RESUMEN
BACKGROUND: Autophagy and genetic predisposition have been suggested to potentially play roles in the development of asthma. However, little is known about the role of autophagy in the pathogenesis of severe asthma. OBJECTIVE: We compared autophagy in the sputum granulocytes, peripheral blood cells (PBCs) and peripheral blood eosinophils (PBEs) between patients with severe asthma and those with non-severe asthma and investigated the functional effects of autophagy. METHODS: We enrolled 36 patients with severe asthma, 14 with non-severe asthma and 23 normal healthy controls in this study. Sputum granulocytes, PBCs and PBEs were isolated from each subject. Autophagy was evaluated based on the expression of microtubule-associated protein light chain 3 (LC3) by Western blot, confocal microscopy, transmission electron microscopy and flow cytometry. IL-8 levels were measured by ELISA. To induce autophagy, HL-60 cells, human primary small airway epithelial cells (SAECs) and A549 cells were treated with IL-5, IL-1ß and TNF-α. To inhibit autophagy, PI3K inhibitors (LY29400 and 3-methyladenine [3-MA]) and hydroxychloroquine (HCQ) were used. Knockdown of ATG5 and Beclin-1 was performed in A549 cells, and the therapeutic effects of dexamethasone were evaluated. RESULTS: Higher autophagy levels were noted in sputum granulocytes, PBCs and PBEs from patients with severe asthma than from patients with non-severe asthma and healthy controls (P < 0.05 for all). IL-5 increased autophagy levels in both PBCs and PBEs (P < 0.05). 3-MA attenuated the increased expression of LC3-II and eosinophil cationic protein in HL-60 cells induced by IL-5 (P = 0.034 for both). Dexamethasone did not affect autophagy levels in PBEs. IL-1ß increased LC3-II expression and IL-8 production (P < 0.01) in SAECs, and this was attenuated by LY294002, 3-MA, HCQ and knockdown of ATG5 and Beclin-1 (in A549 cells) (P < 0.01). CONCLUSIONS AND CLINICAL RELEVANCE: Autophagy could play a role in the pathogenesis of severe asthma. Autophagy modulation may be a novel therapeutic target for conventional therapy-resistant severe asthma.
Asunto(s)
Asma/etiología , Asma/metabolismo , Autofagia , Leucocitos/inmunología , Leucocitos/metabolismo , Esputo/citología , Esputo/inmunología , Adulto , Proteínas Reguladoras de la Apoptosis/genética , Asma/diagnóstico , Asma/terapia , Proteína 5 Relacionada con la Autofagia , Beclina-1 , Estudios de Casos y Controles , Línea Celular , Citocinas , Femenino , Volumen Espiratorio Forzado , Técnicas de Silenciamiento del Gen , Granulocitos/inmunología , Granulocitos/metabolismo , Humanos , Inmunoglobulina E/inmunología , Recuento de Leucocitos , Masculino , Proteínas de la Membrana/genética , Proteínas Asociadas a Microtúbulos/genética , Persona de Mediana Edad , Fagosomas/metabolismo , Índice de Severidad de la Enfermedad , Adulto JovenAsunto(s)
Cimetidina/efectos adversos , Hipersensibilidad a las Drogas/etiología , Antagonistas de los Receptores H2 de la Histamina/efectos adversos , Hipersensibilidad Inmediata/inducido químicamente , Adulto , Cimetidina/inmunología , Hipersensibilidad a las Drogas/inmunología , Femenino , Antagonistas de los Receptores H2 de la Histamina/inmunología , Humanos , Hipersensibilidad Inmediata/inmunologíaRESUMEN
