Preliminary characterization of dexamethasone-loaded cross-linked hyaluronic acid films for topical ocular therapy.

The aim of this work was to design and characterize cross-linked hyaluronic acid (HA)-itaconic acid (IT) films loaded with dexamethasone sodium phosphate salt (DEX) for topical therapy of inflammatory ocular surface diseases. Films were chemically cross-linked with polyethylene glycol diglycidyl ether (PEGDE), then physical and mechanical characterization by stress-strain, X-ray diffraction, X-ray fluorescence spectrometry and swelling assays was conducted. A sequential in vitro therapeutic efficacy model was designed to assess changes in interleukin (IL)-6 production in an inflamed human corneal epithelial (HCE) cell line after film exposure. Changes in cell proliferation after film exposure were assessed using the alamarBlue(®) proliferation assay. Experimental findings showed desirable mechanical properties and in vitro efficacy to reduce cell inflammation. A moderately decreased proliferation rate was induced in HCE cells by DEX-loaded films, compared to commercial DEX eye drops. These results suggest that DEX and HA have opposite effects. The sequential in vitro therapeutic efficacy model arises as an efficient tool to study drug release from delivery systems by indirect measurement of a biological response.

Interaction of IFN-γ with cholinergic agonists to modulate rat and human goblet cell function.

Goblet cells populate wet-surfaced mucosa including the conjunctiva of the eye, intestine, and nose, among others. These cells function as part of the innate immune system by secreting high molecular weight mucins that interact with environmental constituents including pathogens, allergens, and particulate pollutants. Herein, we determined whether interferon gamma (IFN-γ), a Th1 cytokine increased in dry eye, alters goblet cell function. Goblet cells from rat and human conjunctiva were cultured. Changes in intracellular [Ca(2+)] ([Ca(2+)](i)), high molecular weight glycoconjugate secretion, and proliferation were measured after stimulation with IFN-γ with or without the cholinergic agonist carbachol. IFN-γ itself increased [Ca(2+)](i) in rat and human goblet cells and prevented the increase in [Ca(2+)](i) caused by carbachol. Carbachol prevented IFN-γ-mediated increase in [Ca(2+)](i). This cross-talk between IFN-γ and muscarinic receptors may be partially due to use of the same Ca(2+)(i) reservoirs, but also from interaction of signaling pathways proximal to the increase in [Ca(2+)](i). IFN-γ blocked carbachol-induced high molecular weight glycoconjugate secretion and reduced goblet cell proliferation. We conclude that increased levels of IFN-γ in dry eye disease could explain the lack of goblet cells and mucin deficiency typically found in this pathology. IFN-γ could also function similarly in respiratory and gastrointestinal tracts.

Novel bioadhesive hyaluronan-itaconic acid crosslinked films for ocular therapy.

New hyaluronic acid (HA)-itaconic acid (IT) films have been previously synthesized and used as potential topical drug delivery systems (DDS) for ocular administration. In this study we explored homogeneous and heterogeneous crosslinking reactions of HA using glutaraldehyde (GTA) and polyethylene glycol diglycidyl ether (PEGDE) in the presence of IT, a naturally occurring compound that is non-toxic and readily biodegradable. We have studied the morphology, mechanical properties and in vitro biocompatibility between these new materials and ocular surface cells (human corneal epithelial cell line) and evaluated the biopharmaceutical performance of the designed formulations. Although all the synthesized materials exhibited good mechanical properties, the PEGDE modified films exhibited the best biocompatibility, with in vivo assays showing good adhesive performance and minimal irritation. PEGDE films were also tested for their effects in the treatment of intraocular pressure (IOP) in rabbits using timolol maleate (TM) as the model drug. These results may be useful for further design of novel bioadhesive matrix containing drugs by topical application in ophthalmology.

A nanomedicine to treat ocular surface inflammation: performance on an experimental dry eye murine model.

