RESUMEN
The purpose of this research was to determine the potential use of water-soluble anionic and cationic carbosilane dendrimers (generations 1-3) as mucoadhesive polymers in eyedrop formulations. Cationic carbosilane dendrimers decorated with ammonium -NH3(+) groups were prepared by hydrosylilation of Boc-protected allylamine and followed by deprotection with HCl. Anionic carbosilane dendrimers with terminal carboxylate groups were also employed in this study. In vitro and in vivo tolerance studies were performed in human ocular epithelial cell lines and rabbit eyes respectively. The interaction of dendrimers with transmembrane ocular mucins was evaluated with a surface biosensor. As proof of concept, the hypotensive effect of a carbosilane dendrimer eyedrop formulation containing acetazolamide (ACZ), a poorly water-soluble drug with limited ocular penetration, was tested after instillation in normotensive rabbits. The methodology used to synthesize cationic dendrimers avoids the difficulty of obtaining neutral -NH2 dendrimers that require harsher reaction conditions and also present high aggregation tendency. Tolerance studies demonstrated that both prototypes of water-soluble anionic and cationic carbosilane dendrimers were well tolerated in a range of concentrations between 5 and 10 µM. Permanent interactions between cationic carbosilane dendrimers and ocular mucins were observed using biosensor assays, predominantly for the generation-three (G3) dendrimer. An eyedrop formulation containing G3 cationic carbosilane dendrimers (5 µM) and ACZ (0.07%) (289.4 mOsm; 5.6 pH; 41.7 mN/m) induced a rapid (onset time 1 h) and extended (up to 7 h) hypotensive effect, and led to a significant increment in the efficacy determined by AUC0(8h) and maximal intraocular pressure reduction. This work takes advantage of the high-affinity interaction between cationic carbosilane dendrimers and ocular transmembrane mucins, as well as the tensioactive behavior observed for these polymers. Our results indicate that low amounts of cationic carbosilane dendrimers are well tolerated and able to improve the hypotensive effect of an acetazolamide solution. Our results suggest that carbosilane dendrimers can be used in a safe range of concentrations to enhance the bioavailability of drugs topically administered in the eye.
Asunto(s)
Dendrímeros/química , Dendrímeros/farmacocinética , Silanos/química , Silanos/farmacocinética , Acetazolamida/química , Administración Oftálmica , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacos , Dendrímeros/administración & dosificación , Dendrímeros/farmacología , Humanos , Masculino , Conejos , Silanos/administración & dosificación , Silanos/farmacología , Resonancia por Plasmón de SuperficieRESUMEN
Meibomian gland dysfunction is a leading cause of ocular surface disease. However, little is known about the regulatory processes that control the development and maintenance of this sebaceous gland. Here, we identify a novel function for CD147, a transmembrane protein that promotes tissue remodeling through induction of matrix metalloproteinases, in regulating meibocyte differentiation and activity. We found that CD147 localized along basal cells and within discrete membrane domains of differentiated meibocytes in glandular acini containing gelatinolytic activity. Induction of meibocyte differentiation in vitro promoted CD147 clustering and MMP9 secretion, whereas RNAi-mediated abrogation of CD147 impaired MMP9 secretion, concomitant with a reduction in the number of proliferative cells and cytoplasmic lipids. Meibomian glands of CD147 knockout mice had a lower number of acini in both the superior and inferior tarsal plates of the eyelids, and were characterized by loss of lipid-filled meibocytes compared with control mice. Together, our data provide evidence showing that gelatinolytic activity in meibocytes is dependent on CD147, and supports a role for CD147 in maintaining the normal development and function of the meibomian gland.
