RESUMEN
Non-Hodgkin lymphoma (NHL) is a lymphoproliferative disorder derived from either B or T lymphocytes. Among NHL, activated B-cell-like (ABC) diffuse large B-cell lymphoma (DLBCL) and T cell non-Hodgkin lymphomas (T-NHL) are poor prognosis and aggressive subtypes. Macrophages are professional phagocytic cells and dendritic cells (DCs) are professional antigen-presenting cells in immune system. Doxorubicin (Dox) and Etoposide (ET) are the most effective anti-cancer drugs. A20 and CYLD are negative regulators of NF-κB-dependent functions in many cell types. Little is known about the roles of A20 and CYLD in regulating functions of DCs and macrophages from NHL. The present study, therefore, explored whether A20/CYLD expression contributes to functions of DCs and macrophages from NHL. To this end, blood samples of seventy-nine patients with ABC DLBCL and T-NHL were examined. Gene expression profile was determined by quantitative RT-PCR and immunophenotype, cell apoptosis and phagocytosis by flow cytometry. As a result, immunophenotypic analysis showed that the numbers of CD13+CD117-, CD56+CD40+ and CD23+CD40+ expressing cells were significantly elevated in ABC DLBCL cases compared to healthy individuals and T-NHL patients. Interestingly, upon treatment of Dox and ET, the phagocytosis of lymphoma cells was significantly reduced by CD11c+CD123- DCs and the percentage of CD56+ mature DCs was significantly enhanced in ABC DLBCL patients only in the presence of A20 siRNA, but not CYLD siRNA. In conclusion, ABC DLBCL patients with low A20 expression were defective in elimination of lymphoma cells by DCs and linked to killer DC expansion in circulation.
Asunto(s)
Células Dendríticas , Linfoma de Células B Grandes Difuso , Fagocitosis , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa , Humanos , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Fagocitosis/efectos de los fármacos , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/metabolismo , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/genética , Femenino , Linfoma de Células B Grandes Difuso/patología , Linfoma de Células B Grandes Difuso/inmunología , Persona de Mediana Edad , Masculino , Linfoma no Hodgkin/patología , Linfoma no Hodgkin/inmunología , Apoptosis/efectos de los fármacos , Anciano , Adulto , Macrófagos/metabolismo , Macrófagos/inmunología , Doxorrubicina/farmacología , Doxorrubicina/uso terapéutico , Linfocitos B/inmunología , Linfocitos B/metabolismo , InmunofenotipificaciónRESUMEN
Pretilachlor and safener fenclorim are the main components of herbicides widely applied to control weeds. Although some pure cultures of bacteria and fungi which degraded these compounds under aerobic conditions were isolated, no isolated pretilachlor- and fenclorim-degrading bacterial strains under anaerobic condition had been available. In this study, the degradation of these compounds and the effects of them on bacterial community structures were investigated under anaerobic conditions. The dissipation rates of pretilachlor and fenclorim in slurries were in the order: soil from paddy field ≈ sediment from river > sediment from mangrove. Moreover, three pretilachlor-degrading bacterial strains (Pseudomonas sp. Pr1, Proteiniclasticum sp. Pr2 and Paracoccus denitrificans Pr3) and two fenclorim-degrading strains (Dechloromonas sp. Fe1 and Ralstonia pickettii Fe2) isolated from a slurry of paddy soil utilized the substrates as sole carbon and energy sources under anaerobic conditions. The degradation of pure pretilachlor and fenclorim at various concentrations by corresponding mixed pure cultures followed the Michaelis-Menten model, with the maximum degradation was 3.10 ± 0.31 µM/day for pretilachlor, and 2.08 ± 0.18 µM/day for fenclorim. During the degradation, 2-chloro-N-(2,6-diethylphenyl) acetamide and 2,6-dimethylaniline were produced in pretilachlor degradation, and benzene was a product of fenclorim degradation. The synergistic degradation of both substrates by all isolated bacteria reduced the metabolites concentrations accumulated in media. This study provides valuable information on effects of pretilachlor and fenclorim on bacterial communities in soil and sediments, and degradation of these substrates by isolated bacteria under anaerobic condition.