BACKGROUND: Urticaria is the commonest cutaneous reaction caused by aspirin or other nonsteroidal anti-inflammatory drugs. The pathogenesis of aspirin-induced urticaria (AIU) is not fully understood, but appears to involve mast cell activation and neutrophil infiltration. OBJECTIVES: To investigate the genetic contribution of interleukin (IL)-18, which can amplify acute inflammation by promoting mast cell activation, neutrophil migration and cytokine production, to the pathogenesis of AIU. METHODS: A case-control association study was performed using 275 patients with AIU and 196 normal healthy controls in a Korean population. Two promoter polymorphisms of the IL18 gene (-607A/C and -137G/C) were genotyped using the primer extension method. The functional effect of the IL18 gene promoter polymorphism was investigated through in vitro studies including a luciferase reporter assay and electrophoretic mobility shift assays (EMSAs) and ex vivo studies involving neutrophil chemotaxis assays. RESULTS: A significant association was detected between both AIU in general and the aspirin-intolerant acute urticaria (AIAU) phenotype and the IL18 promoter polymorphism -607A/C. Patients with AIAU showed higher frequencies of the C(-607) G(-137) haplotype, ht1 [CG], compared with controls (P=0·02). Moreover, ht1 [CG] showed a high transcript haplotype by the luciferase activity assay, and EMSAs identified a -607C allele-specific DNA-binding protein as CREB2. Neutrophil chemotactic activity was highest in subjects with AIU exhibiting the high transcript haplotype, ht1 [CG] (P=0·019). CONCLUSIONS: The high transcript haplotype ht1 [CG] of the IL18 gene may contribute to the development of acute cutaneous inflammation sensitive to aspirin, leading to the clinical presentation of AIAU.
Asunto(s)
Antiinflamatorios no Esteroideos/efectos adversos , Aspirina/efectos adversos , Interleucina-18/genética , Polimorfismo de Nucleótido Simple/genética , Urticaria/inducido químicamente , Urticaria/genética , Adulto , Estudios de Casos y Controles , Movimiento Celular/genética , Erupciones por Medicamentos/genética , Femenino , Genotipo , Haplotipos , Humanos , Masculino , Persona de Mediana Edad , Regiones Promotoras Genéticas/genética , Transcripción Genética/genética , Células U937 , Adulto JovenRESUMEN
BACKGROUND: Toluene diisocyanate (TDI) is the most important cause of occupational asthma, but the genetic mechanism of TDI-induced asthma is still unknown. OBJECTIVE: The objective of the study was to identify susceptibility alleles associated with the TDI-induced asthma phenotype. METHODS: We conducted a genome-wide association study in 84 patients with TDI-induced asthma and 263 unexposed healthy normal controls using Affymetrix 500K SNPchip. We also investigated the relationships between genetic polymorphisms and transcript levels in Epstein-Barr virus-transformed lymphoblastoid cell lines from patients with TDI-induced asthma enrolled in this study. RESULTS: Genetic polymorphisms of CTNNA3 (catenin alpha 3, alpha-T catenin) were significantly associated with the TDI-induced asthma phenotype (5.84 x 10(-6) for rs10762058, 1.41 x 10(-5) for rs7088181, 2.03 x 10(-5) for rs4378283). Carriers with the minor haplotype, HT2 [GG], of two genetic polymorphisms (rs10762058 and rs7088181) showed significantly lower PC(20) methacholine level (P=0.041) and lower mRNA expression of CTNNA3 than non-carriers (P=0.040). A genetic polymorphism in the 3' downstream region of CTNNA3 (rs1786929), as identified by DNA direct sequencing, was significantly associated with the TDI-induced asthma phenotype (P=0.015 in recessive analysis model) and the prevalence of serum-specific IgG to cytokeratin 19 (P=0.031). CONCLUSION: These findings suggested that multiple genetic polymorphisms of CTNNA3 may be determinants of susceptibility to TDI-induced asthma.