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Basigina/metabolismo , Glándulas Tarsales/citología , Glándulas Tarsales/metabolismo , Animales , Diferenciación Celular/fisiología , Proliferación Celular , Células Epiteliales/citología , Células Epiteliales/metabolismo , Células Epiteliales/patología , Humanos , Glándulas Tarsales/patología , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
In this article we review the mechanism of ocular surface staining. Water-soluble dyes are excluded from the normal epithelium by tight junctions, the plasma membranes and the surface glycocalyx. Shed cells can take up dye. A proportion of normal corneas show sparse, scattered time-dependent, punctate fluorescein uptake, which, we hypothesise, is due to a graded loss of the glycocalyx barrier, permitting transcellular entry into pre-shed cells. In pathological staining, there is little evidence of 'micropooling' at sites of shedding and the term 'punctate erosion' may be a misnomer. It is more likely that the initial event involves transcellular dye entry and, in addition, diffusion across defective tight junctions. Different dye-staining characteristics probably reflect differences in molecular size and other physical properties of each dye, coupled with differences in visibility under the conditions of illumination used. This is most relevant to the rapid epithelial spread of fluorescein from sites of punctate staining, compared to the apparent confinement of dyes to staining cells with dyes such as lissamine green and rose bengal. We assume that fluorescein, with its lower molecular weight, spreads initially by a paracellular route and then by transcellular diffusion. Solution-Induced Corneal Staining (SICS), related to the use of certain contact lens care solutions, may have a different basis, involving the non-pathological uptake of cationic preservatives, such as biguanides, into epithelial membranes and secondary binding of the fluorescein anion. It is transient and may not imply corneal toxicity. Understanding the mechanism of staining is relevant to the standardisation of grading, to monitoring disease and to the conduct of clinical trials.
Asunto(s)
Colorantes , Conjuntiva/metabolismo , Epitelio Corneal/metabolismo , Coloración y Etiquetado/métodos , Animales , Colorantes/metabolismo , Oftalmopatías/diagnóstico , Humanos , Soluciones Oftálmicas/metabolismo , Uniones Estrechas/metabolismoRESUMEN
Epithelial cells lining mucosal surfaces impose multiple barriers to viral infection. At the ocular surface, the carbohydrate-binding protein galectin-3 maintains barrier function by cross-linking transmembrane mucins on the apical glycocalyx. Despite these defense mechanisms, many viruses have evolved to exploit fundamental cellular processes on host cells. Here, we use affinity assays to show that herpes simplex virus type 1 (HSV-1), but not HSV-2, binds human galectin-3. Knockdown of galectin-3 in human corneal keratinocytes by small interfering RNA significantly impaired HSV-1 infection, but not expression of nectin-1, indicating that galectin-3 is a herpesvirus entry mediator. Interestingly, exposure of epithelial cell cultures to transmembrane mucin isolates decreased viral infectivity. Moreover, HSV-1 failed to elute the biological counterreceptor MUC16 from galectin-3 affinity columns, suggesting that association of transmembrane mucins to galectin-3 provides protection against viral infection. Together, these results indicate that HSV-1 exploits galectin-3 to enhance virus attachment to host cells and support a protective role for transmembrane mucins under physiological conditions by masking viral entry mediators on the epithelial glycocalyx.
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Galectina 3/metabolismo , Herpesvirus Humano 1/fisiología , Queratinocitos/virología , Mucinas/metabolismo , Receptores Virales/metabolismo , Acoplamiento Viral , Humanos , Unión ProteicaRESUMEN
PURPOSE: Development of the first in vitro method based on biosensor chip technology designed for probing the interfacial interaction phenomena between transmembrane ocular mucins and adhesive polymers and dendrimers intended for ophthalmic administration. METHODS: The surface plasmon resonance (SPR) technique was used. A transmembrane ocular mucin surface was prepared on the chip surface and characterized by QCM-D (Quartz Crystal Microbalance with Dissipation) and XPS (X-ray photoelectron spectroscopy). The mucoadhesive molecules tested were: hyaluronic acid (HA), carboxymethyl cellulose (CMC), hydroxypropylmethyl cellulose (HPMC), chitosan (Ch) and polyamidoamine dendrimers (PAMAM). RESULTS: While Ch originated interfacial interaction with ocular transmembrane mucins, for HA, CMC and HPMC, chain interdiffusion seemed to be mandatory for bioadherence at the concentrations used in ophthalmic clinical practise. Interestingly, PAMAM dendrimers developed permanent interfacial interactions with transmembrane ocular mucins whatever their surface chemical groups, showing a relevant importance of co-operative effect of these multivalent systems. Polymers developed interfacial interactions with ocular membrane-associated mucins in the following order: Ch(1 %) > G4PAMAM-NH(2)(2 %) = G4PAMAM-OH(2 %) > G3.5PAMAM-COOH(2 %)>> CMC(0.5 %) = HA(0.2 %) = HPMC(0.3 %). CONCLUSIONS: The method proposed is useful to discern between the mucin-polymer chemical interactions at molecular scale. Results reinforce the usefulness of chitosan and dendrimers as polymers able to increase the retention time of drugs on the ocular surface and hence their bioavailability.