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Acetanilidas , Bacterias , Biodegradación Ambiental , Herbicidas , Acetanilidas/metabolismo , Herbicidas/metabolismo , Anaerobiosis , Bacterias/metabolismo , Microbiología del Suelo , Contaminantes del Suelo/metabolismo , AcetamidasRESUMEN
Ethanol is the most commonly encountered substance in forensic toxicology. Determining blood alcohol concentration (BAC) in autopsies accounts for the majority of work in forensic diagnosis. The most common method to assess BAC is the enzymatic oxidation method because of its low cost, easy operation, and high throughput. Still, the elevated lactate and lactate dehydrogenase (LDH) levels in postmortem blood may affect accuracy. This study uses headspace gas chromatography with a flame ionization detector (HS-GC/FID) to assess the interference of lactate and LDH levels on BAC in 110 autopsied blood samples determined by the enzymatic oxidation method. The results showed that lactate and LDH levels in postmortem blood were higher than in normal blood. There was a weak correlation between the lactate levels and BAC difference (r = 0.23, p < 0.05) and a strong correlation between LDH levels and BAC difference (r = 0.67, p < 0.001). The differentiation of BAC between the enzymatic oxidation method and HS-GC/FID was significant (p < 0.001), confirming the interference significantly. All postmortem blood samples with lactate and LDH levels higher than regular lead to a positive error in determining BAC by enzymatic oxidation method. The study results suggest that the HS-GC/FID method should be used to determine BAC in postmortem blood samples instead of the enzymatic oxidation method to avoid mistakes in forensic diagnosis.
RESUMEN
Background and Objectives: Psoriasis is an immune-mediated chronic inflammatory skin disorder and commonly associated with highly noticeable erythematous, thickened and scaly plaques. Deubiquitinase genes, such as tumor necrosis factor-alpha protein 3 (TNFAIP3, A20), the cylindromatosis (CYLD) and Cezanne, function as negative regulators of inflammatory response through the Janus kinase/signal transducers and activators of transcription (JAK-STAT) pathways. In this study, polymorphisms and expressions of A20, CYLD and Cezanne genes as well as immunophenotype in psoriatic patients were determined. Materials and Methods: In total, 82 patients with psoriasis and 147 healthy individuals with well-characterized clinical profiles were enrolled. Gene polymorphisms were determined by direct DNA sequencing, gene expression profile by quantitative real time-polymerase chain reaction (PCR), immunophenotype by flow cytometry, and the secretion of cytokines and cancer antigen (CA) 125 by enzyme-linked Immunosorbent assay (ELISA). Results: The inactivation of A20, CYLD and Cezanne and increased levels of TNF-α, IFN-γ and CA 125 was observed in psoriatic patients. Importantly, patients with low A20 expression had significant elevations of triglyceride and total cholesterol concentrations and higher numbers of CD13+CD117- and CD19+CD23+ (activated B) cells than those with high A20 expression. Genetic analysis indicated that all rs4495487 SNPs in the JAK2 gene, rs200878487 SNPs in the A20 gene and four SNPs (c.1584-375, c.1584-374, rs1230581026 and p.W433R) in the Cezanne gene were associated with significant risks, while the rs10974947 variant in the JAK2 gene was at reduced risk of psoriasis. Moreover, in the Cezanne gene, p.W433R was predicted to be probably damaging by the Polyphen-2 prediction tool and an AA/CC haplotype was associated with a high risk of psoriasis. In addition, patients with higher CA 125 levels than the clinical cutoff 35 U/mL showed increased levels of IFN-γ than those with normal CA 125 levels. Conclusions: A20 expression was associated with lipid metabolism and the recruitment of CD13+ CD117- and activated B cells into circulation in psoriatic patients. Besides this, the deleterious effect of the p.W433R variant in the Cezanne gene may contribute to the risk of psoriasis.
Asunto(s)
Psoriasis , Transducción de Señal , Humanos , Citocinas/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Psoriasis/genética , Janus Quinasa 2/genética , Janus Quinasa 2/metabolismo , Enzima Desubiquitinante CYLD/metabolismoRESUMEN
Chronic myelogenous leukemia (CML) is characterised by the translocation of regions of the BCR and ABL genes, leading to the fusion gene BCR-ABL forming the Philadelphia (Ph) chromosome. Vinblastine (Vinb) and Vincristine (Vinc) are Vinca alkaloids and frequently used in combination chemotherapy in leukemias and lymphomas. Deubiquitinating enzyme (DUB) genes such as A20, Otubain 1 and CYLD are known as inhibitors of functional activation of immune cells mediated through the NF-κB/STAT pathway. Little is known about the regulatory role of Vinb/Vinc on the function of CML cells and the contribution of the DUBs to those effects. In the end, the gene expression profile was determined by quantitative RT-PCR, physiological properties of CML cells by flow cytometry and cytokine production by ELISA. As a result, inactivated expression of the DUBs A20, CYLD, Otubain 1 and Cezanne and enhanced activation of CD11b+ and CD4T cells were observed in CML patients. Importantly, Vinc enhanced the expression of A20 and CYLD and inhibited the proliferation and survival of CML (K562) cells. The effects were abolished in the presence of A20 siRNA, while cell proliferation only depended on the presence of CYLD. In conclusion, the up-regulation of A20 by Vinc could involve inhibitory effects on the proliferation and survival of K562 cells. The events might contribute to the anticancer effect of Vinc on A20-sensitive CML cells.
