RESUMO
This study identified a novel phenomenon that dendritic cells (DCs) produced interleukin (IL)-33 via Toll-like receptor (TLR)-mediated innate pathway. Mouse bone marrow-derived DCs were treated with or without microbial pathogens or recombinant murine IL-33. IL-33 mRNA and protein were found to be expressed by DCs and largely induced by several microbial pathogens, highly by lipopolysaccharide (LPS) and flagellin. Using two mouse models of topical challenge by LPS and flagellin and experimental allergic conjunctivitis, IL-33-producing DCs were observed in ocular mucosal surface and the draining cervical lymph nodes in vivo. The increased expression levels of myeloid differentiation primary-response protein 88 (MyD88), nuclear factor (NF)-κB1, NF-κB2, and RelA accompanied by NF-κB p65 nuclear translocation were observed in DCs exposed to flagellin. IL-33 induction by flagellin was significantly blocked by TLR5 antibody or NF-κB inhibitor quinazoline and diminished in DCs from MyD88 knockout mice. IL-33 stimulated the expression of DC maturation markers, CD40 and CD80, and proallergic cytokines and chemokines, OX40L, IL-4, IL-5, IL-13, CCL17 (C-C motif chemokine ligand 17), TNF-α (tumor necrosis factor-α), and IL-1ß. This stimulatory effect of IL-33 in DCs was significantly blocked by ST2 antibody or soluble ST2. Our findings demonstrate that DCs produce IL-33 via TLR/NF-κB signaling pathways, suggesting a molecular mechanism by which local allergic inflammatory response may be amplified by DC-produced IL-33 through potential autocrine regulation.
Assuntos
Conjuntivite Alérgica/imunologia , Células Dendríticas/imunologia , Mucosa/imunologia , Animais , Anticorpos Bloqueadores/administração & dosagem , Comunicação Autócrina , Diferenciação Celular , Células Cultivadas , Citocinas/metabolismo , Células Dendríticas/efeitos dos fármacos , Flagelina/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Proteína 1 Semelhante a Receptor de Interleucina-1 , Lipopolissacarídeos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Animais , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , NF-kappa B/antagonistas & inibidores , NF-kappa B/genética , NF-kappa B/metabolismo , Quinazolinas/administração & dosagem , Quinazolinas/farmacologia , Receptores de Interleucina/imunologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Receptor 5 Toll-Like/imunologia , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismoRESUMO
Dry eye is a multifactorial condition that results in a dysfunctional lacrimal functional unit. Evidence suggests that inflammation is involved in the pathogenesis of the disease. Changes in tear composition including increased cytokines, chemokines, metalloproteinases and the number of T cells in the conjunctiva are found in dry eye patients and in animal models. This inflammation is responsible in part for the irritation symptoms, ocular surface epithelial disease, and altered corneal epithelial barrier function in dry eye. There are several anti-inflammatory therapies for dry eye that target one or more of the inflammatory mediators/pathways that have been identified and are discussed in detail.
Olho seco é uma doença multifatorial que resulta em disfunção da unidade lacrimal glandular. Evidências sugerem que inflamação está involvida na patogênese da doença. Mudanças na composição das lágrimas, incluindo aumento de citocinas, quimiocinas, metaloproteinases e o número de células T na conjuntiva são encontrados em pacientes com olho seco e em modelos animais. Esta inflamação é responsável em parte pelos sintomas de irritação, doença epitelial de surperfície ocular e função epitelial de barreira alterada em olho seco. Existem várias terapias antiinflamatórias que se direcionam para um ou mais mediadores/vias que foram identificados e são discutidos em detalhe.
Assuntos
Humanos , Anti-Inflamatórios/uso terapêutico , Síndromes do Olho Seco/tratamento farmacológico , Corticosteroides/uso terapêutico , Ciclosporina/uso terapêutico , Síndromes do Olho Seco/metabolismo , Tetraciclina/uso terapêuticoRESUMO
AIM: To evaluate the expression pattern of glial cell line-derived neurotrophic factor (GDNF) with its receptors GDNF family receptor alpha-1 (GFR alpha-1) and Ret in the human corneal and limbal tissues, as well as in the primary human limbal epithelial cultures (PHLEC). METHODS: Expression of GDNF and its receptors, and the co-localisation with stem cell associated and differentiation markers were evaluated by immunofluorescent staining, western blot analysis and real-time PCR in the fresh human corneoscleral tissues, as well as in the PHLEC. Single cell colony-forming and wound-healing assays were also evaluated in PHLEC. RESULTS: GDNF and GFR alpha-1 were found to be expressed by a subset of basal cells and co-localised with ATP-binding cassette, subfamily G (WHITE), member 2 (ABCG2) and p63, but not with cytokeratin 3 in the human limbal basal epithelium. In PHLEC, they were expressed by a small population of cells in the less differentiated stage. The GDNF and GFR alpha-1-positive subpopulations were enriched for the expression of ABCG2 and p63 (p<0.01). Recombinant human GDNF promoted the proliferation and wound healing of epithelial cells in the PHLEC. In contrast, Ret was abundantly located in the human corneal epithelium except for the basal cells of the limbal epithelium. CONCLUSION: These findings indicate that GDNF and GFR alpha-1 may represent a property for the phenotype of human corneal epithelial precursor cells. GDNF may signal independently of Ret through GFR alpha-1 in the stem cell-containing limbal epithelium.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Córnea/metabolismo , Células Epiteliais/metabolismo , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Análise de Variância , Células Cultivadas/metabolismo , Células Epiteliais/citologia , Epitélio Corneano/citologia , Epitélio Corneano/metabolismo , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Humanos , Proteínas Proto-Oncogênicas c-ret/metabolismo , RNA/isolamento & purificação , Transdução de Sinais/genética , Células-Tronco/citologia , Células-Tronco/metabolismoRESUMO
