Determinación de mediadores de la respuesta inmune e inflamación en lágrimas: cambios en ojo seco y glaucoma frente a población sana / Determination of inmune response and inflammation mediators in tears: Changes in dry eye and glaucoma as compared to healthy controls

OBJETIVO: Determinar el perfil de expresión de las moléculas mediadoras de inflamación y respuesta inmune (IRI) en lágrimas de pacientes con ojo seco (OS) y pacientes en tratamiento médico por sospecha o diagnóstico de glaucoma primario de ángulo abierto (GPAA) para compararlos con sujetos sanos. MÉTODO: Estudio prospectivo observacional de cohortes de 107 participantes subdivididos en: pacientes con OS (GOS; n=30), pacientes con sospecha o diagnóstico de GPAA con tratamiento hipotensor ocular (GGPAA; n=41) y controles sanos (GC; n=36). Se obtuvieron muestras de lágrimas mediante capilaridad para analizarlas mediante sistema de multiinmunoanálisis basado en citometría de flujo (Luminex R-200®), determinando diversas interleucinas (IL): 1β, 2, 4, 5, 6 y 10, y también los factores de necrosis tumoral alfa (TNF-α), de crecimiento endotelial vascular (VEGF) y de crecimiento de colonias de granulocitos y macrófagos (GM-CSF). Los datos se procesaron mediante el programa SPSS 20.0. RESULTADOS: Las moléculas que aumentaron significativamente en lágrimas de pacientes en el GOS versus GGPAA fueron: IL-1β (p = 0.01), IL-6 (p = 0,004), IL-10 (p = 0,04), mientras que el VEGF disminuyó significativamente en el GOS. El GGPAA mostró aumento significativo de IL-6 (p < 0,0001) frente al GC. Comparando GOS y GGPAA, observamos diferencias significativas para IL-4 (p = 0,004), IL-6 (p = 0,002), TNF-α (p = 0,03), GM-CSF (p = 0,03) y VEGF (p = 0,002). CONCLUSIONES: El aumento de expresión de los mediadores de IRI en lágrimas de pacientes con OS o GPAA demuestra la importancia de estos procesos en ambas enfermedades, aunque las distintas moléculas implicadas indican diferentes vías de señalización para ambas, que requieren más investigaciones

OBJECTIVE: To determine the expression profile of immune response and inflammation (IRI) mediator molecules in tears from patients with dry eye (DE), and those suspected of having or have primary open-angle glaucoma (POAG) under treatment and compare them with healthy controls. METHODS: A prospective observational cohort study including 107 participants sub-divided into: healthy controls (CG; n=30), patients with DE (DEG; n=41) and patients suspected of having or have POAG and on hypotensive treatment (POAG-G; n=36). Tear samples were collected by capillary to be processed using a multi-immunoassay system based on flow cytometry (Luminex R-200 ®), in order to determine the interleukins (IL): 1β, 2, 4, 5, 6, and 10, and the growth factors: Tumour necrosis alpha (TNF-α), vascular endothelial (VEGF), and granulocyte-macrophage colony stimulating- (GM-CSF). Data were processed using the SPSS 20.0 program. RESULTS: Molecules that significantly increased in tears from DEG vs. POAG-G patients were: IL-1 (P=.01), IL-6 (P=.004), IL-10 (P=.04), whereas VEGF significantly decreased in the DEG. The POAG-G showed significantly higher IL-6 values (P<.0001) as compared to the CG. When comparing both the DEG and POAG-G, significant differences were observed in tear expression of IL-4 (P=.004), IL-6 (P=.002), TNF-α (P=.03), GM-CSF (P=.03), and VEGF (P=.002). CONCLUSIONS: The increased expression of IRI mediators in tears from patients with DE or POAG strongly demonstrated the importance of immune response in both pathologies. However, the different molecules involved also suggest distinct signalling pathways for these processes that still require further research

Determination of inmune response and inflammation mediators in tears: Changes in dry eye and glaucoma as compared to healthy controls. / Determinación de mediadores de la respuesta inmune e inflamación en lágrimas: cambios en ojo seco y glaucoma frente a población sana.

OBJECTIVE: To determine the expression profile of immune response and inflammation (IRI) mediator molecules in tears from patients with dry eye (DE), and those suspected of having or have primary open-angle glaucoma (POAG) under treatment and compare them with healthy controls. METHODS: A prospective observational cohort study including 107 participants sub-divided into: healthy controls (CG; n=30), patients with DE (DEG; n=41) and patients suspected of having or have POAG and on hypotensive treatment (POAG-G; n=36). Tear samples were collected by capillary to be processed using a multi-immunoassay system based on flow cytometry (Luminex R-200 ®), in order to determine the interleukins (IL): 1ß, 2, 4, 5, 6, and 10, and the growth factors: Tumour necrosis alpha (TNF-α), vascular endothelial (VEGF), and granulocyte-macrophage colony stimulating- (GM-CSF). Data were processed using the SPSS 20.0 program. RESULTS: Molecules that significantly increased in tears from DEG vs. POAG-G patients were: IL-1 (P=.01), IL-6 (P=.004), IL-10 (P=.04), whereas VEGF significantly decreased in the DEG. The POAG-G showed significantly higher IL-6 values (P<.0001) as compared to the CG. When comparing both the DEG and POAG-G, significant differences were observed in tear expression of IL-4 (P=.004), IL-6 (P=.002), TNF-α (P=.03), GM-CSF (P=.03), and VEGF (P=.002). CONCLUSIONS: The increased expression of IRI mediators in tears from patients with DE or POAG strongly demonstrated the importance of immune response in both pathologies. However, the different molecules involved also suggest distinct signalling pathways for these processes that still require further research.