Asunto(s)
Leucemia Mielógena Crónica BCR-ABL Positiva , Vinblastina , Humanos , Enzima Desubiquitinante CYLD/genética , Proteínas de Fusión bcr-abl/genética , Expresión Génica , Células K562 , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Vinblastina/farmacología , Vinblastina/uso terapéutico , Vincristina/farmacologíaRESUMEN
Psoriasis is a common chronic, immune-mediated inflammatory disease of the skin. PSORS1C3 is a non-protein coding gene, of which the RNA transcript is found in psoriatic patients. CARD14 is mainly expressed in epidermal keratinocytes. TLR4 is a transmembrane protein to recognize microbial antigens. Our study aimed to assess the relationship among PSORS1C3, CARD14 and TLR4 polymorphisms, inflammatory expression and psoriasis susceptibility. To the end, 71 patients with psoriasis and 46 healthy individuals with the well-characterized clinical profiles were enrolled. Gene polymorphisms were determined by Sanger DNA sequencing and secretion of cytokines by ELISA. As a result, genetic analysis of PSORS1C3 gene identified nine SNPs and three haplotype blocks. Sequencing of the CARD14 gene determined eight SNPs and one haplotype block. Sequencing of TLR4 gene identified nine SNPs, in which a SNP rs1018673641 was found to exert deleterious effect. The linkage disequilibrium analysis showed that seven variants in PSORS1C3 gene and three SNPs in CARD14 gene were in tightly linked. More importantly, a significant association between IL-6 level and rs1018673641 AT genotype in TLR4 gene was detected in psoriatic patients. In conclusion, the PSORS1C3, CARD14 and TLR4 polymorphisms and haplotypes may be correlated with risk of suffering psoriasis and the IL-6-mediated chronic inflammation in psoriasis could be partially regulated by the TLR4 functional variant.
RESUMEN
Acute lymphoblastic leukemia (ALL) is the hematologic malignancy characterized by the aberrant proliferation of immature lymphoid cells. A20 is a deubiquitinase gene that inhibits functional activation of immune cells mediated through NF-κB/STAT pathways and frequently found inactivated in lymphoma. IL-6 is a pro-inflammatory cytokine secreted by immune cells under the pathogenic conditions and regulated by STAT signaling. Little is known about the role of A20 in regulating the function of ALL blasts and underlying molecular mechanisms. The present study, therefore, explored whether A20 expression contributes to IL-6 induced cell migration and activation of myeloid cells in ALL. To this end, blood samples of thirty-five adult ALL patients were examined. Gene expression profile was determined by quantitative RT-PCR, immunophenotype by flow cytometry, secretion of inflammatory cytokines by ELISA, and cell migration by a transwell migration assay. As a result, the expression of A20 was inactivated in ALL. Immunophenotypic analysis indicated that percent of CD11b+CD40+ expressing cells present in ALL was significantly reduced when transfected with PEM-T easy A20. Importantly, IL6-induced CXCL12-mediated migration of ALL blasts was dependent on the presence of A20. The inhibitory effects of A20 on activated myeloid cells and migration of ALL blasts were mediated through the STAT pathway upon IL-6 challenge. In addition, the CA-125 level was much higher in elderly females than either young female or male ALL patients or healthy donors. In conclusion, the inhibitory effects of A20 on activation of ALL blasts are expected to affect the immune response to treatment for adult ALL patients.
Asunto(s)
Regulación Leucémica de la Expresión Génica , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Factores de Transcripción STAT/metabolismo , Transducción de Señal , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/metabolismo , Anciano , Movimiento Celular , Quimiocina CXCL12/metabolismo , Citocinas/metabolismo , Células Dendríticas/citología , Femenino , Perfilación de la Expresión Génica , Humanos , Sistema Inmunológico , Inmunofenotipificación , Inflamación , Interleucina-6/metabolismo , Masculino , Persona de Mediana Edad , Células Mieloides/metabolismo , FN-kappa B/metabolismoRESUMEN
The antiaging protein Klotho is encoded by the Klotho gene first identified as an 'aging suppressor', in mice. Klotho deficiency is involved in premature aging and early death, while its overexpression is related to longevity. Klotho is mostly expressed in the kidney, but also in the brain, and in other organs. Two forms of Klotho, the cell membrane and secreted form, have pleiotropic activities that include regulation of general metabolism, oxidative stress, and mineral metabolism that correlates with its effect on accelerating aging. Membrane Klotho serves as an obligate co-receptor for the fibroblast growth factor (FGF), while secreted Klotho plays its role as a humoral factor. Klotho protein participates in the regulation of several biological activities, including regulation of calcium-phosphate homeostasis and PTH as well as vitamin D metabolism. The active form of vitamin D, 1,25(OH)2D3 (1,25-dihydroxy-vitamin D3 = calcitriol), acts as a neurosteroid that participates in the regulation of multiple brain functions. It provides neuroprotection and suppresses oxidative stress, inhibits inflammation and inflammatory mediators, and stimulates various neurotrophins. Calcitriol is involved in many brain-related diseases, including multiple sclerosis, Alzheimer´s disease, Parkinson´s disease, and schizophrenia. This review covers the most recent advances in Klotho research and discusses Klotho-dependent roles of calcitriol in neuro-psycho-pathophysiology.