MUC5AC is a glycoprotein with gel-forming properties, whose altered expression has been implicated in the pathogenesis of dry eye disease. The aim of our study was to achieve an efficient in vivo transfection of MUC5AC, restore its normal levels in an inflamed ocular surface and determine whether restored MUC5AC levels improve ocular surface inflammation. Cationized gelatin-based nanoparticles (NPs) loaded with a plasmid coding a modified MUC5AC protein (pMUC5AC) were instilled in healthy and experimental dry eye (EDE) mice. MUC5AC expression, clinical signs, corneal fluorescein staining and tear production were evaluated. Ocular specimens were processed for histopathologic evaluation, including goblet cell count and CD4 immunostaining. Neither ocular discomfort nor irritation was observed in vivo after NP treatment. Expression of modified MUC5AC was significantly higher in ocular surface tissue of pMUC5AC-NP-treated animals than that of controls. In healthy mice, pMUC5AC-NPs had no effect on fluorescein staining or tear production. In EDE mice, both parameters significantly improved after pMUC5AC-NP treatment. Anterior eye segment of treated mice showed normal architecture and morphology with lack of remarkable inflammatory changes, and a decrease in CD4+ T-cell infiltration. Thus, pMUC5AC-NPs were well tolerated and able to induce the expression of modified MUC5A in ocular surface tissue, leading to reduction of the inflammation and, consequently improving the associated clinical parameters, such as tear production and fluorescein staining. These results identify a potential application of pMUC5AC-NPs as a new therapeutic modality for the treatment of dry eye disease.

Branched-chain fatty acids, increased in tears of blepharitis patients, are not toxic for conjunctival cells.

AIM: The composition of the meibum of blepharitis patients is characterised by increased levels of branched-chain fatty acids (BCFAs) that return to normal values in patients treated with cyclins and lid hygiene. The aim of this study was to determine if BCFAs had toxic effects on conjunctival cells related to the disease. METHODS: Chang and IOBA-NHC conjunctival human cells were treated with BCFAs (isoC16 and isoC20) or palmitic acid as a control for 4 h or 24 h at 50 microM or 100 microM. Morphological and functional changes were investigated by measuring mitochondrial dehydrogenase activity, cell permeability, mitochondrial depolarisation, chromatin condensation, IL-1beta and reactive oxygen species production. RESULTS: None of the fatty acids modified the parameters of cytotoxicity in conjunctival cells in Chang or IOBA-NHC cell lines. Only the mitochondrial dehydrogenase activity was significantly decreased in relation to the isoC20 concentration increase. CONCLUSIONS: The increase in BCFAs in the tears of blepharitis patients does not consistently participate in the conjunctival cell changes throughout the course of the disease. Instead, it is likely an adaptive response of the ocular surface to the lack of tears, possibly increasing meibum fluidity, thus enhancing lacrimal film stability.

The need for continuous immunosuppression with cyclosporin A to maintain an experimental model of uveal melanoma.

We investigated the need for continuous immunosuppression to maintain experimental tumours derived from human uveal melanoma cells implanted in the choroid of pigmented rabbits. Two groups of pigmented rabbits immunosuppressed with cyclosporin A (CsA) were implanted with human uveal melanoma cells in the suprachoroidal space. After 5 weeks, CsA was discontinued in group 2. Animals were treated with prophylactic antibiotics and examined weekly for tumour growth, weight and secondary effects; blood urea nitrogen levels were measured every two weeks. Autopsies and histopathological studies were performed after death or euthanasia at the end of week 12. The difference between the groups in the development of ophthalmoscopic tumours was not statistically significant 5 weeks after implantation. Tumours in group 1 grew progressively throughout the experiment, whereas group 2 tumours showed marked regression 3-4 weeks after discontinuing CsA. Tumours in group 1 were significantly larger and had greater mitotic activity and showed more ciliary body, optic nerve and extrascleral invasion than tumours in group 2, which showed massive fibrosis, minimal mitotic activity and marked inflammatory cell infiltration. Continuous immunosuppression with CsA seems to be necessary to maintain tumour growth in this experimental model of uveal melanoma.

[In vitro toxicity of non-preserved artificial-tear formulations]. / Lágrimas artificiales sin conservantes: estudio comparativo in vitro.