Sistemas genéticos para un nuevo abordaje del riesgo de progresión de la retinopatía diabética / Genetic systems for a new approach to risk of progression of diabetic retinopathy

OBJETIVO: Evaluar el riesgo de progresión de la retinopatía diabética (RD) utilizando nuevas estrategias para obtener información genética en diabéticos tipo 2 (DT2) basadas en interferencia por ácido ribonucleico (ARN). MATERIAL Y MÉTODOS: Estudio multicéntrico, prospectivo de casos-controles en 132 participantes divididos en: grupo DT2 (GDT2) con RD (+RD) y sin RD (-RD) (n = 77) y grupo control (GC) (n = 55). Tras entrevista personal y examen oftalmológico, se extrajeron lágrimas para análisis molecular (expresión de micro-ARN [miARN] [miRCURY™ ARN Isolation Kit, Qiagen]). En 18 muestras (GDT2+RD = 6; GDT2-RD = 6; GC = 6) obtuvimos librerías de 137 vs. 140 pares de bases (GeneMapper, Applied Biosystems) y realizamos secuenciación de próxima generación (NGS). El programa SPSS 15.0 vehiculizó el análisis estadístico. RESULTADOS: Edad media: 67 ± 12 años en GDT2 vs. 55 ± 21 años en GC. Distribución hombres/mujeres: 51/28 en GDT2 vs. 25/30 en GC. Los antecedentes familiares de DM, cumplir dieta, fumar, beber y realizar ejercicio mostraron diferencias significativas entre grupos (p < 0,001). Con 20-25 μL de lágrimas extrajimos 9,42 ± 3,30 ng/mL de ARN purificado, con diferencias significativas entre GDT2/GC (p = 0,002) y GDT2+RD/GC (p = 0,004). La expresión lagrimal de miARN en GDT2 correlacionó directamente con: edad/obesidad/duración de DM (p < 0,05), e indirectamente con: agudeza visual (p < 0,05). Hemos identificado 14 miARN relacionados con la presencia, mecanismos patogénicos y factores de riesgo para la progresión de la RD. CONCLUSIONES: Proponemos utilizar lágrimas como fuente de información genética para la DM. Los miARN específicos implicados en desarrollo o progresión de la RD pueden utilizarse como biomarcadores moleculares y, a partir de ellos, desarrollar futuras bioterapias

OBJECTIVE: To evaluate the risk of progression of diabetic retinopathy (DR) using new strategies to obtain genetic information in type 2 diabetes (T2D) based on interfering ribonucleic acid (RNA). MATERIAL AND METHODS: A prospective multicentre case-control study of 132 participants was distributed into: T2D with (+DR) or without (-DR) (T2DG; n = 77), and a control group (CG; n = 55). After an eye examination and personal interview, tears were collected for molecular analysis (expression of microRNAs [miRNAs] (miRCURY(TM) ARN Isolation Kit, Qiagen)]. Libraries, 137 vs. 140 bp (GeneMapper, Applied Biosystems), were obtained in 18 samples (T2DG+DR = 6; T2DG-DR = 6; CG = 6) by performing next-generation sequencing (NGS). SPSS 15.0 statistical program was used to perform data analysis. RESULTS: The mean age was 67 ± 12 years in the T2DG vs. 55 ± 21 years in the CG. Distribution men/women: 25/30 in T2DG vs. 51/28 in CG. A family history of DM, diet compliance, smoking, drinking and exercise, showed significant differences between groups (P<.001). A 20-25 microlitre sample of tears contained a mean of 9.42 ± 3.30 ng/mL of purified ARN, with significant differences between T2DG/CG (P=.002) and T2DG+RD/CG (P=.004). Tear expression of miARNs in T2DG directly correlated with age/obesity/T2D duration (P<.05), and indirectly with visual acuity (P<.05). A total of 14 miRNAs related to the presence, pathogenic mechanisms and risk factors for the progression of diabetic retinopathy, were identified. CONCLUSIONS: We propose to use tears as a source of genetic information for DM. Specific miRNAs involved in DR development and/or progression can be used as molecular biomarkers, and based on these, for developing future biotherapies

Metabolómica de la lágrima / Human tear metabolome

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Genetic systems for a new approach to risk of progression of diabetic retinopathy. / Sistemas genéticos para un nuevo abordaje del riesgo de progresión de la retinopatía diabética.

