RESUMEN
OBJECTIVES: The proprotein convertase enzyme FURIN is a critical regulator of the anti-inflammatory TGFß-1 cytokine and peripheral immune tolerance. In T cells, FURIN is co-regulated with IFN-γ and thus highly expressed in T helper 1 type cells. Previous studies have demonstrated that FURIN is upregulated in inflammatory conditions, including atherosclerosis, rheumatoid arthritis and systemic lupus erythematosus. Here, we evaluated the levels of FURIN in the plasma and peripheral blood mononuclear cells (PBMCs) of patients with primary Sjögren's syndrome (pSS) and in healthy controls. METHODS: FURIN plasma levels were determined by ELISA, and the mRNA expression in PBMCs was quantitated using qPCR. FURIN levels in the plasma were correlated with the clinical and demographic characteristics of the patients. RESULTS: FURIN was found to be significantly upregulated at both the protein and mRNA level in pSS patients compared to healthy controls. In pSS patients, high FURIN protein levels were significantly associated with elevated IFN-γ levels in the plasma as well as a longer duration of sicca symptoms in the eyes. pSS patients with high FURIN levels in their plasma showed a trend towards lower levels of serum beta-2 microglobulin, ESR and a lower systemic disease activity index ESSDAI. CONCLUSIONS: The proprotein convertase FURIN is significantly upregulated in pSS. Elevated FURIN levels associate with high levels of the Th1 type cytokine IFN-γ and long duration of dry eye symptoms. Patients with high FURIN levels show signs of lower disease activity suggesting that FURIN might have a protective role in pSS.
Asunto(s)
Furina/sangre , Leucocitos Mononucleares/enzimología , Síndrome de Sjögren/enzimología , Adulto , Biomarcadores/sangre , Sedimentación Sanguínea , Estudios de Casos y Controles , Ensayo de Inmunoadsorción Enzimática , Femenino , Furina/genética , Humanos , Interferón gamma/sangre , Masculino , Persona de Mediana Edad , ARN Mensajero/genética , ARN Mensajero/metabolismo , Índice de Severidad de la Enfermedad , Síndrome de Sjögren/sangre , Síndrome de Sjögren/diagnóstico , Regulación hacia Arriba , Xeroftalmia/sangre , Xeroftalmia/diagnóstico , Xeroftalmia/enzimología , Microglobulina beta-2/sangreRESUMEN
OBJECTIVE: To explore the expression of SIRT1 with oxidative stress and observe physiological and pathological changes in the corneas as well as the association between SIRT1 and oxidative stress of diabetic dry eyes in mice. METHOD: Forty-eight C57BL/6Jdb/db mice at eight weeks of age were divided randomly into two groups: the diabetic dry eye group and the diabetic group. An additional forty-eight C57BL/6J mice at eight weeks of age were divided randomly into two groups: the dry eye group and the control group. Every mouse in the dry eye groups (diabetic and normal) was injected with scopolamine hydrobromide three times daily, combined with low humidity to establish a dry eye model. After the intervention, phenol red cotton string tests and corneal fluorescein staining were performed. In addition, HE staining and immunofluorescence were done. Expression of SIRT1 in the cornea was examined by real-time PCR and Western Blot and expression of FOXO3 and MnSOD proteins was detected by Western Blot. RESULTS: At one, four, and eight weeks post intervention, all of the groups except the controls showed significant decreases in tear production and increases in the corneal fluorescein stain (P<0.05 vs control). Between the experimental groups, the diabetic dry eye group had the least tear production and the highest corneal fluorescein stain score (P<0.05). As the disease progressed, all of the experimental groups showed obviously pathological changes in HE staining, particularly the diabetic dry eye group. In the 1(st) and 4(th) week, the expression of SIRT1, FOXO3, and MnSOD were significantly higher in the diabetic DE and DM groups but lower in the DE group compared to the controls (P<0.05). In the 8(th) week, the expression of SIRT1, FOXO3, and MnSOD was significantly down-regulated in the diabetic DE group and the DM group (P<0.05). Immunofluorescence showed similar results. CONCLUSION: In the condition of diabetic dry eye, tear production declined markedly coupled with seriously wounded corneal epithelium. Oxidative stress in the cornea was enhanced significantly and the expression of SIRT1 was decreased.