PURPOSE: To analyse the putative toxic effect of three commercially available non-preserved artificial tear formulations on in vitro human conjunctival cells. MATERIAL AND METHOD: A conjunctival human epithelial cell line was exposed to Cellufresh, Oculotect and Acuolens formulations during 1, 3 and 24 hours. Cytotoxicity was measured by calculating the percentage of cell viability examination and scanning electron microscopy (SEM). Controls underwent exposure to supplement free DMEM-F12 (negative control) and exposure to 0.005% benzalkonium chloride solution (positive control). RESULTS: Cell viability after 1 or 3 hours incubation with Cellufresh and Oculotect was similar to that obtained for negative controls. With Acuolens incubation however, cell viability showed significant reduction after 3 and 24 hours compared to control. SEM showed that Cellufresh and Oculotect exposed cells presented similar behavior to control cells. All three cell lines presented evidence of cellular surface alteration after incubation for 1 or 3 hours compared to controls, Acuolens showing the highest rate of alterations in exposed cells and an additional increment in cell loss was observed. CONCLUSION: In the present study, non preserved artificial tears formulations showed a different degree in their in vitro toxicity, Acuolens being more toxic than Cellufresh or Oculotect.

Lágrimas artificiales sin conservantes: estudio comparativo in vitro / In vitro toxicity of non-preserved artificial-tear formulations

Objetivo: Analizar los posibles efectos tóxicos de tres preparados comerciales de lágrimas artificiales sin conservantes sobre células conjuntivales humanas in vitro. Métodos: Cultivos de células epiteliales de conjuntiva humana fueron expuestos a la acción de los preparados comerciales Cellufresh, Oculotect y Acuolens durante 1, 3 y 24 horas. Transcurrido dicho tiempo se estudió su posible efecto tóxico, expresado como porcentaje de Viabilidad Celular, y se analizó la presencia de alteraciones en la superficie de las células mediante microscopia electrónica de barrido (MEB). El estudio incluyó como control negativo de toxicidad células expuestas a medio de cultivo DMEM-F12 sin suplementos y como control positivo, células expuestas a una solución de cloruro de benzalconio al 0,005 por ciento en dicho medio de cultivo. Resultados: La Viabilidad Celular obtenida tras incubar 1 ó 3 horas con Cellufresh y Oculotect fue similar a la del control negativo, si bien disminuyó algo cuando el tiempo de incubación fue de 24 horas. Sin embargo, la Viabilidad Celular tras incubar con Acuolens fue significativamente menor para todos los tiempos. La MEB mostró que con Cellufresh y Oculotect el aspecto general de las células era muy similar al observado en las células control. Sin embargo, con Acuolens se observaron alteraciones acusadas, tanto tras 1 hora como tras 3 horas de incubación, y una notable pérdida celular. Conclusión: De las lágrimas artificiales sin conservantes en estudio, Acuolens resultó ser la más tóxica in vitro, tanto a tiempos cortos como largos (AU)

Presence of nerves and their receptors in mouse and human conjunctival goblet cells.

PURPOSE: To determine whether neural pathways for controlling goblet cell secretion are present in mouse and human conjunctiva. METHODS: Mouse conjunctiva was homogenized and subjected to electrophoresis and Western blotting to detect PGP 9.5 (indicates nerves), muscarinic receptor subtypes (indicates parasympathetic pathway), and adrenergic receptors (indicates sympathetic pathway). Mouse eyes and human conjunctival tissue were analyzed by immunofluorescence microscopy. Antibodies to vasoactive intestinal peptide (VIP), tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH), and muscarinic and alpha(1)- and beta-adrenergic receptor subtypes were used. RESULTS: Western blot demonstrated PGP 9.5, M(1), M(2), and M(3) muscarinic receptors and alpha(1A)-, beta(1)-, beta(2)-, and beta(3)-adrenergic receptors in mouse conjunctiva. Immunoreactivity for VIP, TH, and DBH was found adjacent to mouse and human goblet cells. M(1) and M(2) muscarinic receptors were identified throughout mouse conjunctiva, but M(3) receptor was predominantly on goblet cells. All three muscarinic receptor subtypes were detected on goblet cells in human conjunctiva. alpha(1A)-Adrenergic receptors were found on epithelial cells and on goblet cells in mouse and human conjunctiva. beta(1)- and beta(2)-Adrenergic receptors were found on both epithelial and goblet cells in mouse conjunctiva, but not on human conjunctival cells. beta(3)-Adrenergic receptors were found on both epithelial and goblet cells in human conjunctiva but not on mouse conjunctival cells. CONCLUSIONS: The following conclusions were drawn: parasympathetic nerves and M(1), M(2), and M(3) muscarinic receptors, as well as sympathetic nerves are present on mouse and human goblet cells. The adrenergic receptors beta(1) and beta(2,) but not alpha(1A) and beta(3) are present on mouse conjunctival goblet cells, whereas alpha(1A) and beta(3,) but not beta(1) and beta(2) are present on human conjunctival goblet cells, suggesting that these nerves and receptors could activate goblet cell secretion in mouse and humans.