PURPOSE: To compare the expression of the pro- and anti-inflammatory forms of interleukin (IL)-1 in the tear fluid and conjunctival epithelium of normal eyes and those with dry-eye disease. METHODS: The concentrations of IL-1 alpha, IL-1 beta (precursor and mature forms), and IL-1 receptor antagonist (IL-1Ra) were measured by ELISA in tear fluid samples obtained from normal individuals and patients with dry eye who had rosacea-associated meibomian gland disease (MGD) or Sjögren's syndrome (SS) aqueous tear deficiency (ATD). These cytokines were also measured in normal tear fluid before and after nasal stimulation to induce reflex tearing. The relative expression of these cytokines was evaluated in conjunctival impression cytology specimens and conjunctival biopsy tissue obtained from normal subjects and SS ATD-affected patients using immunofluorescent staining. Matrix metalloproteinase (MMP)-9 concentration and activity in the tear fluid were evaluated with gelatin zymography and with an MMP-9 activity assay kit, respectively. RESULTS: Compared with normal subjects, the concentration of IL-1 alpha and mature IL-1 beta in the tear fluid was increased, and the concentration of precursor IL-1 beta was decreased in patients with MGD (P < 0.05, P = 0.02, and P < 0.01, respectively) and SS ATD (P < 0.001, P = 0.02, and P < 0.001, respectively). There was no significant change in the concentration of IL-1 alpha, precursor IL-1 beta, and IL-1Ra in reflex tear fluid, indicating that the lacrimal glands may secrete these cytokines. The activity of MMP-9, a physiological activator of IL-1 beta, was significantly elevated in the tear fluid of both dry-eye groups compared with normal subjects. A strong positive correlation was observed between the intensity of corneal fluorescein staining and the tear fluid IL-1 alpha concentration (r(2) = 0.17, P < 0.02) and the mature-to-precursor IL-1 beta ratio (r(2) = 0.46, P < 0.001). Positive immunofluorescent staining for IL-1 alpha, mature IL-1 beta, and IL-1Ra was observed in a significantly greater percentage of conjunctival cytology specimens from eyes with SS ATD than in those from normal eyes (P < 0.01 for IL-1 alpha, P < 0.009 for mature IL-1 beta, and P < 0.05 for IL-1Ra). CONCLUSIONS: Dry-eye disease is accompanied by an increase in the proinflammatory forms of IL-1 (IL-1 alpha and mature IL-1 beta) and a decrease in the biologically inactive precursor IL-1 beta in tear fluid. Increased protease activity on the ocular surface may be one mechanism by which precursor IL-1 beta is cleaved to the mature, biologically active form. The conjunctival epithelium appears to be one source of the increased concentration of IL-1 in the tear fluid of patients with dry-eye disease. These results suggest that IL-1 may play a key role in the pathogenesis of keratoconjunctivitis sicca.
Assuntos
Túnica Conjuntiva/metabolismo , Síndromes do Olho Seco/metabolismo , Proteínas do Olho/metabolismo , Interleucina-1/metabolismo , Sialoglicoproteínas/metabolismo , Lágrimas/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Síndromes do Olho Seco/patologia , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Fluorofotometria , Humanos , Proteína Antagonista do Receptor de Interleucina 1 , Lactoferrina/metabolismo , Masculino , Metaloproteinase 9 da Matriz/metabolismo , Pessoa de Meia-IdadeRESUMO
PURPOSE: A case of inferior corneal thinning and high astigmatism with features of keratoconus in a patient with long-standing ocular rosacea is described. METHODS: Axial curvature mapping was performed with the Tomey TMS-1 videokeratoscopy instrument and corneal thickness mapping was performed with the Orbscan Corneal Topography System (CTS). Tear clearance was assessed by measuring the concentration of fluorescein in the tear fluid with a fluorometer. RESULTS: There were inferior corneal thinning and opacification in both eyes. Tear fluorescein clearance was markedly delayed in the right eye. There was asymmetric inferior corneal steepening in both eyes with I-S values of 1 in the right eye and 5.9 in the left eye. There were 5.9 diopters of astigmatism at 85 degrees in the right eye and 7.3 diopters of astigmatism at 73 degrees in the left eye. Corneal pachymetry mapping with the Orbscan CTS showed a normal central corneal thickness and maximal thinning in the inferotemporal periphery of the right cornea and inferonasal periphery of the left cornea. CONCLUSION: Chronic ocular rosacea can produce inferior corneal thinning and high astigmatism with some features of keratoconus. The inferior pattern of thinning in rosacea may be related to chronic exposure of the inferior cornea to inflammatory and matrix-degrading factors in the inferior tear meniscus.