Asunto(s)
Calcitriol , Glucuronidasa , Animales , Encéfalo/metabolismo , Calcio de la Dieta , Glucuronidasa/metabolismo , Proteínas Klotho , RatonesRESUMEN
A20 is a negative regulator of nuclear factor (NF)-κB-dependent inflammatory reaction in response to different stimuli by immune cells including dendritic cells (DCs), the most potent antigen-presenting cells involved in both the innate and adaptive immune response. Dendritic cells use glucose as carbon source to synthesize fatty acid and generate energy. Glucose enhances cell apoptosis mediated through PI3K/Akt, ERK1/2, and Bax/Bcl-2 pathways. The protein kinase Akt2/PKBß is expressed in DCs and a regulator of Ca2+ influx, Na+/H+ exchanger activity, and migration of DCs. This study explored whether regulation of high glucose-induced DC function through Akt2 signaling is influenced by overexpression of A20. To this end, A20 protein expression was determined by western blotting and immunoprecipitation, secretion of inflammatory cytokines by ELISA, and expression of apoptotic markers by flow cytometry. As a result, treatment of mice with 10% high glucose enriched water increased secretion of insulin/IGF1 and reduced A20 protein level, the effects were blunted in Akt2-/- mice. Incubation of DCs with high glucose significantly decreased A20 protein expression in both control and Akt1-silenced DCs, but not in Akt2-/- DCs. Importantly, treatment of DCs with high glucose increased ceramide synthesis, caspase 8 activity, and annexin V binding in control DCs, the effects were abolished in Akt2-/- DCs or by A20 overexpression. In conclusion, regulation of A20 sensitive DC function by high glucose is mediated through insulin/IGF-1/Akt2 signaling.
Asunto(s)
Inflamación/genética , Factor I del Crecimiento Similar a la Insulina/genética , Insulina/genética , Proteínas Proto-Oncogénicas c-akt/genética , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/genética , Inmunidad Adaptativa/genética , Animales , Células Presentadoras de Antígenos/inmunología , Apoptosis/genética , Movimiento Celular/genética , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Células Dendríticas/patología , Citometría de Flujo , Regulación de la Expresión Génica/genética , Glucosa/farmacología , Inmunidad Innata/genética , Inflamación/metabolismo , Inflamación/patología , Insulina/metabolismo , Ratones Noqueados , FN-kappa B/genética , Agua/metabolismoRESUMEN
Insulin or insulin-like growth factor 1 (IGF-1) promotes the activation of phosphoinositide 3 kinase (PI3K)/Akt signaling in immune cells including dendritic cells (DCs), the most potent professional antigen-presenting cells for naive T cells. Klotho, an anti-aging protein, participates in the regulation of the PI3K/Akt signaling, thus the Ca2+-dependent migration is reduced in klotho-deficient DCs. The present study explored the effects of insulin/IGF-1 on DC function through klotho expression. To this end, the mouse bone marrow cells were isolated and cultured with GM-CSF to attain bone marrow-derived DCs (BMDCs). Cells were treated with insulin or IGF-1 and followed by stimulating with lipopolysaccharides (LPS). Tumor necrosis factor (TNF)-α formation was examined by enzyme-linked immunosorbent assay (ELISA). Phagocytosis was analyzed by FITC-dextran uptake assay. The expression of klotho was determined by quantitative PCR, immunoprecipitation and western blotting. As a result, treatment of the cells with insulin/IGF-1 resulted in reducing the klotho expression as well as LPS-stimulated TNF-α release and increasing the FITC-dextran uptake but unaltering reactive oxygen species (ROS) production in BMDCs. The effects were abolished by using pharmacological inhibition of PI3K/Akt with LY294002 and paralleled by transfecting DCs with klotho siRNA. In conclusion, the regulation of klotho sensitive DC function by IGF-1 or insulin is mediated through PI3K/Akt signaling pathway in BMDCs.
