-specific colonization of these cells: extracts of liver promote the growth of F9 cells, in contrast to extracts of lung or kidney which have no effect. These data suggest that preferential formation of metastases in the liver following the intravenous injection of F9 cells is the result of both their adhesive abilities and their growth response to local microenvironment.

The aim of this study was to provide support for epigallocatechin gallate (EGCG), a component of green tea, to be considered in the context for neuroprotection in glaucoma, where administration by an oral route is required for adequate penetration into the retina. Ischemia was delivered to one eye of a number of rats by raising the intraocular pressure. EGCG was present in the drinking water of half of the animals 3 days before ischemia and also during the next 5 days of reperfusion. The electroretinograms (ERGs) of both eyes from all rats were recorded before ischemia and 5 days following ischemia. Seven days after ischemia retinas from both eyes of all rats were either analysed for the localisation of various antigens or extracts prepared for analysis for the level of specific proteins and mRNAs. Ischemia/reperfusion to the retina affected a number of parameters. These included the localisation of Thy-1 and choline acetyltransferase, the a- and b-wave amplitudes of the ERG, the content of certain retinal and optic nerve proteins and various mRNAs. Significantly, EGCG statistically blunted many of the effects induced by ischemia/reperfusion which included the activation of caspases. These studies demonstrate conclusively that orally administered EGCG attenuates injury to the retina caused by ischemia/reperfusion where caspases were activated. Studies were also conducted on a cell line (RGC-5 cells) where it was shown that white light (1000 lx, 48 h)-induced apoptosis is caspase-independent and can be blunted by EGCG. The present studies support the view for the use of EGCG in the treatment of glaucoma based on the premise that any potential neuroprotective agent must be administered orally, have a safe profile and poses a broad spectrum of properties that allows various risk factors (that include ischemia and light) to be attenuated.

The aim of this study was to examine whether the antioxidant epigallocatechin gallate (EGCG), a catechin-base flavonoid derived from green tea protects retina neurones in situ from ischemia/reperfusion and in vitro from an oxidative stress insult of hydrogen peroxide (H(2)O(2)). Similar results were obtained when rats were injected by two different regimes of EGCG. Ischemia was delivered by raising the intraocular pressure above the systolic blood pressure (120 mm Hg) generally for 45 min. The electroretinogram (ERG) was measured prior to ischemia and 5 days after reperfusion. Rats were killed 7 days after ischemia and processed for immunohistochemistry and for determining of mRNA and protein levels by RT-PCR and electrophoresis/western blotting, respectively. In addition, optic nerves 7 days after ischemia were subjected to protein analysis. Ischemia/reperfusion caused a significant reduction in the a- and b-wave amplitudes of the ERGs, a decrease in retinal ganglion cell and photoreceptor specific proteins and mRNAs, an increase in retinal caspase-3 mRNA and protein, an increase in retinal caspase-8 mRNA, an increase in retinal GFAP protein and mRNA and a decrease in optic nerve proteins associated with ganglion cell axons. All these changes were significantly counteracted by EGCG. Moreover, EGCG clearly blunted ischemia/reperfusion-induced changes in the localisation of retinal Thy-1 and ChAT immunoreactivities. EGCG also significantly reduced the apoptosis to retinal ganglion cells (RGC-5 cells) in culture caused by H(2)O(2). The results of the study demonstrate that EGCG provides protection to retinal neurones from oxidative stress and ischemia/reperfusion.

Tear lactoferrin, mainly secreted by the lachrymal glands, exerts a protective effect on the ocular surface, and an abnormal decrease of its production may lead to an increased risk of infection and pathological alterations of ocular surface epithelia. In this study we analyzed whether corneal and conjunctival epithelia could be an additional source of tear lactoferrin, and whether conjunctival epithelial cells in culture could be a suitable model system to address regulation of lactoferrin gene expression. Real-time PCR and Western immunoblotting showed that in bovines lactoferrin is indeed produced by these epithelia, and that the human lactoferrin promoter can direct the expression of a CAT reporter gene, thus indicating that these cells are a true source of lactoferrin, and may be used in vitro to study the regulation of lactoferrin expression.

Epigallocatechin gallate (EGCg), the main polyphenol component of green tea, has several antibacterial properties. Here we show that sub-MICs of EGCg appear to decrease slime production, therefore inhibiting biofilm formation by ocular staphylococcal isolates previously characterized for the presence of ica genes by the Congo red agar plate assay and for adhesion to microtiter plates.

Purpose This study determined whether adenovirus-mediated transfer of the murine interferon-beta (AdCMVIFN-beta) gene protects against liver metastases arising from intraocular melanomas in mice. Methods A replication-deficient adenovirus vector (AdCMVIFN-beta) was used for the in vivo transfer of the murine IFN-beta gene into intraocular melanoma-bearing mice. AdCMVIFN-beta was injected either intravenously or directly into the intraocular melanomas. The effect of gene transfer on liver metastases was ascertained by histopathologic analysis of the livers and by measuring serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT), which are two enzymes associated with liver metastases in patients with uveal melanoma. Results Mice treated with two intratumoral injections of AdCMVIFN-beta had a 68% reduction in metastatic liver lesions (P = 0.016) and a 51% reduction in liver enzyme levels compared with control mice (P = 0.02). However, the antimetastatic effect of AdCMVIFN-beta was not directly attributable to the adenovirus vector or virus-mediated cytolysis of tumor cells. Intravenous treatment with AdCMVIFN-beta resulted in an 86% reduction in the number of metastatic foci in the liver (P = 0.014) and a 61% reduction of serum AST levels compared with mice treated with AdCMVLacZ (P = 0.015). AdCMVIFN-beta treatment produced a sharp increase in the NK cell activity that was demonstrable in vivo and in vivo. In vivo depletion of NK cells by anti-asialo GM1 antibody abrogated the antimetastatic effects of AdCMVIFN-beta. Conclusions The results support the feasibility of activation of NK cell function through gene transfer as one possible therapeutic strategy for reducing hepatic metastases of uveal melanomas.