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Carboximetilcelulosa de Sodio/metabolismo , Quitosano/metabolismo , Dendrímeros/metabolismo , Ácido Hialurónico/metabolismo , Metilcelulosa/análogos & derivados , Mucinas/metabolismo , Soluciones Oftálmicas/metabolismo , Línea Celular , Córnea/citología , Córnea/metabolismo , Humanos , Derivados de la Hipromelosa , Metilcelulosa/metabolismo , Mucinas/aislamiento & purificación , Espectroscopía de Fotoelectrones , Tecnicas de Microbalanza del Cristal de Cuarzo , Resonancia por Plasmón de Superficie/métodosRESUMEN
No disponible
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Investigación/normas , Investigación/tendencias , Nanotecnología/educación , Ciencia/educación , Universidades/organización & administración , Acreditación/métodos , Empleos en Salud/educación , Empleos en Salud/estadística & datos numéricos , Tecnología/educación , Evaluación de la Tecnología Biomédica/normas , Universidades/ética , Universidades/tendencias , Estudiantes , Estudiantes del Área de la SaludRESUMEN
PURPOSE: To assess the sensitivity, specificity, and reliability of peroxidase labeled lectin staining in the diagnosis of fungal keratitis in an experimental rabbit model. METHODS: Fungal keratitis by Candida albicans, Aspergillus fumigatus, and Fusarium solani was induced in rabbits. WGA-peroxidase staining of 660 corneal sections was performed. Fungal staining was evaluated independently by two observers. The test sensitivity, specificity, and reliability indexes were calculated. RESULTS: The sensitivity of the lectin staining test for Candida albicans was 100% (95% CI: 93.51-100.00), and specificity was 100% (95% CI: 93.51-100.00). The sensitivity of the test for Aspergillus fumigatus was 96.36% (95% CI: 86.46-99.35), and specificity was 100% (95% CI: 93.51-100.00). The sensitivity of the test for Fusarium solani was 96.36% (95% CI: 86.46-99.35) and specificity was 96.15% (95% CI: 85.74-99.31). There was also a high degree of test-retest and inter-rater concordance for all three fungi tested. The test-retest k reliability indexes were 0.9455, 0.9636, and 0.8879, for Candida albicans, Aspergillus fumigatus, and Fusarium solani, respectively. The inter-rater k reliability indexes were 0.9636, 0.9818, and 0.9252, for Candida albicans, Aspergillus fumigatus, and Fusarium solani, respectively. CONCLUSIONS: WGA-peroxidase staining is a very sensitive, specific, and reliable test for the identification of fungi in an experimental rabbit model of fungal keratitis.
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Infecciones Fúngicas del Ojo/diagnóstico , Queratitis/diagnóstico , Sondas Moleculares , Micosis/diagnóstico , Aglutinina del Germen de Trigo-Peroxidasa de Rábano Silvestre Conjugada , Animales , Aspergillus fumigatus/fisiología , Candida albicans/fisiología , Sustancia Propia/microbiología , Sustancia Propia/patología , Infecciones Fúngicas del Ojo/microbiología , Fusarium/fisiología , Técnicas para Inmunoenzimas , Queratitis/microbiología , Metenamina , Modelos Animales , Micosis/microbiología , Variaciones Dependientes del Observador , Mitógenos de Phytolacca americana , Conejos , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Coloración y Etiquetado/métodosRESUMEN
PURPOSE: To evaluate the ability of topical hyaluronic acid to decrease corneal opacity after excimer laser photorefractive keratectomy (PRK) in hens. METHODS: Twenty-four white hens underwent bilateral 193-nm excimer laser PRK to correct -9.00 D of myopia. One eye received postoperative treatment with topical 1% hyaluronic acid six times daily for 3 days; the other eye received phosphate buffered saline. Slit-lamp evaluation by a masked observer was performed for 6 months after PRK, and electron microscopy was carried out at the end of the study. RESULTS: There were no significant differences in postoperative haze between the eyes treated with hyaluronic acid and those treated with phosphate buffered saline. CONCLUSION: Topical administration of hyaluronic acid had no effect on the development of corneal haze following PRK in hens.