Asunto(s)
Células Dendríticas/metabolismo , Glucuronidasa/biosíntesis , Factor I del Crecimiento Similar a la Insulina/genética , Insulina/genética , Proteína Oncogénica v-akt/genética , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Cromonas/administración & dosificación , Células Dendríticas/efectos de los fármacos , Dextranos/administración & dosificación , Fluoresceína-5-Isotiocianato/administración & dosificación , Fluoresceína-5-Isotiocianato/análogos & derivados , Regulación de la Expresión Génica/efectos de los fármacos , Glucuronidasa/genética , Humanos , Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Proteínas Klotho , Lipopolisacáridos/administración & dosificación , Ratones , Morfolinas/administración & dosificación , Proteína Oncogénica v-akt/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Especies Reactivas de Oxígeno , Transducción de Señal/efectos de los fármacosRESUMEN
DCs contribute to immune homeostasis under physiological conditions and regulate the immune activation during infection. The deubiquitinase A20 inhibits the activation of NF-κB-dependent immune reactions, and prevents the hyperactivation of DCs under steady-state conditions. However, the role of DC-specific A20 under pathological conditions is unknown. Here, we demonstrate that upon injection of low-dose LPS, mice with DC-specific A20 deletion (CD11c-Cre A20(fl/fl) ) died within 6 h, whereas A20(fl/fl) controls survived. LPS-induced mortality in CD11c-Cre A20(fl/fl) mice was characterized by increased serum levels of IL-2, IL-10, IL-12, IFN-γ, and TNF. Upon LPS stimulation, the activation of NF-κB and ERK-NFATc3 pathways were enhanced in A20-deficient DCs, resulting in an increased production of IL-2, IL-12, and TNF both in vitro and in vivo. Targeted inhibition of ERK in A20-deficient DCs abolished the increased production of IL-2. A20-deficient DCs failed to induce LPS tolerance, which was independent of T cells and the intestinal flora, since T-cell depletion and decolonization of CD11c-Cre A20(fl/fl) mice could not prevent death of LPS-challenged CD11c-Cre A20(fl/fl) mice. In conclusion, these findings show that DC-specific A20 preserves immune homeostasis in steady-state conditions and is also required for LPS tolerance.
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Cisteína Endopeptidasas/inmunología , Células Dendríticas/inmunología , Regulación de la Expresión Génica/efectos de los fármacos , Péptidos y Proteínas de Señalización Intracelular/inmunología , Lipopolisacáridos/toxicidad , Animales , Cisteína Endopeptidasas/genética , Citocinas/genética , Citocinas/inmunología , Células Dendríticas/patología , Quinasas MAP Reguladas por Señal Extracelular/genética , Quinasas MAP Reguladas por Señal Extracelular/inmunología , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/inmunología , Péptidos y Proteínas de Señalización Intracelular/genética , Ratones , Ratones Transgénicos , Linfocitos T/inmunología , Linfocitos T/patología , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfaRESUMEN
BACKGROUND: Dendritic cells (DCs) are the most potent professional antigen-presenting cells for naive T cells to link innate and acquired immunity. Klotho, an anti-aging protein, participates in the regulation of Ca2+ dependent migration in DCs. Vitamin E (VitE) is an essential antioxidant to protect cells from damage and elicits its inhibitory effects on NF-κB-mediated inflammatory response. However, the roles of VitE on mouse DC functions and the contribution of klotho to those effects both are unknown. The present study explored the effects of VitE on klotho expression, maturation, ROS production and migration in DCs. METHODS: The mouse bone marrow cells were isolated and cultured with GM-CSF to attain bone marrow-derived DCs (BMDCs). Cells were stimulated with LPS (100 ng/ml) in the presence or absence of VitE (500 µM). RT-PCR and immunoprecipitation methods were employed to determine klotho expression, ELISA to determine cytokine release, flow cytometry to analyze number of CD86+CD11c+ cells, the intracellular expression of cytokines and reactive oxygen species (ROS) production and a transwell migration assay to trace migration. RESULTS: Klotho transcript level and this hormone secretion in DC supernatant were enhanced by VitE treatment and further increased in the presence of NF-κB inhibitor Bay 11-7082 (10 µM). Moreover, VitE treatment inhibited IL-12p70 protein expression of, ROS accumulation in and CCL21-dependent migration of LPS-triggered mature DCs, these effects were reversed following klotho silencing. CONCLUSION: The up-regulation of klotho by VitE could contribute to the inhibitory effects of VitE on NF-κB-mediated DC functional maturation. The events might contribute to immunotherapeutic effect of VitE on the pathophysiology of klotho-related disease.