Asunto(s)
Asma/inducido químicamente , Asma/genética , Estudio de Asociación del Genoma Completo , Polimorfismo de Nucleótido Simple/genética , 2,4-Diisocianato de Tolueno/efectos adversos , alfa Catenina/genética , Adulto , Linfocitos B/metabolismo , Pruebas de Provocación Bronquial , Línea Celular Transformada , Femenino , Expresión Génica/genética , Frecuencia de los Genes , Predisposición Genética a la Enfermedad/genética , Genotipo , Humanos , Inmunoglobulina E/sangre , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Queratina-19/inmunología , Masculino , Persona de Mediana Edad , Enfermedades Profesionales/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Factores de RiesgoRESUMEN
BACKGROUND: It has been known that interleukin (IL)-10 promoter polymorphisms at -1082A/G, -819T/C and -592A/C, may influence IL-10 expression and associate with asthma. Interleukin-10 facilitates the regulatory function of transforming growth factor (TGF)-beta. The goal of this study was to investigate a gene-gene interaction between IL-10 and TGF-beta1 polymorphisms in Korean asthmatics with aspirin hypersensitivity. METHODS: Single-nucleotide polymorphism genotyping of IL-10 and TGF-beta1 genes was performed and the functional effect of the IL-10 polymorphisms was analysed applying a luciferase reporter assay and an electrophoretic mobility shift assay. RESULTS: Among the patients with asthma, polymorphism at -1082A/G was significantly associated with the phenotype of aspirin-intolerant asthma, AIA (P = 0.007, P(c) = 0.021). Moreover, a synergistic effect between the TGF-beta1-509C/T and IL-10-1082A/G polymorphisms on the phenotype of AIA was noted; when stratified by the presence of rhinosinusitis, the frequency of rare alleles (the CT or TT genotype of TGF-beta1-509C/T and AG or GG genotype of IL-10-1082A/G) was significantly higher in the patients with AIA (15.2%) when compared with those with ATA (6.3%, P = 0.031; odds ratio 4.111; 95% confidence interval 1.504-11.235). In an in vitro functional assay, the -1082G reporter plasmid exhibited significantly greater promoter activity when compared with the -1082A construct in Jurkat T cells (P = 0.011). Moreover, we found that the transcription factor Myc-associated zinc-finger protein preferentially bound the -1082G allele. CONCLUSION: Our results suggest that IL-10 promoter polymorphisms contribute to the development of AIA and that rhinosinusitis may interact genetically with TGF-beta1.
Asunto(s)
Aspirina/efectos adversos , Asma/genética , Hipersensibilidad a las Drogas/genética , Interleucina-10/genética , Rinitis Alérgica Perenne/genética , Sinusitis/genética , Factor de Crecimiento Transformador beta1/genética , Adulto , Alelos , Aspirina/inmunología , Asma/inmunología , Hipersensibilidad a las Drogas/inmunología , Epistasis Genética , Femenino , Frecuencia de los Genes , Predisposición Genética a la Enfermedad , Humanos , Células Jurkat , Corea (Geográfico) , Masculino , Polimorfismo Genético , Regiones Promotoras Genéticas/genética , Rinitis Alérgica Perenne/inmunología , Sinusitis/inmunologíaRESUMEN
We previously reported that a methanolic extract of Coptis japonica, which is a well-known traditional oriental medicine, inhibits morphine-induced conditioned place preference (CPP) in mice. Berberine is a major component of Coptis japonica extract, and it has been established that the adverse effects of morphine on the brain involve dopamine (DA) receptors. However, to our knowledge, no study has investigated the inhibitory effects of berberine on morphine-induced locomotor sensitization and analgesic tolerance in mice. Here, we investigated the effects of berberine on morphine-induced locomotor sensitization and on the development of analgesic tolerance. Furthermore, we examined the effects of berberine treatment on N-methyl-D-aspartate (NMDA) receptor channel activity expressed in Xenopus laevis oocytes. Berberine was found to completely block both morphine-induced locomotor sensitization and analgesic tolerance, and reduce D(1) and NMDA receptor bindings in the cortex. Moreover, berberine markedly inhibited NMDA current in Xenopus laevis oocytes expressing NMDA receptor subunits. Our results suggest that the inhibitory effects of berberine on morphine-induced locomotor sensitization and analgesic tolerance are closely related to the modulation of D1 and NMDA receptors, and that berberine should be viewed as a potential novel means of attenuating morphine-induced sensitization and analgesic tolerance.