Assuntos
Astigmatismo/etiologia , Ceratocone/etiologia , Rosácea/complicações , Administração Oral , Antibacterianos/uso terapêutico , Astigmatismo/tratamento farmacológico , Topografia da Córnea , Doxiciclina/uso terapêutico , Feminino , Humanos , Ceratocone/tratamento farmacológico , Pessoa de Meia-Idade , Rosácea/tratamento farmacológico , Acuidade VisualRESUMO
PURPOSE: To investigate the distribution and relative level of expression of the receptor tyrosine kinases, epidermal growth factor receptor (EGFR), ErbB2 and ErbB3, in human ocular surface epithelia. METHODS: Immunofluorescent staining was performed to identify expression of the EGFR, ErbB2 and ErbB3 in the corneal, limbal and conjunctival epithelium in tissue sections and impression cytologies taken from normal human eyes. Western blotting was undertaken to confirm the results of immunofluorescent staining. RESULTS: The three receptor tyrosine kinases, EGFR, ErbB2 and ErbB3, were detected in human corneal, limbal and conjunctival epithelia by immunofluorescent staining. Strong staining for the EGFR was observed in the basal epithelial cells at all 3 sites and throughout the corneal epithelium. Minimal or no staining for the EGFR was observed in the superficial conjunctival and limbal epithelia. The strongest staining for ErbB2 and ErbB3 was observed in the superficial ocular surface epithelium. All three receptors were detected in the corneal, limbal and conjunctival epithelium by western blot. CONCLUSION: EGFR, ErbB2 and ErbB3 are expressed by the ocular surface epithelia. EGFR is preferentially expressed by the basal epithelial cells that have the greatest proliferative potential. In contrast, ErbB2 and ErbB3 are preferentially expressed by the superficial differentiated ocular surface epithelia.
Assuntos
Túnica Conjuntiva/metabolismo , Epitélio Corneano/metabolismo , Receptores ErbB/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Receptor ErbB-2/metabolismo , Receptor ErbB-3/metabolismo , Animais , Biomarcadores , Western Blotting , Túnica Conjuntiva/citologia , Epitélio Corneano/citologia , Imunofluorescência , Humanos , RatosRESUMO
The matrix metalloproteinases, MMP-2 and MMP-9, are known to be critical extracellular-remodeling enzymes in wound healing and other diseases of the ocular surface. This study investigated the regulation of MMP-2 and MMP-9 in human corneal epithelial cells by growth factors and pro-inflammatory cytokines (IL-1beta and TNF-alpha) they are exposed to, and by doxycycline, a medication used to treat ocular surface disease. Primary human corneal epithelial cell cultures were treated with one of the following cytokines (IL-1alpha, IL-1beta, IL-6, IL-8, TNF-alpha) or growth factors (EGF, HGF, KGF, PDGF-BB, TGF-alpha, TGF-beta), with or without their corresponding inhibitors. The conditioned media were collected after 24 hr for gelatin zymography and MMP-9 activity assay. Total RNA was extracted from the cells treated for 6 hr and was subjected to RT-PCR and Northern hybridization. Between the two gelatinases, MMP-2 and MMP-9, detected by zymography, the 92 kDa MMP-9 in the conditioned medium was markedly up-regulated by the pro-inflammatory cytokines, IL-1beta and TNF-alpha. The MMP-9 protein and activity were dose-dependently stimulated by IL-1beta or TNF-alpha at 0.1, 1.0 and 10 ng ml(-1). This up-regulation was nearly abolished by neutralizing antibodies (IL-1beta and TNF-alpha) and by IL-1 receptor antagonist. Semi-quantitative RT-PCR and Northern hybridization disclosed that the MMP-9 transcript was also markedly up-regulated in a dose-dependent manner by IL-1beta and TNF-alpha. Doxycycline (10 microg ml(-1)) suppressed MMP-9 protein level and activity, but not its mRNA, that was stimulated by IL-1beta and TNF-alpha (1 ng ml(-1)). In contrast, the 72 kDa MMP-2 was not significantly modulated by any of these cytokines. In conclusion, production of MMP-9 is stimulated by the pro-inflammatory cytokines, IL-1beta and TNF-alpha. These factors may play a role in the pathogenesis of MMP-9 mediated corneal matrix degradation. The efficacy of doxycycline in treating ocular surface diseases may be related to its ability to suppress MMP-9 production in the corneal epithelium.
Assuntos
Epitélio Corneano/enzimologia , Metaloproteinase 9 da Matriz/biossíntese , Técnicas de Cultura de Células , Meios de Cultivo Condicionados/metabolismo , Citocinas/farmacologia , Epitélio Corneano/efeitos dos fármacos , Regulação da Expressão Gênica , Humanos , Interleucina-1/farmacologia , Metaloproteinase 2 da Matriz/biossíntese , Metaloproteinase 9 da Matriz/genética , RNA Mensageiro/genética , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
PURPOSE: To evaluate interleukin-6 (IL-6) levels in the conjunctival epithelium of patients with moderate to severe dry eye disease before and after treatment with cyclosporin A ophthalmic emulsion (CsA) or its vehicle. METHODS: Conjunctival cytology specimens were obtained from a subset of patients enrolled in a 6-month randomized, double-masked clinical trial of the efficacy and safety of topical CsA at baseline and after 3 and 6 months of B.I.D. treatment with 0.05% cyclosporine emulsion (n = 13), 0.1% cyclosporine emulsion (n = 8), or vehicle (n = 10). RNA was extracted and a competitive reverse transcriptase polymerase chain reaction (RT-PCR) was used to evaluate the levels of mRNA encoding the inflammatory cytokine IL-6 and a housekeeping gene, G3PDH. Levels of IL-6 and G3PDH were measured and compared. RESULTS: There was no change from baseline in the level of G3PDH after 3 or 6 months in any group. IL-6 normalized for G3PDH (IL-6/G3PDH ratio) was not different from baseline at 3 months but showed a significant decrease from baseline in the group treated with 0.05% CsA (p = 0.048) at 6 months. No significant between-group differences were noted and no correlation was observed between the change in IL-6/G3PDH and corneal fluorescein staining. CONCLUSIONS: This preliminary, small-cohort study showed a decrease in IL-6 in the conjunctival epithelium of moderate to severe dry eye patients treated with 0.05% CsA for 6 months. The observed decrease suggests that dry eye disease involves immune-mediated inflammatory processes that may be decreased by treatment with topical ophthalmic cyclosporine.