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Células Dendríticas/efectos de los fármacos , Glucuronidasa/efectos de los fármacos , Vitamina E/farmacología , Vitaminas/farmacología , Análisis de Varianza , Animales , Células de la Médula Ósea/citología , Movimiento Celular/efectos de los fármacos , Células Dendríticas/fisiología , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Glucuronidasa/fisiología , Immunoblotting , Interleucina-12/análisis , Péptidos y Proteínas de Señalización Intracelular , Proteínas Klotho , Lipopolisacáridos , Ratones Endogámicos BALB C , Proteínas/análisis , Proteínas/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Reproducibilidad de los Resultados , Factores de Tiempo , Factor de Necrosis Tumoral alfa/análisis , Regulación hacia ArribaRESUMEN
BACKGROUND/AIMS: Dendritic cells (DCs), antigen-presenting cells linking innate and adaptive immunity, are required for initiation of specific T cell-driven immune responses. Phosphoinositide-3-kinase (PI3K) suppresses proinflammatory cytokine production in DCs, which limits T helper (Th1) polarization. PI3K is in part effective by downregulation of transcription factor NF-κB. Downstream signaling elements of PI3K include serum- and glucocorticoid-inducible kinase 1 (SGK1) and its phosphorylation target N-myc downstream regulated gene 1 (NDRG1). The present study explored whether SGK1 and NDRG1 play a role in the regulation of NF-κB and DC-maturation. METHODS: DCs were isolated from bone marrow (BMDCs) or spleen of mice lacking functional SGK1 (sgk1(-/-)) and corresponding wild type mice (sgk1(+/+)). Protein abundance was determined by Western blotting. Transcription was inhibited by siRNA. Abundance of maturation markers was quantified by flow cytometry. FITC-dextran uptake was determined to quantify phagocytosis. RESULTS: NDRG1 was similarly expressed in sgk1(+/+) and sgk1(-/-)BMDCs, but SGK1-dependent phosphorylation of NDRG-1 was decreased in sgk1(-/-)BMDCs. Silencing of NDRG1 in sgk1(+/+)BMDCs as compared to control empty vector-treated BMDCs enhanced nuclear abundance of NF-κB subunit p65. Moreover, the abundance of phosphorylated NF-κB inhibitor IκBα, of phosphorylated IκB kinase (IKKα/ß) and of nuclear p65 were significantly higher in sgk1(-/-)BMDCs than in sgk1(+/+)BMDCs. Expression of maturation markers, MHC II, and CD86, was significantly larger and phagocytic capacity was significantly lower in sgk1(-/-) than in sgk1(+/+)BMDCs. Expression of CD86 and MHCII was also significantly higher in DCs isolated from the spleen of sgk1(-/-) mice than those from sgk1(+/+)mice. CONCLUSION: SGK1 and NDRG1 participate in the regulation of NF-κB signaling in and maturation of DCs.
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Células Dendríticas/metabolismo , Proteínas Inmediatas-Precoces/metabolismo , FN-kappa B/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Secuencia de Bases , Proteínas de Ciclo Celular/metabolismo , Diferenciación Celular , Cartilla de ADN , Células Dendríticas/enzimología , Células Dendríticas/inmunología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fagocitosis , Fosforilación , Reacción en Cadena en Tiempo Real de la Polimerasa , Regulación hacia ArribaRESUMEN
Alterations in signaling pathways and modulation of cell metabolism are associated with the pathogenesis of cancers, including hepatocellular carcinoma (HCC). Small ubiquitin-like modifier (SUMO) proteins and NF-κB family play major roles in various cellular processes. The current study aims to determine the expression profile of SUMO and NF-κB genes in HCC tumors and investigate their association with the clinical outcome of HCC. The expression of 5 genes - SUMO1, SUMO2, SUMO3, NF-κB p65, and NF-κB p50 - was quantified in tumor and adjacent non-tumor tissues of 58 HBV-related HCC patients by real-time quantitative PCR and was analyzed for the possible association with clinical parameters of HCC. The expression of SUMO2 was significantly higher in HCC tumor tissues compared to the adjacent non-tumor tissues (Pâ =â .01), while no significant difference in SUMO1, SUMO3, NF-κB p65, and NF-κB p50 expression was observed between HCC tumor and non-tumor tissues (Pâ >â .05). In HCC tissues, a strong correlation was observed between the expression of SUMO2 and NF-κB p50, between SUMO3 and NF-κB p50, between SUMO3 and NF-κB p65 (Spearman rhoâ =â 0.83; 0.82; 0.772 respectively; Pâ <â .001). The expression of SUMO1, SUMO2, SUMO3, NF-κB p65, and NF-κB p50 was decreased in grade 3 compared to grades 1 and 2 in HCC tumors according to the World Health Organization grades system. Our results highlighted that the SUMO2 gene is upregulated in tumor tissues of patients with HCC, and is related to the development of HCC, thus it may be associated with the pathogenesis of HCC.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina , Humanos , Carcinoma Hepatocelular/virología , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/virología , Neoplasias Hepáticas/metabolismo , Masculino , Femenino , Persona de Mediana Edad , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/genética , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Proteína SUMO-1/genética , Proteína SUMO-1/metabolismo , FN-kappa B/metabolismo , Adulto , Factor de Transcripción ReIA/metabolismo , Factor de Transcripción ReIA/genética , Virus de la Hepatitis B/genética , Subunidad p50 de NF-kappa B/genética , Subunidad p50 de NF-kappa B/metabolismo , Anciano , Regulación Neoplásica de la Expresión Génica , Ubiquitinas/genética , Ubiquitinas/metabolismo , Hepatitis B/complicaciones , Hepatitis B/genéticaRESUMEN