Asunto(s)
Berberina/farmacología , Encéfalo/efectos de los fármacos , Tolerancia a Medicamentos/fisiología , Dependencia de Morfina/tratamiento farmacológico , Morfina/antagonistas & inhibidores , Actividad Motora/efectos de los fármacos , Animales , Berberina/química , Encéfalo/metabolismo , Encéfalo/fisiopatología , Modelos Animales de Enfermedad , Dopamina/metabolismo , Femenino , Ácido Glutámico/metabolismo , Masculino , Ratones , Ratones Endogámicos ICR , Estructura Molecular , Morfina/efectos adversos , Dependencia de Morfina/fisiopatología , Actividad Motora/fisiología , Narcóticos/efectos adversos , Oocitos , Subunidades de Proteína/efectos de los fármacos , Subunidades de Proteína/metabolismo , Receptores de Dopamina D1/efectos de los fármacos , Receptores de Dopamina D1/metabolismo , Receptores de N-Metil-D-Aspartato/efectos de los fármacos , Receptores de N-Metil-D-Aspartato/metabolismo , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiología , XenopusRESUMEN
DNA ligase IV (Lig4) and the DNA-dependent protein kinase (DNA-PK) function in nonhomologous end joining (NHEJ). However, although Lig4 deficiency causes late embryonic lethality, deficiency in DNA-PK subunits (Ku70, Ku80, and DNA-PKcs) does not. Here we demonstrate that, similar to p53 deficiency, ataxia-telangiectasia-mutated (ATM) gene deficiency rescues the embryonic lethality and neuronal apoptosis, but not impaired lymphocyte development, associated with Lig4 deficiency. However, in contrast to p53 deficiency, ATM deficiency enhances deleterious effects of Lig4 deficiency on growth potential of embryonic fibroblasts (MEFs) and genomic instability in both MEFs and cultured progenitor lymphocytes, demonstrating significant differences in the interplay of p53 vs. ATM with respect to NHEJ. Finally, in dramatic contrast to effects on Lig4 deficiency, ATM deficiency causes early embryonic lethality in Ku- or DNA-PKcs-deficient mice, providing evidence for an NHEJ-independent role for the DNA-PK holoenzyme.
Asunto(s)
Antígenos Nucleares , Ataxia Telangiectasia/genética , ADN Helicasas , ADN Ligasas/genética , Proteínas Serina-Treonina Quinasas/genética , Proteína p53 Supresora de Tumor/genética , Animales , Apoptosis , Proteínas de la Ataxia Telangiectasia Mutada , Linfocitos B/citología , Proteínas de Ciclo Celular , Diferenciación Celular , Aberraciones Cromosómicas , ADN Ligasa (ATP) , ADN Ligasas/fisiología , Proteína Quinasa Activada por ADN , Proteínas de Unión al ADN/genética , Autoantígeno Ku , Ratones , Ratones Noqueados , Neuronas/citología , Proteínas Nucleares/genética , Linfocitos T/citología , Proteína p53 Supresora de Tumor/fisiología , Proteínas Supresoras de TumorRESUMEN
The Atm-/- mice recapitulate most of the defects observed in ataxia-telangiectasia (A-T) patients, including a high incidence of lymphoid tumors and immune defects characterized by defective T cell differentiation, thymus hypoplasia, and defective T-dependent immune responses. To understand the basis of the T cell developmental defects in Atm-/- mice, a functional TCR alpha beta transgene was introduced into these mutant mice. Analysis of the Atm-/-TCR alpha beta+ mice indicated that the transgenic TCR alpha beta can rescue the defective T cell differentiation and partially rescue the thymus hypoplasia in Atm-/- mice, indicating that thymocyte positive selection is normal in the Atm-/- mice. In addition, cell cycle analysis of the thymocytes derived from Atm-/-TCR alpha beta+ and control mice suggested that Atm is involved in the thymocyte expansion. Finally, evaluation of the T-dependent immune responses in Atm-/-TCR alpha beta+ mice indicated that Atm is dispensable for normal T cell function. Therefore, the defective T-dependent immune responses in Atm-/- mice must be secondary to greatly reduced T cell numbers in these mutant mice.
