RESUMEN
BACKGROUND: Spinal muscular atrophy (SMA) is a rare autosomal-recessive neurodegenerative disorder caused by mutations in survival motor neuron 1 (SMN1) gene, and consequent loss of function of SMN protein, which results in progressive loss of lower motor neurons, and muscular wasting. Antisense oligonucleotide (ASO) nusinersen (Spinraza®) modulates the pre-mRNA splicing of the SMN2 gene, allowing rebalance of biologically active SMN. It is administered intrathecally via lumbar puncture after removing an equal amount of cerebrospinal fluid (CSF). Its effect was proven beneficial and approved since 2017 for SMA treatment. Given the direct effect of nusinersen on RNA metabolism, the aim of this project was to evaluate cell-free RNA (cfRNA) in CSF of SMA patients under ASOs treatment for biomarker discovery. METHODS: By RNA-sequencing approach, RNA obtained from CSF of pediatric SMA type 2 and 3 patients was processed after 6 months of nusinersen treatment, at fifth intrathecal injection (T6), and compared to baseline (T0). RESULTS: We observed the deregulation of cfRNAs in patients at T6 and we were able to classify these RNAs into disease specific, treatment specific and treatment dependent. Moreover, we subdivided patients into "homogeneous" and "heterogeneous" according to their gene expression pattern. The "heterogeneous" group showed peculiar activation of genes coding for ribosomal components, meaning that in these patients a different molecular effect of nusinersen is observable, even if this specific molecular response was not referable to a clinical pattern. CONCLUSIONS: This study provides preliminary insights into modulation of gene expression dependent on nusinersen treatment and lays the foundation for biomarkers discovery.
Asunto(s)
Atrofia Muscular Espinal , ARN , Humanos , Niño , Atrofia Muscular Espinal/tratamiento farmacológico , Atrofia Muscular Espinal/genética , Oligonucleótidos/uso terapéutico , MutaciónRESUMEN
Extracellular adenosine 5'-triphosphate (ATP) has significant effects on a variety of pathological conditions and it is the main physiological agonist of P2X7 purinergic receptor (P2X7R). It is known that ATP acting via purinergic receptors plays a relevant role on skin inflammation, and P2X7R is required to neutrophil recruitment in a mice model of irritant contact dermatitis (ICD).The present study investigated the effects of chemical irritant croton oil (CrO) upon ATP, ADP, and AMP hydrolysis in mice blood serum, and the potential involvement of P2X7R. The topical application CrO induced a decrease on soluble ATP/ADPase activities (~50 %), and the treatment with the selective P2X7R antagonist, A438079, reversed these effects to control level. Furthermore, we showed that CrO decreased cellular viability (52.6 % ± 3.9) in relation to the control and caused necrosis in keratinocytes (PI positive cells). The necrosis induced by CrO was prevented by the pre-treatment with the selective P2X7R antagonist A438079. The results presented herein suggest that CrO exerts an inhibitory effect on the activity of ATPDase in mouse serum, reinforcing the idea that ICD has a pathogenic mechanism dependent of CD39. Furthermore, it is tempting to suggest that P2X7R may act as a controller of the extracellular levels of ATP.
Asunto(s)
Nucleótidos de Adenina/sangre , Dermatitis por Contacto/genética , Dermatitis Irritante/genética , Receptores Purinérgicos P2X7/genética , Animales , Antígenos CD/sangre , Apirasa/sangre , Aceite de Crotón/toxicidad , Dermatitis por Contacto/sangre , Dermatitis por Contacto/patología , Dermatitis Irritante/sangre , Dermatitis Irritante/patología , Modelos Animales de Enfermedad , Humanos , Hidrólisis , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Ratones , Nucleótido Desaminasas/sangre , Antagonistas del Receptor Purinérgico P2X/administración & dosificación , Receptores Purinérgicos P2X7/sangreRESUMEN
A quercetina é um flavonoide, amplamente encontrada em frutas, vegetais, grãos, flores, com elevada concentração no vinho tinto, e tem sido caracterizada funcionalmente pela atividade antioxidante. Para avaliação da maturação nuclear e do desenvolvimento embrionário bovino, os oócitos foram maturados por 22h na presença de quercetina (0,4, 2, 10 e 50µM), cisteamina (100µM) e na ausência dos antioxidantes. Os oócitos maturados foram corados com Hoechst para avaliação da maturação in vitro. Para avaliação do desenvolvimento embrionário, os oócitos foram fertilizados e cultivados in vitro, as taxas de desenvolvimento embrionário foram determinadas no sétimo dia de cultivo e o percentual de eclosão e o número de células dos embriões no oitavo dia. Os níveis de glutationa (GSH) dos oócitos foram mensurados por emissão de fluorescência com CMF2HC. A porcentagem de maturação nuclear (±89%) não diferiu entre os grupos. O desenvolvimento embrionário variou entre os tratamentos, o percentual de blastocisto foi superior (P<0,05) nos grupos tratados com 0,4, 2, 10 e 50∝M de quercetina (56,9, 59,5, 53,6 e 49,6%, respectivamente) e com 100∝M de cisteamina (50,4%) em relação ao grupo controle (42,3%). Na comparação entre os dois antioxidantes, a quercetina (0,4 e 2µM) foi superior na produção de embriões (56,9 e 59,5%, respectivamente) em comparação com cisteamina (50,4%). As taxas de embriões eclodidos foram similares (P>0,05) entre os grupos (±63,0%). O número médio de células dos embriões também foi similar entre os grupos (±233). Os níveis intracelulares de GSH foram superiores nos oócitos maturados com cisteamina, mas similares entre os oócitos tratados com quercetina e o controle. A suplementação da maturação in vitro com antioxidantes melhora as taxas de blastocistos. A quercetina foi superior à cisteamina, que, por sua vez, foi superior ao controle. Mas os níveis de GSH foram superiores somente nos oócitos tratados com cisteamina.