BACKGROUND: Prostate cancer (PCa) is one of the most common malignant tumors of the male urinary system, and its incidence and mortality rates have been increasing worldwide. Benign prostatic hyperplasia (BPH) represents stromal and epithelial cell proliferation in the prostate in elderly males. Abnormal activation of inflammation-related signalling molecules, such as toll-like receptor 4 (TLR4) and Janus kinase/signal transducers and activators of transcription (JAK/STAT) has been linked to the initiation and progression of various human diseases including PCa and BPH. Cylindromatosis (CYLD) gene alterations are associated with PCa progression. In this study, the contribution of CYLD, JAK2, and TLR4 gene variants to PCa and BPH risks and their associations with prostate-specific antigen (PSA) levels, immunophenotype, and clinical features in Vietnamese men were determined. METHODS: A total of 102 patients with PCa, 65 with BPH, and 114 healthy controls were enrolled. The immunophenotype was analyzed by flow cytometry, cytokine secretion by enzyme-linked immunosorbent assay (ELISA), and gene variants by DNA sequencing. RESULTS: Lower levels of transforming growth factor ß (TGF-ß) and higher numbers of CD13+CD117- and CD56+CD25+ cells were observed in the PCa group than in the BPH group. Genetic analysis of the CYLD gene identified five single nucleotide polymorphisms (SNPs), of which c.2351-47 C>T, c.2351-46A>T, and rs1971432171 T>G had significantly higher frequencies in PCa patients than in the control and BPH groups. Sequencing of the TLR4 gene revealed five nucleotide changes, in which the rs2149356 SNP showed an increased risk for both PCa and BPH and the c.331-206 SNP had a reduced risk for PCa. Importantly, the expansion of activated natural killer (NK) cells and higher levels of PSA were found in PCa patients carrying the CT genotype of the CYLD c.2351-47 compared to those with the wild-type genotype. CONCLUSION: Activation of NK cells in CYLD-sensitive PCa patients was associated with serum PSA release and the CYLD c.2351-47 variant may be a significant risk factor for prostatitis in PCa patients.
Asunto(s)
Enzima Desubiquitinante CYLD , Janus Quinasa 2 , Antígeno Prostático Específico , Hiperplasia Prostática , Neoplasias de la Próstata , Receptor Toll-Like 4 , Humanos , Masculino , Hiperplasia Prostática/genética , Hiperplasia Prostática/sangre , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/inmunología , Neoplasias de la Próstata/sangre , Receptor Toll-Like 4/genética , Enzima Desubiquitinante CYLD/genética , Enzima Desubiquitinante CYLD/metabolismo , Antígeno Prostático Específico/sangre , Anciano , Janus Quinasa 2/genética , Persona de Mediana Edad , Inmunofenotipificación , Genotipo , Polimorfismo de Nucleótido Simple , Estudios de Casos y ControlesRESUMEN
The function of dendritic cells (DCs), antigen-presenting cells regulating naïve T-cells, is regulated by cytosolic Ca²âº concentration ([Ca²âº]i). [Ca²âº]i is increased by store-operated Ca²âº entry and decreased by Kâº-independent (NCX) and Kâº-dependent (NCKX) Naâº/Ca²âº exchangers. NCKX exchangers are stimulated by immunosuppressive 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], the biologically active form of vitamin D. Formation of 1,25(OH)2D3 is inhibited by the antiaging protein Klotho. Thus 1,25(OH)2D3 plasma levels are excessive in Klotho-deficient mice (klothohm). The present study explored whether Klotho deficiency modifies [Ca²âº]i regulation in DCs. DCs were isolated from the bone marrow of klothohm mice and wild-type mice (klotho+/+) and cultured for 7-9 days with granulocyte-macrophage colony-stimulating factor. According to major histocompatibility complex II (MHC II) and CD86 expression, differentiation and lipopolysaccharide (LPS)-induced maturation were similar in klothohm DCs and klotho+/+ DCs. However, NCKX1 membrane abundance and NCX/NCKX-activity were significantly enhanced in klothohm DCs. The [Ca²âº]i increase upon acute application of LPS (1 µg/ml) was significantly lower in klothohm DCs than in klotho+/+ DCs, a difference reversed by the NCKX blocker 3',4'-dichlorobenzamyl (DBZ; 10 µM). CCL21-dependent migration was significantly less in klothohm DCs than in klotho+/+ DCs but could be restored by DBZ. NCKX activity was enhanced by pretreatment of klotho+/+ DC precursors with 1,25(OH)2D3 the first 2 days after isolation from bone marrow. Feeding klothohm mice a vitamin D-deficient diet decreased NCKX activity, augmented LPS-induced increase of [Ca²âº]i, and enhanced migration of klothohm DCs, thus dissipating the differences between klothohm DCs and klotho+/+ DCs. In conclusion, Klotho deficiency upregulates NCKX1 membrane abundance and Naâº/Ca²âº-exchange activity, thus blunting the increase of [Ca²âº]i following LPS exposure and CCL21-mediated migration. The effects are in large part due to excessive 1,25(OH)2D3 formation.