Assuntos
Túnica Conjuntiva/metabolismo , Ciclosporina/uso terapêutico , Síndromes do Olho Seco/tratamento farmacológico , Epitélio/metabolismo , Imunossupressores/uso terapêutico , Interleucina-6/metabolismo , Administração Tópica , Biomarcadores , Túnica Conjuntiva/patologia , Primers do DNA/química , Método Duplo-Cego , Síndromes do Olho Seco/metabolismo , Síndromes do Olho Seco/patologia , Emulsões , Epitélio/patologia , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Humanos , Interleucina-6/genética , Estudos Prospectivos , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
PURPOSE: To evaluate human corneal epithelial culture supernatant and tear fluid for the presence of activators and inhibitors of matrix metalloproteinase (MMP)-9, MMP-3, and tissue inhibitor of metalloproteinase (TIMP)-1, respectively, and to evaluate the effect of MMP-3 on the activation of MMP-9 in these specimens. METHODS: Unstimulated tear fluid was collected from patients with ocular rosacea and normal control subjects. Levels of MMP-9, MMP-3, and TIMP-1 were determined by enzyme-linked immunosorbent assay (ELISA) and/or immunoblot analysis. Supernatants from primary human corneal epithelial cultures and human tear fluid were incubated with MMP-3. Cultured epithelial cells and their supernatants were also treated with doxycycline before MMP-3 was added. Gelatin zymography was used to identify activated 82-kDa MMP-9. MMP-9 activity was assessed with a commercial MMP-9 activity assay system. RESULTS: MMP-9 and TIMP-1 were detected at significantly higher concentrations in rosacea-affected than in normal tear fluids. MMP-3 was detected exclusively in the tear fluid of patients with ocular rosacea who had corneal epithelial disease. Treatment of the supernatant and tear fluid with MMP-3 resulted in two bands with molecular weights of 92 kDa and 82 kDa, representing pro-MMP-9 and activated MMP-9, respectively. Doxycycline added to the conditioned media did not affect activation of MMP-9 by MMP-3. However, 24-hour treatment of corneal epithelial cultures with doxycycline resulted in a lower concentration and activity of MMP-9 in their supernatants. CONCLUSIONS: MMP-9 and TIMP-1 are produced by the human corneal epithelium and are present in tear fluid. MMP-3 alone is sufficient to activate MMP-9 on the ocular surface. Doxycycline does not directly inhibit this activation by MMP-3, but it decreases MMP-9 activity when added to corneal epithelial cultures.
Assuntos
Epitélio Corneano/enzimologia , Metaloproteinase 9 da Matriz/metabolismo , Lágrimas/enzimologia , Western Blotting , Células Cultivadas , Doxiciclina/farmacologia , Ativação Enzimática/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Epitélio Corneano/efeitos dos fármacos , Epitélio Corneano/patologia , Doenças Palpebrais/enzimologia , Doenças Palpebrais/etiologia , Humanos , Metaloproteinase 3 da Matriz/metabolismo , Metaloproteinase 3 da Matriz/farmacologia , Glândulas Tarsais/enzimologia , Glândulas Tarsais/patologia , Rosácea/complicações , Rosácea/enzimologia , Inibidor Tecidual de Metaloproteinase-1/metabolismoRESUMO
PURPOSE: To evaluate human ocular surface epithelium and tear fluid for the presence of sialomucin complex (MUC4), a high-molecular-weight heterodimeric glycoprotein composed of mucin (ASGP-1) and transmembrane (ASGP-2) subunits. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) and Northern blot analysis assays were used to identify sialomucin complex RNA in ocular surface epithelia. Immunoprecipitation and immunoblot analysis were used to identify immunoreactive species in human tears and in the corneal and conjunctival epithelia using antibodies specific for carbohydrate and peptide epitopes on the sialomucin complex subunits. Immunofluorescence staining was used to detect sialomucin complex in frozen sections and impression cytology specimens of human cornea and conjunctival epithelia. RESULTS: ASGP-1- and ASGP-2-specific sequences were amplified from RNA extracted from both conjunctival and corneal epithelial biopsies by RT-PCR. Sialomucin complex transcripts were also detected in these tissues by Northern blot analysis, with a greater level of RNA detected in the peripheral than the central corneal epithelium. Sialomucin complex was immunoprecipitated from tear fluid samples and both corneal and conjunctival epithelia and detected by immunoblot analysis with specific anti-ASGP-1 and anti-ASGP-2 antibodies. The ASGP-1 peptide antibody HA-1 stained the full thickness of the corneal and conjunctival epithelia. In contrast, antibody 15H10, which reacts against a carbohydrate epitope on ASGP-1, stained only the superficial epithelial layers of these tissues. No staining was observed in the conjunctival goblet cells. CONCLUSIONS: Sialomucin complex was originally identified in rat mammary adenocarcinoma cells and has recently been shown to be produced by the ocular surface epithelia of rats. Furthermore, it has been identified as the rat homologue of human MUC4 mucin. The present studies show that it is expressed in the stratified epithelium covering the surface of the human eye and is present in human tear fluid. Expression of a carbohydrate-dependent epitope on the mucin subunit (ASGP-1) of sialomucin complex occurs in a differentiation-dependent fashion. Sialomucin complex joins MUC1 as another membrane mucin produced by the human ocular surface epithelia but is also found in the tear fluid, presumably in a soluble form, as found on the rat ocular surface.