Uveal melanoma model with metastasis in rabbits: effects of different doses of cyclosporine A.

OBJECTIVE: To compare the toxicity and efficacy of different doses of cyclosporine A (CsA) in a rabbit model of uveal melanoma. METHODS: We used four experimental groups: control, no CsA; group 1, 15 to 10 mg/kg/day; group 2, 15 mg/kg/day; and group 3, 20 mg/kg/day. The MKT-BR cell line was implanted in the choroid. All animals underwent ophthalmoscopic evaluation; the animals were weighed and blood levels of CsA, blood urea nitrogen (BUN), creatinine and alanine aminotransferase (ALT) were measured weekly. Necropsies and histologic study were performed to detect intraocular tumors and metastasis. RESULTS: A difference in survival rates was found between groups 2 and 3 (p = 0.0042). Differences were observed in the mean BUN and creatinine levels (p < 0.001 and p < 0.003, respectively) between groups but not in the ALT. Intraocular tumors were detected ophthalmoscopically in 50%, 65%, and 70% of the animals in groups 1, 2, and 3 respectively, and histologically in 70%, 90%, and 100% of the same groups. Lung metastases were found in 26.8% of animals with intraocular tumors. Differences were observed in mean CsA blood levels between animals with and without histologically demonstrated uveal tumors (p = 0.001) but not in animals with or without metastasis. CONCLUSIONS: Different doses of CsA affect survival, tumor development and renal toxicity. Metastatic disease is independent of CsA dose and the subsequent CsA blood levels. A blood level of CsA ranging from 500 to 1000 ng/ml and doses of 10 to 15 mg/kg/day may effectively develop this model of uveal melanoma.

Development of conjunctival goblet cells and their neuroreceptor subtype expression.

PURPOSE: To investigate expression of muscarinic, cholinergic, and adrenergic receptors on developing conjunctival goblet cells. METHODS: Eyes were removed from rats 9 to 60 days old, fixed, and used for microscopy. For glycoconjugate expression, sections were stained with Alcian blue/periodic acid-Schiffs reagent (AB/PAS) and with the lectins Ulex europeus agglutinin I (UEA-I) and Helix pomatia agglutinin (HPA). Goblet cell bodies were identified using anti-cytokeratin 7 (CK7). Nerve fibers were localized using anti-protein gene product 9.5. Location of muscarinic and adrenergic receptors was investigated using anti-muscarinic and beta-adrenergic receptors. RESULTS: At days 9 and 13, single apical cells in conjunctival epithelium stained with AB/PAS, UEA-I, and CK7. At days 17 and 60, increasing numbers of goblet cells were identified by AB/PAS, UEA-I, HPA, and CK7. Nerve fibers were localized around stratified squamous cells and at the epithelial base at days 9 and 13, and around goblet cells and at the epithelial base at days 17 and 60. At days 9 and 13, M2- and M3-muscarinic and beta2-adrenergic receptors were found in stratified squamous cells, but M1-muscarinic and beta1-adrenergic receptors were not detected. At days 17 and 60, M2- and M3-muscarinic receptors were found in goblet cells, whereas M1-muscarinic receptors were in stratified squamous cells. Beta1- and beta2-adrenergic receptors were found on both cell types. Beta3-adrenergic receptors were not detected. CONCLUSIONS: In conjunctiva, nerves, M2- and M3-muscarinic, and beta1- and beta2-adrenergic receptors are present on developing goblet cells and could regulate secretion as eyelids open.