Quercetin is a flavonoid widely found in fruit, vegetables, grains and flowers, with a high concentration in red wine, and has been functionally characterized by its antioxidant activity. For assessment of nuclear maturation and bovine embryo, oocytes were matured for 22h in the presence of quercetin (0.4, 2, 10 and 50µM), cysteamine (100µM) and in the absence of antioxidants. The matured oocytes were stained with Hoechst to evaluate the in vitro maturation. To assess embryonic development, oocytes were fertilized and cultured in vitro and rates of embryo development were obtained in the seventh day of culture and the percentage of hatching and the number of cells on eighth day embryos. The levels of glutathione (GSH) of the oocytes were measured by fluorescence emission with CMF2HC. The percentage of nuclear maturation (±89%) did not differ between groups. Embryonic development varied between treatments, the percentage of blastocyst was higher (P<0.05) in the groups treated with 0.4, 2, 10 and 50∝M of quercetin (56.9, 59.5, 53.6 and 49.6%, respectively) and 100 ∝M cysteamine (50.4%) compared to the control group (42.3%). Comparing the two antioxidants, quercetin (0.4 to 2µM) was superior in embryo production (56.9 and 59.5% respectively) compared with cysteamine (50.4%). The rates of hatched embryos were similar (P>0.05) between groups (±63.0%). The average number of embryo cells was also similar in both groups (±233). The intracellular GSH levels were higher in oocytes matured with cysteamine, but similar between the oocytes treated with quercetin and control. The supplementation of matured in vitro with antioxidants improves blastocyst rates. Quercetin was greater than cysteamine, which in turn was superior to the control. However, GSH levels were higher in oocytes treated only with cysteamine.
Asunto(s)
Animales , Bovinos , Antioxidantes , Embrión de Mamíferos/embriología , Oocitos/citología , Bovinos/clasificación , Técnicas de Maduración In Vitro de los OocitosRESUMEN
Kinins and their receptors have been recently implicated in cancer. Using functional and molecular approaches, we investigated the relevance of kinin B1 and B2 receptors in bladder cancer. Functional studies were conducted using bladder cancer cell lines, and human biopsies were employed for molecular studies. Both B1 des-Arg(9)-BK and B2 BK receptor agonists stimulated the proliferation of grade 3-derived T24 bladder cancer cells. Furthermore, treatment with B1 and B2 receptor antagonists (SSR240612 and HOE140) markedly inhibited the proliferation of T24 cells. Only higher concentrations of BK increased the proliferation of the grade 1 bladder cancer cell line RT4, while des-Arg(9)-BK completely failed to induce its proliferation. Real-time PCR revealed that the mRNA expression of kinin receptors, particularly B1 receptors, was increased in T24 cells relative to RT4 cells. Data from bladder cancer human biopsies revealed that B1 receptor expression was increased in all tumor samples and under conditions of chronic inflammation. We also show novel evidence demonstrating that the pharmacological inhibition of PI3Kγ (phosphatidylinositol 3-kinase) with AS252424, concentration-dependently reduced T24 cell proliferation induced by BK or des-Arg(9)-BK. Finally, the incubation of T24 cells with kinin agonists led to a marked activation of the PI3K/AKT and ERK 1/2 signaling pathways, whereas p38 MAP kinase remained unaffected. Kinin receptors, especially B1 receptors, appear to be implicated in bladder cancer progression. It is tempting to suggest that selective kinin antagonists might represent potential alternative therapies for bladder cancer.