Assuntos
Túnica Conjuntiva/química , Epitélio Corneano/química , Mucinas/análise , Lágrimas/química , Especificidade de Anticorpos , Northern Blotting , Epitélio/química , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Mucina-4 , Mucinas/genética , RNA/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sialoglicoproteínas/análiseRESUMO
PURPOSE: To investigate the expression of type 1 growth factor receptors (epidermal growth factor receptor, ErbB2, and ErbB3) in the conjunctival epithelium of patients with keratoconjunctivitis sicca. METHODS: Immunofluorescent staining and Western blotting were performed to grade the level of expression of the epidermal growth factor receptor ErbB2, and ErbB3 in conjunctival epithelial impression cytologies taken from both eyes of seven normal subjects and 22 patients with keratoconjunctivitis sicca. RESULTS: Epidermal growth factor receptor staining was observed in a greater percentage of keratoconjunctivitis sicca than normal samples (P <.05). ErbB2 and ErB3 staining in the apical conjunctival epithelium was observed in both groups, but stronger ErbB2 and ErbB3 staining was noted in keratoconjunctivitis sicca conjunctival samples (P <.05). The relative levels of expression of these receptor proteins on immunoblots were consistent with immunofluorescent staining. On immunoblots, epidermal growth factor receptor protein was detected in 50% of keratoconjunctivitis sicca samples, but none of the normal samples (P <.025). The expression of ErbB2 and ErbB3 on immunoblots was also greater in the keratoconjunctivitis sicca samples (P <.05). Immunofluorescent staining scores for these receptors were correlated with conjunctival lissamine green staining scores (r =. 574, P <.01 for epidermal growth factor receptor; r =.620, P <.0025 for ErbB2; r =.502, P <.025 for ErbB3) and with corneal fluorescein staining (r =.409, P <.05 for ErbB2; r =.588, P <.005 for ErbB3). CONCLUSION: The expression of the type 1 growth factor receptors is significantly greater in the conjunctival epithelium of eyes with keratoconjunctivitis sicca than normal eyes. The increased expression of these receptors was positively correlated with ocular surface dye staining. The increased expression of these receptors may contribute to the abnormal growth and differentiation of the conjunctival epithelium that occurs in keratoconjunctivitis sicca.
Assuntos
Túnica Conjuntiva/metabolismo , Ceratoconjuntivite Seca/metabolismo , Receptor ErbB-2/biossíntese , Receptor ErbB-3/biossíntese , Adulto , Idoso , Anticorpos Monoclonais , Western Blotting , Túnica Conjuntiva/patologia , Epitélio/metabolismo , Epitélio/patologia , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Ceratoconjuntivite Seca/patologia , Pessoa de Meia-IdadeRESUMO
OBJECTIVE: To evaluate the expression of type 1 growth factor receptor family [epidermal growth factor receptor (EGFR), ErbB2 and ErbB3] by the epithelium in pterygium so as to understand the pathogenesis of this disorder. METHODS: The immunofluorescent staining and Western blotting were performed in 15 patients with primary pterygium, compared to normal conjunctival epithelium, to determine the expression of EGFR, ErbB2 and ErbB3 proteins. RESULTS: The EGFR was present at the basal cells while the ErbB2 and ErbB3 were expressed by the superficial cells in normal conjunctival epithelium in immunofluorescent staining. Of 15 pterygia, 11 pterygia expressed the stains of EGFR, ErbB2 and ErbB3 in the full thickness of epithelium and with stronger staining compared to the control group, 4 of them showed the same staining pattern as that in the normal conjunctiva. Compared to normal conjunctiva, the stronger density of protein bands of EGFR, ErbB2 and ErbB3 was also demonstrated by western blotting in 11 pterygia with strong staining of these antibodies. CONCLUSIONS: The expression of EGFR, ErbB2 and ErbB3 is increased in pterygium, showing that the pterygium is a disorder with proliferation. The abnormal expression of EGFR, ErbB2 and ErbB3 by epithelium and their communicating with cytokines at stroma in pterygium may be one of the key pathogenetic factors in this disorder.
Assuntos
Receptores ErbB/biossíntese , Pterígio/metabolismo , Receptor ErbB-2/biossíntese , Receptor ErbB-3/biossíntese , Adulto , Epitélio/metabolismo , Epitélio/patologia , Feminino , Humanos , Hiperplasia , Masculino , Pessoa de Meia-Idade , Pterígio/patologiaRESUMO
PURPOSE: To correlate tear fluorescein clearance with interleukin-1alpha (IL-1alpha) concentration and gelatinase B (matrix metalloproteinase [MMP]-9) activity in the tear fluid of patients with ocular rosacea and normal control subjects. METHODS: Gelatinase activity was evaluated by gelatin zymography in tear fluid obtained from 13 patients with ocular rosacea (including 1 patient with recurrent epithelial erosion, 2 with recurrent peripheral corneal infiltrates and vascularization, and 2 patients with epithelial basement membrane dystrophy) and 13 normal subjects with normal aqueous tear production and no irritation symptoms. Tear fluorescein clearance was evaluated by measuring fluorescence in tear fluid collected from the inferior meniscus 15 minutes after instillation of 5 microl of 2% Na-fluorescein with a CytoFluor II fluorometer. Pro-MMP-9 and IL-1alpha concentrations in the tear fluid were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: Compared with normal control subjects, patients with ocular rosacea had a greater delay of tear fluorescein clearance (P < 0.001), a higher tear IL-1alpha concentration (P < 0.001), and a greater pro-gelatinase B (92 kDa) activity (P < 0.001) in their tear fluid. The 84-kDa active form of gelatinase B was observed in 46% of the rosacea tear samples and none of the controls. The zymographic results were confirmed by ELISA that showed a significantly greater concentration of pro-MMP-9 (92 kDa) in the tear fluid of rosacea patients than controls. Delayed tear clearance was correlated with elevated tear IL-1alpha concentration (p=0.67, P < 0.001) and increased tear gelatinase B activity (p=0.84, P < 0.001). Tear IL-1alpha concentration was correlated with tear gelatinase B activity (p=0.58, P < 0.002). CONCLUSIONS: Gelatinase B (MMP-9) activity is greater in patients with ocular rosacea than in normal eyes. The majority of this activity is due to 92-kDa proform of this enzyme. This activity is correlated with delayed tear clearance and tear fluid concentration of interleukin-1alpha, a proinflammatory cytokine that has been reported to stimulate gelatinase B production. Elevated gelatinase B activity in ocular rosacea may be involved in the pathogenesis of the irritation symptoms, recurrent epithelial erosions, vascularization, and epithelial basement membrane dystrophy that develops in the corneas of patients with this condition.