Asunto(s)
Córnea/enzimología , Complicaciones de la Diabetes/enzimología , Estrés Oxidativo , Sirtuina 1/metabolismo , Xeroftalmia/enzimología , Animales , Western Blotting , Córnea/patología , Complicaciones de la Diabetes/inducido químicamente , Complicaciones de la Diabetes/genética , Complicaciones de la Diabetes/patología , Modelos Animales de Enfermedad , Femenino , Técnica del Anticuerpo Fluorescente , Proteína Forkhead Box O3 , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Ratones Endogámicos C57BL , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Escopolamina , Sirtuina 1/genética , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Lágrimas/metabolismo , Factores de Tiempo , Xeroftalmia/inducido químicamente , Xeroftalmia/genética , Xeroftalmia/patologíaRESUMEN
PURPOSE: An acidic mammalian chitinase (AMCase) seems to be implicated in allergic asthma and allergic ocular pathologies. The aim of this work was to investigate the role of AMCase during Sjögren's Syndrome (SS) and Meibomian Gland Dysfunction (MGD) dry eye diseases. METHODS: Six patients with MGD dry eye (20-58 years, median 40) and six patients with dry eye associated to SS (32-60 years, median 47) were enrolled in this study. AMCase activity was measured in tears and AMCase mRNA expression was evaluated by real-time polymerase chain reaction from RNA extracted from epithelial cells of the conjunctiva. Six healthy adult subjects of the same age (34-44 years, median 39) were also studied as the control group. RESULTS: AMCase activity was significantly increased in patients affected by MGD dry eye (18.54 +/- 1.5 nmol/ml/h) and SS dry eye (8.94 +/- 1.0 nmol/ml/h) respectively, compared to healthy controls (1.6 +/- 0.2 nmol/ml/h). AMCase activity was higher in the tears of subjects with MGD dry eye (P < 0.001). AMCase mRNA was detected in conjunctival epithelial cells and the expression was significantly higher in MGD dry eye than SS dry eye. A significant correlation between AMCase activity in the tears and mRNA in conjunctival epithelial cells was found. CONCLUSION: AMCase may be an important marker in the pathogenesis of dry eye, suggesting the potential role of AMCase as a therapeutic target in these frequent pathologies.
Asunto(s)
Quitinasas/metabolismo , Enfermedades de los Párpados/complicaciones , Glándulas Tarsales , Síndrome de Sjögren/complicaciones , Xeroftalmia/enzimología , Xeroftalmia/etiología , Adulto , Biomarcadores/metabolismo , Quitinasas/genética , Conjuntiva/enzimología , Células Epiteliales/enzimología , Femenino , Humanos , Masculino , Persona de Mediana Edad , ARN Mensajero/metabolismo , Lágrimas/enzimología , Adulto JovenRESUMEN
Although xerophthalmia due to severe vitamin A deficiency is the leading cause of childhood blindness in the underdeveloped countries, little is known about the proteases (other than collagenase) that are involved in the degradative mechanism. The degree of cellular autolysis and stromal degradation observed histologically in early stages of xerophthalmia and in ulcerating corneas in vitamin A deficient rabbits in this study were, in general, proportional to the levels of the proteases studied. The only major histologic and ultrastructural alteration observed in early xerophthalmic corneas was autolysis of superficial epithelial and stromal cells. In contrast, in the ulcerating corneas the stroma was infiltrated heavily with inflammatory cells and extensive stromal degradation was observed in the central necrotic region of the lesions. Maximal proteolytic activity toward hemoglobin was observed at pH 3.3 for corneal extracts from normal (N) and pair-fed control (C) rabbits and rabbits with early xerophthalmia (X) and ulcerating xerophthalmia (U) corneas. This activity was a cathepsin D-like enzyme per cornea that had a ratio of 1:1:3:16 in the N, C, X, and U corneas. The ratio of cathepsin B-like activity per cornea for N, C, X, and U corneas was 1:2:2:10.
Asunto(s)
Úlcera de la Córnea/patología , Endopeptidasas/análisis , Deficiencia de Vitamina A/patología , Animales , Ácido Aspártico Endopeptidasas , Catepsina D/análisis , Úlcera de la Córnea/enzimología , Conejos , Deficiencia de Vitamina A/enzimología , Xeroftalmia/enzimología , Xeroftalmia/patologíaRESUMEN
Previously described methods for measuring human tear lysozyme are fraught with shortcomings. A new method has been devised. Tear fluid was collected on Whatman filter paper discs. Each disc was placed in a tightly capped tube containing sodium phosphate buffer. Fluid from each tube was placed directly into a well of the lysozyme immunodiffusion plate. After the precipitation rings had reached maximum size, their diameters were measured. A linear standard curve was constructed, and lysozyme concentration was expressed as micrograms per milliliter. The tear lysozyme concentration was obtained from the standard curve and corrected for the assay dilution factor. The mean tear lysozyme concentration in 15 normal patients was 1.4 +/- 0.5 mg/mL. In ten patients with dry eyes, the mean was 0.7 +/- 0.5 mg/mL. The method used to collect, store, and transport tears is easily performed in the clinic and readily tolerated by patients. The technique of radial immunodiffusion is reliable and simple, compared with other assays.
Asunto(s)
Muramidasa/análisis , Lágrimas/enzimología , Adulto , Anciano , Humanos , Inmunodifusión , Persona de Mediana Edad , Xeroftalmia/enzimologíaRESUMEN
We have developed a method for assaying the concentration of tear lysozyme using eluates of tear fluid collected on filter paper discs. Specimens can be stored and transported to remote laboratories for assay. We have shown that the 'indirect' or eluate method gives statistically comparable results to the 'direct' method using fresh, neat tear fluid.