Asunto(s)
Fosfatidilinositol 3-Quinasas/metabolismo , Receptor de Bradiquinina B1/metabolismo , Receptor de Bradiquinina B2/metabolismo , Neoplasias de la Vejiga Urinaria/enzimología , Anciano , Anciano de 80 o más Años , Biopsia , Antagonistas del Receptor de Bradiquinina B1 , Antagonistas del Receptor de Bradiquinina B2 , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Sistema de Señalización de MAP Quinasas , Persona de Mediana Edad , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor de Bradiquinina B1/agonistas , Receptor de Bradiquinina B1/genética , Receptor de Bradiquinina B2/agonistas , Receptor de Bradiquinina B2/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Tiazolidinedionas/farmacología , Vejiga Urinaria/efectos de los fármacos , Vejiga Urinaria/enzimología , Vejiga Urinaria/patología , Neoplasias de la Vejiga Urinaria/genética , Neoplasias de la Vejiga Urinaria/patologíaRESUMEN
OBJECTIVES: Conflicting evidence indicates an increased risk for cardiovascular disease in postmenopausal women suffering from hot flushes. In this study, we tested whether, beyond hot flushes, menopausal symptoms are associated with biochemical and biophysical risk factors for cardiovascular disease. METHODS: Retrospective cross-sectional analysis on 951 women in surgical or physiological postmenopause, recruited at the menopause outpatient service of our university hospital between April 2002 and December 2009. The Greene Climacteric Scale and its subscales for anxiety, depression, somatic symptoms, vasomotor symptoms and sexuality were used to evaluate menopausal complaints. Blood pressure, fasting glucose and lipids levels were evaluated as risk factors for cardiovascular disease. Anthropometric parameters and those derived by remote and reproductive medical history were used as possible confounders. All data were anonymously retrieved from an electronic database. RESULTS: By multiple regression analysis, high density lipoprotein (HDL) cholesterol was inversely related to body mass index, the Greene Climacteric Scale score and years since menopause (R = 0.390; p = 0.0001). The total cholesterol/HDL cholesterol ratio was positively related to waist circumference and the Greene Climacteric Scale score (R = 0.356; p = 0.0001). Triglycerides (R = 0.353; p = 0.0001) and triglyceride/HDL cholesterol (R = 0.425; p = 0.0001) were positively related to waist circumference, the Greene Climacteric Scale score and the Greene vasomotor subscore. Glucose was positively related to waist circumference, years since menopause and the Greene Climacteric Scale score (R = 0.390; p = 0.0001). Blood pressure was not related to menopausal symptoms. The 10-year risk for cardiovascular disease calculated by the Framingham formula was related independently and directly to body mass index and the Greene Climacteric Scale score (R = 0.183; p = 0.0001). CONCLUSIONS: Menopausal symptoms evaluated by a validated climacteric scale are associated with a worsening of biochemical risk factors for atherosclerosis and cardiovascular disease.
Asunto(s)
Índice de Masa Corporal , Enfermedades Cardiovasculares/etiología , Posmenopausia , Índice de Severidad de la Enfermedad , Anciano , Glucemia , Presión Sanguínea , Enfermedades Cardiovasculares/sangre , Enfermedades Cardiovasculares/fisiopatología , Colesterol/sangre , HDL-Colesterol/sangre , Estudios Transversales , Femenino , Sofocos/sangre , Sofocos/fisiopatología , Humanos , Persona de Mediana Edad , Posmenopausia/sangre , Posmenopausia/fisiología , Análisis de Regresión , Estudios Retrospectivos , Factores de Riesgo , Factores de Tiempo , Triglicéridos/sangre , Circunferencia de la CinturaRESUMEN
Apyrase and 5'-nucleotidase activities were analyzed in an ethidium bromide (EB) demyelinating model associated with interferon-beta (IFN-beta). The animals were divided in groups: I, control (saline); II, saline and IFN-beta; III, EB and IV, EB and IFN-beta. After 7, 15 and 30 days the animals (n = 5) were sacrificed and the cerebral cortex was removed for synaptosome preparation and enzymatic assays. Apyrase activity using ATP as substrate increased in groups II, III and IV (P < 0.001) after 7 days and in groups III and IV (P < 0.001) after 15 days. Using ADP as substrate, an activation of this enzyme was observed in group III (P < 0.05) after seven and 15 days. The 5'-nucleotidase activity increased in group III (P < 0.05) after 7 days and in groups II, III and IV (P < 0.001) after 15 days. After 30 days treatment, no significant alteration was observed in enzyme activities. Results showed that apyrase and 5'-nucleotidase activities are altered in demyelination events and that IFN-beta was able to regulate the adenine nucleotide hydrolysis.