Assuntos
Colagenases/metabolismo , Doenças da Túnica Conjuntiva/metabolismo , Doenças da Córnea/metabolismo , Fluoresceína/farmacocinética , Interleucina-8/metabolismo , Rosácea/metabolismo , Lágrimas/enzimologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Ensaio de Imunoadsorção Enzimática , Feminino , Fluorofotometria , Humanos , Masculino , Metaloproteinase 9 da Matriz , Pessoa de Meia-IdadeRESUMO
PURPOSE: To compare epidermal growth factor (EGF) concentration in tear fluid and levels of inflammatory cytokines in the conjunctival epithelium of patients with Sjögren's syndrome keratoconjunctivitis sicca with those of normal controls. METHODS: Schirmer 1 tear testing, corneal fluorescein staining and conjunctival impression cytology for quantitation of goblet cell density were performed in ten patients with Sjögren's syndrome-associated keratoconjunctivitis sicca and ten asymptomatic normal controls. ELISA was used to detect the concentration of EGF in tear fluid and interleukin 6 in lysates of conjunctival cytology specimens obtained from all subjects. The levels of RNA transcripts encoding inflammatory cytokines [interleukin 1alpha_(IL-1alpha), interleukin 6 (IL-6), interleukin 8 (IL-8), tumor necrosis factor alpha_(TNF-alpha), and transforming growth factor beta1 (TGF-beta1)] as well as a housekeeping gene (G3PDH) were evaluated in conjunctival cytology specimens taken from all subjects by semiquantitative competitive reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: Decreased tear fluid EGF concentration was noted in Sjögren's syndrome patients (mean 0.68 +/- 0.59 ng/ml) compared to controls (mean 1.66 +/- 0.45 ng/ml, P = 0.004). Significantly increased levels of IL-1alpha, IL-6, IL-8, TNF-alpha and TGF-beta1 RNA transcripts were found in the conjunctival epithelium of Sjögren's syndrome patients compared to controls (P < 0.05), while the level of G3PDH was similar in both groups. The concentration of IL-6 protein was significantly higher in Sjögren's syndrome conjunctiva samples (P = 0.012). Tear EGF concentration correlated with Schirmer 1 scores (rho 0.767, P < 0.001), corneal fluorescein staining scores (rho -0.562, P = 0.01), conjunctival goblet cell density (rho 0.661, P = 0.001) and the levels of IL-1alpha_and IL-8 RNA in the conjunctival epithelium (rho -0.677 and -0.747, respectively, P = 0.001). Both IL-1alpha_and IL-8 RNA in the conjunctival epithelium increased as Schirmer 1 scores decreased (P = 0.001). IL-8 RNA level correlated with corneal fluorescein staining (rho 0.690, P = 0.001) and conjunctival goblet cell density (rho -0.767, P < 0.001). A significant decrease in IL-8 RNA level, corresponding to improvement in irritation symptoms and ocular surface disease, was observed in six eyes after two weeks of topical corticosteroid therapy. CONCLUSIONS: The balance of cytokines in the tear fluid and conjunctival epithelium is altered in Sjögren's syndrome. The severity of keratoconjunctivitis sicca in this condition increases as tear fluid EGF concentration decreases and levels of inflammatory cytokines in the conjunctival epithelium increase. These findings provide new insight into the pathogenesis of keratoconjunctivitis and provide potential targets for therapy.