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Córnea/cirugía , Opacidad de la Córnea/prevención & control , Ácido Hialurónico/administración & dosificación , Miopía/cirugía , Queratectomía Fotorrefractiva/efectos adversos , Administración Tópica , Animales , Pollos , Córnea/ultraestructura , Opacidad de la Córnea/etiología , Opacidad de la Córnea/patología , Modelos Animales de Enfermedad , Láseres de Excímeros , Miopía/patología , Cicatrización de Heridas/efectos de los fármacosRESUMEN
The physical character and amount of mucus secreted by the endocervix changes dramatically during the menstrual cycle to facilitate sperm migration at the time of midcycle ovulation. Mucins are highly glycosylated, high-molecular-weight proteins, which are the major structural components of the protective mucus gel covering all wet-surfaced epithelia, including that of the endocervix. We have previously demonstrated that the endocervical epithelium expresses messenger RNA (mRNA) of three of the large gel-forming mucins, designated MUC5AC, MUC5B, and MUC6, with mRNA of MUC5B predominating. Because mucin protein levels may be regulated posttranscriptionally, measurement of MUC5B protein levels with cycle are needed for correlation to mRNA levels. Measurement of specific mucin gene products within mucus secretions has been limited by availability of specific, well-characterized antibodies and by volume requirements of the isolation protocols for mucins, which include CsCl density centrifugation and fraction isolation. To measure MUC5B protein within the cervical mucus through the hormone cycle, we developed a polyclonal antibody specific to the mucin. The antibody, designated no. 799, is to a synthetic peptide mimicking a 19-amino-acid segment of an intercysteine-rich region within the D4 domain in the 3' region of the MUC5B protein. It recognizes native as well as denatured MUC5B on immunoblot, is preadsorbable with its peptide, and binds to apical secretory vesicles of epithelia expressing MUC5B. We used the MUC5B antibody along with a cervical mucin standard cervical mucin isolate in enzyme-linked immunosorbent assay to determine the relative amount of MUC5B mucin in samples of human cervical mucus taken through the menstrual cycle. We demonstrate a peak of MUC5B mucin in human cervical mucus collected at midcycle, compared with mucus from early or late in the cycle. This peak in MUC5B content coincides with the change in mucus character that occurs at midcycle, suggesting that this large mucin species may be important to sperm transit to the uterus.
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Moco del Cuello Uterino/fisiología , Regulación de la Expresión Génica , Ciclo Menstrual/fisiología , Mucinas/genética , Secuencia de Aminoácidos , Especificidad de Anticuerpos , Moco del Cuello Uterino/citología , Ensayo de Inmunoadsorción Enzimática , Epítopos/química , Epítopos/inmunología , Femenino , Humanos , Hormona Luteinizante/metabolismo , Datos de Secuencia Molecular , Mucina 5B , Mucinas/análisis , Mucinas/sangre , ARN Mensajero/análisis , Análisis de Regresión , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Saliva/químicaRESUMEN
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.
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Carboximetilcelulosa de Sodio/toxicidad , Córnea/efectos de los fármacos , Soluciones Oftálmicas/toxicidad , Polímeros/toxicidad , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Córnea/ultraestructura , Geles , Microscopía Electrónica de Rastreo , ConejosRESUMEN
Mature leaves of dwarf elder (Sambucus ebulus L.) contain the non-toxic type 2 ribosome-inactivating protein ebulin 1 (Girbés et al., 1993b, J. Biol. Chem. 268: 18195-18199). We have now found that the green fruits of dwarf elder contain both free and polymerized forms of ebulin (ebulin f) and a new homodimeric D-galactose-binding lectin (SELfd). Polymerized material containing ebulin and lectin is composed of aggregates of variable relative molecular mass, some of them being close to 250,000. These aggregate forms are maintained in part by reducible disulphide bridges and reconstitute from reductant-free dialyzed material previously reduced with 2-mercaptoethanol. Direct incubation of free ebulin f with the free SELfd did not lead to polymerization, thus indicating that polymerization triggers some kind of substantial and perhaps catalyzed change in the structure of these proteins. Ebulin-containing polymerized material reacts with anti-ebulin f antibodies. Our results indicate that ebulin f is a fruit-form of ebulin 1. In contrast to green fruits, mature fruits lack both polymerized material and ebulin f, thus indicating some kind of reserve role for them in green fruits. Polymerization of ebulin and the dimeric lectin may represent a novel means of storing the non-toxic type 2 ribosome-inactivating proteins and lectins found in highly metabolic tissues, such as green fruits.