Ultrastructural evidence of mucus in human conjunctival epithelial cultures.

PURPOSE: To demonstrate by ultrastructural techniques that human conjunctival epithelium cells in vitro can produce mucin-like secretion. METHODS: Primary cultures of human conjunctival epithelial cells were grown in different culture media. Cultures were allowed to grow and were processed after 5 days and 1, 2, 3, 4, or 5 weeks for transmission and scanning electron microscopy, according to the method of Nichols et al. modified in our laboratory. RESULTS: Marked differences were seen between primary cultures grown with or without hydrocortisone. A thick tannic acid-stained layer was observed when hydrocortisone was present in the culture medium; however, that layer was virtually absent in cultures grown with hydrocortisone-free media. Scanning electron microscopy revealed a dense deposit showing a network-like structure. Moreover, the age of the cultures clearly influenced the thickness of the tannic acid-stained deposit, which thickened as the cultures aged. CONCLUSIONS: These results strongly suggest that the layer growing in the presence of hydrocortisone is mucus. The fact that this material became more abundant as the cultures aged indicates that mucus is actively produced and secreted by conjunctival epithelial cells in vitro. This study might contribute to the knowledge of mucus-deficient pathologies of the ocular surface.

Expression of ICAM-1 and HLA-DR by human conjunctival epithelial cultured cells and modulation by nedocromil sodium.

It is unclear whether conjunctival epithelial cells participate in the development of immune-mediated events. Using a previously reported in vitro system of human conjunctival epithelium, we determined whether conjunctival epithelial cells express two relevant markers in the antigenic presentation process. Moreover, the potential capability of nedocromil sodium, an antiallergic and antiinflammatory drug, to modulate such expression was investigated. Primary cultures of human conjunctival epithelium and Chang conjunctival cells, incubated with or without 100 U/ml IL-1beta and/or IFNgamma for 1, 3 or 6 h, were simultaneously exposed to 10(-5) M nedocromil sodium. The expression of the intercellular adhesion molecule-1 (ICAM-1) and the human leukocyte antigen-DR (HLA-DR) was determined immunocytochemically. Constitutive expression of ICAM-1 and HLA-DR was observed in primary cultures and Chang cells and was minimally affected by incubation with IL-1beta and/or IFNgamma. The addition of nedocromil sodium resulted in complete abolition of HLA-DR expression and a notable reduction in ICAM-1 expression in primary cultures and Chang cells. These results suggest that epithelial cells from human conjunctiva constitutively express ICAM-1 and HLA-DR in vitro and that such expression is downregulated by nedocromil sodium. This may indicate that conjunctival epithelial cells may be another target for this drug.

Carbomer- versus cellulose-based artificial-tear formulations: morphologic and toxicologic effects on a corneal cell line.

PURPOSE: Frequent instillation of artificial tears is the primary disadvantage of the treatment for dry-eye syndrome. Recently gel formulations have been proposed as an alternative to classic cellulose formulations. The higher viscosity of these gels presumably prolongs tear-retention time in the eye and results in fewer daily applications. However, no toxicologic studies with gel formulations have been performed. Our aim was to study the toxic effects of these formulations on corneal cells. METHODS: SIRC cells from rabbit cornea were exposed for 30 min, and 1, 3, and 6 h to five commercially available artificial tears, three of them carbomer gel formulations (Lacrivisc, Lacrivisc unit-doses, and Viscotears) and two carboxymethylcellulose (CMC) formulations (Celluvisc and Cellufresh). A cytotoxicity assay and a scanning electron microscopy (SEM) study were used to analyze the putative toxic effects of the formulations. The preservatives of the gel formulations also were tested. RESULTS: Carbomer gel formulations, both with and without preservatives, caused more in vitro toxic effects in the corneal cells than did CMC formulations and caused severe damage even after 30 min of exposure. SEM revealed dramatic cell-surface alterations. Preservatives added to Lacrivisc and Viscotears also had toxic effects on cells, whose effects were not significantly different from those of the commercial preparations. CONCLUSION: These results demonstrate that in the in vitro study, CMC artificial tears are less toxic than carbomer gel formulations. Questions about the benefits of using high-viscosity gels in the treatment of dry-eye syndrome still remain.