Assuntos
Túnica Conjuntiva/metabolismo , Citocinas/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Ceratoconjuntivite Seca/metabolismo , Síndrome de Sjogren/metabolismo , Lágrimas/metabolismo , Adulto , Idoso , Contagem de Células , Ensaio de Imunoadsorção Enzimática , Células Epiteliais/metabolismo , Feminino , Humanos , Interleucinas/metabolismo , Ceratoconjuntivite Seca/etiologia , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Síndrome de Sjogren/complicações , Fator de Crescimento Transformador beta/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
OBJECTIVE: To correlate and compare the Schirmer 1 test and a new method of measuring tear fluorescein clearance with the CytoFluor II fluorometer with the severity of ocular irritation symptoms, clinical signs of meibomian gland disease, corneal fluorescein staining scores, and corneal and conjunctival sensitivity. DESIGN: Case-control study. PARTICIPANTS: Forty patients presenting with a chief complaint of ocular irritation, and 40 asymptomatic control subjects of similar age distribution. INTERVENTION: All subjects completed a symptom questionnaire, a baseline ocular examination, fluorescein clearance test (FCT), and Schirmer 1 test. MAIN OUTCOME MEASURES: The FCT was performed with a CytoFluor II fluorophotometer by measuring the fluorescein concentration in minimally stimulated tear samples collected from the inferior tear meniscus 15 minutes after instillation of 5 microl of 2% sodium fluorescein. Severity of ocular irritation was assessed with a symptom questionnaire. Schirmer 1 test, biomicroscopic meibomian gland evaluation, corneal fluorescein staining score, and corneal and conjunctival sensation scores were assessed with the Cachet-Bonnet anesthesiometer in all subjects. RESULTS: Irritation symptoms correlated with higher log tear fluorescein concentration (symptomatic 3.08 +/- 0.62 units/,microl, normal control 1.89 +/- 0.7 units/microl, P < 0.005) and lower Schirmer 1 test scores (symptomatic 12.6 mm, normal control 22.3 mm, P < 0.005). The FCT showed greater predictive value for identifying ocular irritation than the Schirmer 1 test. A fluorescein concentration of 274 units//microl eliminated 80% of the normal subjects (specificity) and identified 85% of the abnormal subjects (sensitivity). Log of tear fluorescein concentration and the Schirmer 1 test correlated with meibomian gland orifice metaplasia (2.81 +/- 0.78 units/microl and 14.47 +/- 9.53 mm in those with metaplasia vs. 1.83 +/- 0.71 units/microl and 23.14 +/- 7.67 mm in those without metaplasia, P < 0.001) and with the percentage of acinar dropout. Both log of tear fluorescein concentration and the Schirmer 1 test correlated with corneal fluorescein staining (Pearson correlation of 0.394 P < 0.0001 for Schirmer 1 test and 0.312 P < 0.005 for log of tear fluorescein). In addition, log of tear fluorescein and Schirmer 1 test scores correlated with corneal and conjunctival sensation scores (Spearman's rho for corneal sensation: log of tear fluorescein -0.38, P < 0.003, Schirmer 1 test -0.39, P < 0.002, and for conjunctival sensation: log of tear fluorescein -0.391, P < 0.001, Schirmer 1 test -0.23, P < 0.061). CONCLUSIONS: The FCT shows a greater predictive value for ocular irritation than the Schirmer 1 test. It correlates better with age, meibomian gland dysfunction, and decreased corneal and conjunctival sensation. Decreased tear clearance was identified as a risk factor for ocular irritation, even in subjects with normal Schirmer scores. This simple technique may provide new clues into the mechanism and therapy of ocular irritation.
Assuntos
Síndromes do Olho Seco/metabolismo , Doenças Palpebrais/metabolismo , Fluoresceína/metabolismo , Glândulas Tarsais/metabolismo , Lágrimas/metabolismo , Adulto , Estudos de Casos e Controles , Córnea/metabolismo , Síndromes do Olho Seco/diagnóstico , Doenças Palpebrais/diagnóstico , Fluorofotometria/métodos , Humanos , Glândulas Tarsais/patologia , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
OBJECTIVE: This study aimed to review the clinical features, therapeutic response, and histopathology of cases of nontuberculous mycobacterial keratitis at the Bascom Palmer Eye Institute. DESIGN AND PARTICIPANTS: Retrospective review of medical records, clinical photographs, histopathology, and microbiology of 24 cases of nontuberculous acid-fast keratitis over the past 15 years. RESULTS: Causal organisms included Mycobacterium chelonae (16), M. fortuitum (3), M. avium-intracellulare (2), M. nonchromogenicum (1), M. triviale (1), and M. asiaticum (1). Clinically, the keratitis had a superficial location except in those patients with a history of surgery. Amikacin was the most commonly used antibiotic (63%). Three patients were treated with Clarithromycin. In one patient, it was stopped because of toxicity; the other two had resolution of their infiltrates. Fifty-five percent did not respond to topical antimicrobial therapy. The organisms as a group were sensitive to amikacin and Clarithromycin and resistant to the fluoroquinolones. Sixty-four percent of the group that failed to respond to medical treatment were treated with steroids after the diagnosis was known, in comparison to 44% of the group treated successfully with medications. The histopathology of the patients treated with steroids showed minimal inflammation despite a large number of organisms, in contrast to the dense infiltrates seen in the specimens from patients not treated with topical steroids. CONCLUSION: Nontuberculous mycobacterial keratitis is a chronic insidious infection that is often unresponsive to medical therapy. The authors recommend that steroids be withheld. Based on the authors' experience of three patients, topical Clarithromycin may hold promise as a therapeutic agent. Lamellar keratectomy or penetrating keratoplasty should be considered in those patients who do not respond to medical therapy or those who have recurrent exacerbations on attempted weaning of topical antibiotic therapy.
Assuntos
Infecções Oculares Bacterianas/epidemiologia , Ceratite/epidemiologia , Infecções por Mycobacterium não Tuberculosas/epidemiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Antibacterianos , Córnea/microbiologia , Córnea/patologia , Quimioterapia Combinada/uso terapêutico , Infecções Oculares Bacterianas/tratamento farmacológico , Infecções Oculares Bacterianas/microbiologia , Feminino , Florida/epidemiologia , Humanos , Ceratite/tratamento farmacológico , Ceratite/microbiologia , Ceratoplastia Penetrante , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Infecções por Mycobacterium não Tuberculosas/tratamento farmacológico , Infecções por Mycobacterium não Tuberculosas/microbiologia , Micobactérias não Tuberculosas/isolamento & purificação , Estudos RetrospectivosRESUMO
In summary, tear EGF levels correlate most strongly with tear production in normals, and it is likely that some form of homeostatic mechanism exists to provide a constant supply to the ocular surface. Commercial ELISA kits appear to measure EGF in tears with good consistency and may be useful in the future to improve comparability of data from different studies. In addition, in ocular rosacea, which mimics keratoconjunctivitis sicca in a number of respects, there is a differential increase in the level of the inflammatory cytokine IL-1 alpha in the tear fluid. Much of this elevation appears to be the result of reduced tear turnover, which may form an important positive feedback mechanism encouraging tear stagnation and the perpetuation of ocular surface inflammation.