Asunto(s)
Ataxia Telangiectasia/genética , Proteínas Serina-Treonina Quinasas/genética , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Linfocitos T/inmunología , Linfocitos T/patología , Transgenes/inmunología , Animales , Ataxia Telangiectasia/inmunología , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Ciclo Celular , Diferenciación Celular/genética , Diferenciación Celular/inmunología , División Celular/genética , División Celular/inmunología , Células Cultivadas , Proteínas de Unión al ADN , Modelos Animales de Enfermedad , Activación de Linfocitos/genética , Ratones , Ratones Noqueados , Ratones Transgénicos , Proteína Básica de Mielina/inmunología , Fragmentos de Péptidos/inmunología , Proteínas Serina-Treonina Quinasas/fisiología , Linfocitos T/metabolismo , Timo/patología , Proteínas Supresoras de TumorRESUMEN
Disruption of the mouse Atm gene, whose human counterpart is consistently mutated in ataxia-telangiectasia (A-T) patients, creates an A-T mouse model exhibiting most of the A-T-related systematic and cellular defects. While ATM plays a major role in signaling the p53 response to DNA strand break damage, Atm-/- p53(-/-) mice develop lymphomas earlier than Atm-/- or p53(-/-) mice, indicating that mutations in these two genes lead to synergy in tumorigenesis. The cell cycle G1/S checkpoint is abolished in Atm-/- p53(-/-) mouse embryonic fibroblasts (MEFs) following gamma-irradiation, suggesting that the partial G1 cell cycle arrest in Atm-/- cells following gamma-irradiation is due to the residual p53 response in these cells. In addition, the Atm-/- p21(-/-) MEFs are more severely defective in their cell cycle G1 arrest following gamma-irradiation than Atm-/- and p21(-/-) MEFs. The Atm-/- MEFs exhibit multiple cellular proliferative defects in culture, and an increased constitutive level of p21 in these cells might account for these cellular proliferation defects. Consistent with this notion, Atm-/- p21(-/-) MEFs proliferate similarly to wild-type MEFs and exhibit no premature senescence. These cellular proliferative defects are also rescued in Atm-/- p53(-/-) MEFs and little p21 can be detected in these cells, indicating that the abnormal p21 protein level in Atm-/- cells is also p53 dependent and leads to the cellular proliferative defects in these cells. However, the p21 mRNA level in Atm-/- MEFs is lower than that in Atm+/+ MEFs, suggesting that the higher level of constitutive p21 protein in Atm-/- MEFs is likely due to increased stability of the p21 protein.
Asunto(s)
Ciclinas/fisiología , Proteínas Serina-Treonina Quinasas , Proteínas/fisiología , Proteína p53 Supresora de Tumor/fisiología , Animales , Proteínas de la Ataxia Telangiectasia Mutada , Pruebas de Carcinogenicidad , Proteínas de Ciclo Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/genética , Proteínas de Unión al ADN , Fase G1 , Rayos gamma , Humanos , Linfoma/etiología , Ratones , Fenotipo , Proteínas/genética , ARN Mensajero , Proteína p53 Supresora de Tumor/genética , Proteínas Supresoras de TumorRESUMEN
Methyl esters of polyunsaturated fatty acids labeled with tritium were prepared by partial stereoselective reduction of the corresponding acetylenic esters with tritiated disiamylborane, followed by protonolysis with tritiated acetic acid. The labeling was strictly specific, and the tritium atoms were located only at the carbon atoms forming the unsaturated bond(s). Synthesis of some tritiated fatty acid methyl esters with methylene-interrupted trans-cis double bonds is also reported.