OBJECTIVE: To evaluate the risk of progression of diabetic retinopathy (DR) using new strategies to obtain genetic information in type 2 diabetes (T2D) based on interfering ribonucleic acid (RNA). MATERIAL AND METHODS: A prospective multicentre case-control study of 132 participants was distributed into: T2D with (+DR) or without (-DR) (T2DG; n=77), and a control group (CG; n=55). After an eye examination and personal interview, tears were collected for molecular analysis (expression of microRNAs [miRNAs] (miRCURY ™ ARN Isolation Kit, Qiagen)]. Libraries, 137 vs. 140bp (GeneMapper, Applied Biosystems), were obtained in 18 samples (T2DG+DR=6; T2DG-DR=6; CG=6) by performing next-generation sequencing (NGS). SPSS 15.0 statistical program was used to perform data analysis. RESULTS: The mean age was 67±12 years in the T2DG vs. 55±21 years in the CG. Distribution men/women: 25/30 in T2DG vs. 51/28 in CG. A family history of DM, diet compliance, smoking, drinking and exercise, showed significant differences between groups (P<.001). A 20-25 microlitre sample of tears contained a mean of 9.42±3.30 ng/mL of purified ARN, with significant differences between T2DG/CG (P=.002) and T2DG+RD/CG (P=.004). Tear expression of miARNs in T2DG directly correlated with age/obesity/T2D duration (P<.05), and indirectly with visual acuity (P<.05). A total of 14 miRNAs related to the presence, pathogenic mechanisms and risk factors for the progression of diabetic retinopathy, were identified. CONCLUSIONS: We propose to use tears as a source of genetic information for DM. Specific miRNAs involved in DR development and/or progression can be used as molecular biomarkers, and based on these, for developing future biotherapies.

Los miRNAs como potenciales biomarcadores de las enfermedades oculares / MicroRNAs as potential biomarkers of eye diseases

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Reduced retinal nerve fibre layer thickness in children with severe obesity.

BACKGROUND: Some optic nerve diseases are silent and insidious. Recently, reduced thickness of retinal nerve fibre layer (RNFL) has been associated with increasing body mass index in adults. OBJECTIVES: To investigate the association of childhood obesity with RNFL measured by optical coherence tomography imaging. METHODS: Ninety-seven children aged 5-14 years classified according to standard deviation score of body mass index (SDS-BMI) were included. Parameters of metabolic risk, adipocytokines (leptin, adiponectin) and interleukin-6 were analyzed. All subjects underwent a comprehensive ophthalmologic examination with direct ophthalmoscopy. Evaluation of RNFL with optical coherence tomography of the head of the nerve was performed. RESULTS: RNFL thickness on the average and inferior, superior and nasal quadrants were decreased in severely obese children (SDS-BMI > 4) with respect to the other groups. However, no statistically significant association was found between the different groups of children and RNFL thickness in the temporal quadrant. There was a significant inverse correlation of RNFL thickness with adiposity indices (P = 0.016), leptin (P = 0.029) and interleukin-6 (P = 0.030) in overweight and obese children. CONCLUSIONS: These findings suggest that adiposity and obesity-related inflammatory factors may be associated with the loss of retinal ganglion cells in children.

Glutathione-dependent formaldehyde dehydrogenase (ADH3) and low km mitochondrial aldehyde dehydrogenase (ALDH2). New evidence for differential expression in the rat retina in response to oxidative stress.

OBJECTIVES: Epidemiological and experimental studies support the involvement of lipid peroxidation (LPO) in retinal diseases. In addition to other pathogenic mechanisms not fully understood, the possibility remains that peroxidic aldehydes, acting as cytotoxic chemicals, mediate in the progression of chronic ocular disorders. METHODS: To test proper mechanisms involved in removing peroxidic aldehydes from the retina, in an attempt to understand long-lasting changes induced by LPO, the oxidative and antioxidant enzymatic activities, as well as the retinal distribution and activity of glutathione-dependent formaldehyde dehydrogenase (ADH3) and low km mitochondrial aldehyde dehydrogenase (ALDH2), were studied and compared with induced LPO sites in the adult rat retina. Biochemical enzymatic-colorimetric assays, histochemical and immunocytochemical analyses were carried out in the mature rat retinal tissues. Statistics were performed by the SPSS 15.0 program. RESULTS: Data revealed (1) the noticeable LPO and glutathione (GSH) enzymatic system retinal and optic nerve activities; (2) the retinal expression and distribution of both the ADH3 and ALDH2; and (3) the co-localisation of iron/nicotine adenine dinucleotide phosphate (Fe/NADPH)-induced LPO, mainly in the outermost and innermost retinal strata, as compared to the rest of the retinal layers (p < 0.001). CONCLUSIONS: Changes in the GSH and GSH enzymatic system, and in the ADH3 and ALDH2 retinal expression and distribution might be crucial in assessing the intrinsic mechanisms of LPO-mediated retinopathies. Further research is needed to evaluate these findings and their application to new ophthalmological therapy.