Characterization of epithelial primary cultures from human conjunctiva.

Primary cultures of human epithelial cells from normal conjunctiva were developed and characterized to determine whether they retained epithelial characteristics. Conjunctival explants were obtained from the upper fornix of healthy donors and cultured in supplemented DMEM/F-12 medium for 5 days. The epithelial outgrowth was maintained for an additional 10 days. Primary cultures were then processed for light microscopy, transmission and scanning electron microscopy (TEM, SEM), and immunocytochemistry. They exhibited typical features of conjunctival epithelium on light microscopy (polygonal morphology, intimate cohesion, production of mucins), TEM (abundant desmosomes, keratin bundles, granules, microvilli), SEM (polygonal shape, microvilli, intimate cohesion), and immunocytochemistry (positivity for the receptor of epidermal growth factor, desmosomal proteins, and cytokeratins). In conclusion, primary cultures developed from normal human conjunctiva maintained the epithelial characteristics in vitro. Because the conjunctiva is a major component of the anterior ocular surface, we propose this in vitro system as suitable for physiopathologic and toxicologic studies.

Morphologic and immunocytochemical characterization of four human uveal cell lines (melanoma- and melanocytes-derived).

PURPOSE: To characterize three cell lines from human uveal melanomas and one ocular melanocyte cell line to study the specificity of several antigens in the malignant transformation of melanocytic uveal cells. METHODS: Light microscopy (LM), transmission electron microscopy (TEM), and immunocytochemical techniques were used in the characterization of OCM-1, SP 6.5, and MKT-BR human uveal melanoma cell lines and UW-1 normal melanocyte cell line from human uvea. Several monoclonal antibodies (MoAbs) S-100, HMB-45, MNF-116, PAL-M1, NK1/C-3, IND-1, and MAAMA were used. RESULTS: All cell lines showed an epithelioid/spindle morphology with occasional multinucleated cells, and nuclear pleomorphism. TEM showed intracytoplasmatic premelanosomes. Incubation with HMB-45 MoAb was positive in all cell lines. PAL-M2, NK1/C-3, MAAMA, and IND-1 MoAbs stainings were positive with variable intensity. MNF-116 MoAb showed negative staining in the four lines, and S-100 MoAb was also negative except for the UW-1 cell line. CONCLUSIONS: Human uveal melanoma cell lines OCM-1, SP 6.5, and MKT-BR and the ocular melanocyte cell line UW-1 exhibited maintenance of some structural and ultrastructural characteristics of melanocytic cells. All four MoAbs, PAL-M2, NK1/C3, IND-1, and MAAMA against cutaneous melanoma-associated antigens stained positively all melanoma cell lines as well as the melanocytic cell line, suggesting that in vitro proliferation of melanocytes could modify their antigenic expression.

Effects of maternal bilateral adrenalectomy and betamethasone administration on fetal rat encephalic development.

The present study examined the effects of maternal bilateral adrenalectomy and betamethasone treatment on fetal encephalic development, in terms of fetal body weight, brain weight, DNA, protein and lipid content and morphological development. Both influenced the developmental time patterns of fetal brain and cerebellum. Fetuses of adrenalectomized rats had decreased body weights, whereas brain weight was not affected. Maternal adrenalectomy produces in fetal brain a decreased number of cells and increased cell size, while betamethasone treatment of adrenalectomized rats increased cell number, which was not different from control values; cell size remained lower than in control fetuses. Lipid content was increased in the fetuses of betamethasone-treated rats. In terms of morphological development, laminated structures (hippocampus and brain and cerebellar cortex) were the ones most affected.