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Proteínas de Plantas/química , Árboles/química , Secuencia de Aminoácidos , Biopolímeros , Reacciones Cruzadas , Galactosa/metabolismo , Datos de Secuencia Molecular , Proteínas de Plantas/metabolismo , Proteínas de Plantas/farmacología , Unión Proteica , Biosíntesis de Proteínas/efectos de los fármacos , Proteínas Inactivadoras de Ribosomas Tipo 2 , Homología de Secuencia de AminoácidoRESUMEN
PURPOSE: Our goal was to establish the characteristic migration pattern on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of high molecular weight mucins from human ocular mucus and the effects of treatment with exo- and endoglycosidases. METHODS: Chromatography by gel filtration with Sepharose CL-4B was performed on samples collected from normal subjects. Human ocular mucins from the high molecular weight fraction were digested with exoglycosidases (neuraminidase, N-acetyl-beta-D-glucosaminidase, beta-D-glucosidase) and endoglycosidases (chitinase, lysozyme); and the resulting products were analyzed by electrophoresis. Carbohydrate identification was performed using lectin probes. RESULTS: The migration of the ocular mucins on SDS-PAGE stopped after treatment with neuraminidase, which removes the terminal negatively charged sialic acid residues from mucin. Chitinase (beta(1-4)N-acetylglucosaminidase) treatment increased the electrophoretic migration of mucins. Staining with wheat germ agglutinin and Maackia amurensis agglutinin lectins showed that these mucins contain beta(1-4)NAcGlc and SAa(2-3)Gal linkages. CONCLUSIONS: These studies demonstrate that the mobility of human ocular mucins on SDS-PAGE is determined by their intrinsic total negative charge and is not dependent on SDS treatment. It is interesting to note that human ocular mucus contains chitinous material resistant to lacrimal lysozyme, which is accessible to chitinase, an enzyme now found to degrade human ocular mucins. These chitinous linkages could be in part responsible for the mucus resistance.
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Quitinasas/farmacología , Aparato Lagrimal/química , Mucinas/análisis , Moco/química , Neuraminidasa/farmacología , Adolescente , Adulto , Cromatografía en Gel , Electroforesis en Gel de Poliacrilamida , Femenino , Humanos , Lectinas , Masculino , Persona de Mediana Edad , Moco/efectos de los fármacos , Ácido N-Acetilneuramínico/análisis , Polisacáridos/análisis , Sefarosa/análogos & derivadosRESUMEN
A new N-glycosidase ribosome-inactivating protein (RIP) belonging to the novel family of the nontoxic type 2 RIPs from Sambucaceae has been isolated from rhizomes of dwarf elder (Sambucus ebulus L.) and named ebulin r. Dwarf elder rhizomes also contain a novel monomeric N-Ac-galactosamine-binding lectin that we named SEAII. Ebulin r and SEAII have two isoforms each one, which were readily resolved by ion exchange. Both isoforms of ebulin (ebulins r1 and r2) strongly inhibited protein synthesis in mammalian but not in plant ribosomes by promoting depurination of sensitive ribosomes. Ebulin r and SEAII have apparent molecular masses of 56 and 33.5 kDa, respectively. Ebulins r1 and r2 are composed of two dissimilar subunits (types A-B) of apparent molecular masses of 26 and 30 kDa by disulphide bridges. The rhizome SEAII and the lectins SNA II and SNA III from elder (Sambucus nigra L.) share good amino acid sequence homology. This rhizome ebulin-A chain is more sequence-related to RIP members of cucurbitaceae than to any other plant family. The rhizome ebulin B chain shares a large homology in amino acid sequence with ebulin 1-B chain and SEAII. Anti-ebulin 1 polyclonal antibodies raised in rabbits reacted better with ebulin r1 than with ebulin r2, thus suggesting that both RIP isoforms could have some differences.