Assuntos
Citocinas/fisiologia , Oftalmopatias/fisiopatologia , Rosácea/fisiopatologia , Lágrimas/química , Lágrimas/fisiologia , Adulto , Idoso , Envelhecimento , Citocinas/análise , Ensaio de Imunoadsorção Enzimática , Fator de Crescimento Epidérmico/análise , Oftalmopatias/complicações , Feminino , Humanos , Interleucina-1/análise , Masculino , Valores de Referência , Rosácea/complicações , Caracteres SexuaisAssuntos
Túnica Conjuntiva/fisiologia , Aparelho Lacrimal/metabolismo , Lágrimas/fisiologia , Divisão Celular , Túnica Conjuntiva/patologia , Síndromes do Olho Seco/patologia , Síndromes do Olho Seco/fisiopatologia , Fator de Crescimento Epidérmico/metabolismo , Células Epiteliais/fisiologia , Epitélio/patologia , Humanos , Inflamação , Metaplasia , Modelos Biológicos , Propriedades de Superfície , Lágrimas/química , Fator de Crescimento Transformador beta/metabolismoRESUMO
PURPOSE: To determine which subjective assessments and objective tests have clinical utility as diagnostic tools in ocular irritation associated with Sjögren's syndrome-related aqueous tear deficiency (ATD), non-Sjögren ATD, inflammatory meibomian gland disease (MGD) associated with rosacea, and atrophic MGD. METHODS: Forty adults with ocular irritation and 10 with normal ocular surfaces were enrolled in a nonrandomized, nonblinded clinical trial. Symptoms were evaluated. Tests included biomicroscopy; evaluation of tear-film integrity, production, and clearance; fluorescein and rose bengal staining; and serum autoantibody screening. RESULTS: Symptoms were similar among groups and most severe in the Sjögren's group. Fluorescein tear break-up time was significantly faster in the ATD and MGD groups than that in controls. Schirmer scores were significantly lower in the ATD group than those in MGD and control groups. Tear clearance was delayed in the ATD and atrophic MGD groups. Xeroscope grid distortion was noted only with ATD. The Sjögren's group had greater loss of naso-lacrimal reflex, slower fluorescein clearance, and greater ocular-surface fluorescein and rose bengal staining than did the others. More MGD subjects had meibomian gland orifice metaplasia and acinar dropout than did those with Sjögren-related ATD and controls. Schirmer scores correlated inversely with rose bengal staining, corneal fluorescein staining, and grid distortion. Rose bengal staining correlated with grid distortion and loss of nasal-lacrimal reflex, but not with MGD. CONCLUSION: Subjective assessments and objective diagnostic tests have clinical utility as diagnostic tools in tear-film disorders. ATD is correlated with ocular-surface disease. An algorithm summarizing the diagnostic utility of these tests is included.
Assuntos
Técnicas de Diagnóstico Oftalmológico , Síndromes do Olho Seco/diagnóstico , Doenças Palpebrais/diagnóstico , Doenças do Aparelho Lacrimal/diagnóstico , Glândulas Tarsais/patologia , Lágrimas/fisiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Síndromes do Olho Seco/complicações , Síndromes do Olho Seco/fisiopatologia , Oftalmopatias/etiologia , Doenças Palpebrais/complicações , Doenças Palpebrais/fisiopatologia , Feminino , Humanos , Doenças do Aparelho Lacrimal/complicações , Doenças do Aparelho Lacrimal/fisiopatologia , Masculino , Pessoa de Meia-Idade , Estudos RetrospectivosRESUMO
OBJECTIVE: The purpose of the study is to compare tear fluid concentrations of interleukin-1alpha (IL-1alpha), tumor necrosis factor-alpha (TNF-alpha), and epidermal growth factor (EGF) in ocular rosacea with those in control subjects and to examine the relation between tear functions, such as production and clearance rate, and the concentrations of cytokines in tear fluid. PARTICIPANTS AND INTERVENTION: Fourteen patients with severe meibomian gland disease, facial rosacea, and symptoms of ocular irritation were examined for ocular surface disease, tear production, and tear clearance rate (TCR). Twelve control subjects, frequency-matched for age, and 15 ideal normal subjects with no ocular symptoms and normal tear function were assessed using the same parameters. Minimally stimulated tear samples (20 microl) were drawn from each subject and analyzed using a sandwich enzyme-linked immunosorbent assay to detect IL-1alpha, TNF-alpha, and EGF. RESULTS: Tear IL-1alpha concentration was significantly higher in patients with rosacea than in age-matched (P = 0.003) and ideal control subjects (P < 0.001). Tumor necrosis factor-alpha was not detected in patients or control subjects, indicating levels of less than 10 pg/ml. Epidermal growth factor was not significantly higher in patients with rosacea than in age-matched control subjects. Tear turnover LN(TCR) was lower in patients with rosacea than in both age-matched (P = 0.048) and ideal control subjects (P = 0.002). Schirmer I scores were statistically lower in patients with rosacea than in ideal control subjects (P = 0.013), but not age-matched control subjects. Interleukin-1alpha was correlated inversely with LN(TCR) (r= -0.58, P < 0.0001) and Schirmer I (r = -0.39, P = 0.012). CONCLUSIONS: Concentrations of IL-1alpha are present in normal tears but are elevated in ocular rosacea, whereas TNF-alpha is not present in either case. The reduced tear turnover, LN(TCR), its inverse correlation with IL-1alpha, and the absence of TNF-alpha in the tears of these patients suggest that the increased concentration of IL-1alpha observed may be largely because of clearance failure of cytokine normally produced at the ocular surface.