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Calcio/metabolismo , Citosol/metabolismo , Células Dendríticas/metabolismo , Glucuronidasa/metabolismo , Animales , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Movimiento Celular/fisiología , Células Cultivadas , Quimiocina CCL21 , Citosol/química , Regulación de la Expresión Génica/fisiología , Glucuronidasa/genética , Proteínas Klotho , Lipopolisacáridos , Macrófagos/citología , Macrófagos/metabolismo , Ratones , Ratones NoqueadosRESUMEN
Single-nucleotide polymorphisms in the tumor necrosis factor, alpha-induced protein 3 gene, which encodes the ubiquitin-modifying protein A20, are linked to susceptibility to multiple sclerosis (MS), a demyelinating autoimmune disease of the central nervous system (CNS). Since it is unresolved how A20 regulates MS pathogenesis, we examined its function in a murine model of MS, namely experimental autoimmune encephalomyelitis (EAE). Deletion of A20 in neuroectodermal cells (astrocytes, neurons, and oligodendrocytes; Nestin-Cre A20fl/fl mice) or selectively in astrocytes (GFAP-Cre A20fl/fl mice) resulted in more severe EAE as compared to control animals. In Nestin-Cre A20fl/fl and GFAP-Cre A20fl/fl mice demyelination and recruitment of inflammatory leukocytes were increased as compared to A20fl/fl control mice. Importantly, numbers of encephalitogenic CD4+ T cells producing interferon (IFN)-γ, interleukin (IL)-17, and granulocyte-macrophage colony-stimulating factor (GM-CSF), respectively, as well as mRNA production of IFN-γ, IL-17, tumor necrosis factor (TNF), GM-CSF, IL-6, CXCL1, CCL2, and CXCL10 were significantly increased in spinal cords of Nestin-Cre A20fl/fl and GFAP-Cre A20fl/fl mice, respectively. Compared to A20-sufficient astrocytes, A20-deficient astrocytes displayed stronger activation of nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB) in response to TNF, IL-17, and GM-CSF, and of signal transducer and activator of transcription 1 (STAT1) upon IFN-γ stimulation. Due to NF-κB and STAT1 hyperactivation, A20-deficient astrocytes produced significantly more chemokines in response to these key encephalitogenic cytokines of autoimmune CD4+ T cells resulting in an amplification of CD4+ T cell recruitment to the CNS. Thus, astrocytic A20 is an important inhibitor of autoimmune-mediated demyelination in the CNS.
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Background: Harlequin ichthyosis (HI) is a severe rare genetic disease that mainly affects the skin. Neonates with this disease are born with thick skin and large diamond-shaped plates covering most of their bodies. Affected neonates lose the ability to control dehydration and regulate temperature and are more susceptible to infections. They also face respiratory failure and feeding problems. These clinical symptoms are factors associated with high mortality rates of neonates with HI. Until now, there are still no effective treatments for HI patients and most patients die in the newborn period. Mutation in the ABCA12 gene, which encodes an adenosine triphosphate-binding cassette (ABC) transporter, has been demonstrated as the major cause of HI. Case presentation: In this study, we report the case who is one infant that was born prematurely at 32 gestational weeks with the whole body covered with thick plate-like scales of skin. The infant was severely infected with mild edema, multiple cracked skins full of the body, yellow discharge, and necrosis of fingers and toes. The infant was suspected to be affected by HI. Whole exome sequencing (WES) was performed as a tool for detecting the novel mutation in one prematurely born Vietnam infant with HI phenotype. And after that, the mutation was confirmed by the Sanger sequencing method in the patient and the members of his family. In this case, one novel mutation c.6353C > G (p.S2118X, Hom) in the ABCA12 gene, was detected in the patient. The mutation has not been reported in any HI patients previously. This mutation was also found in a heterozygous state in the members of the patient's family, including his parents, an older brother, and an older sister who are no symptoms. Conclusions: In this study, we identified a novel mutation in a Vietnamese patient with HI by whole exome sequencing. The results for the patient and the members of his family will be helpful in understanding the etiology of the disease, diagnosing carriers, assisting in genetic counseling, and emphasizing the need for DNA-based prenatal screening for families with a history of the disease.
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Background: Limb-girdle muscular dystrophy (LGMD) is a group of inherited neuromuscular disorders characterized by atrophy and weakness in the shoulders and hips. Over 30 subtypes have been described in five dominant (LGMD type 1 or LGMDD) and 27 recessive (LGMD type 2 or LGMDR). Each subtype involves a mutation in a single gene and has high heterogeneity in age of onset, expression, progression, and prognosis. In addition, the lack of understanding of the disease and the vague, nonspecific symptoms of LGMD subtypes make diagnosis difficult. Even as next-generation sequencing (NGS) genetic testing has become commonplace, some patients remain undiagnosed for many years. Methods: To identify LGMD-associated mutations, Targeted sequencing was performed in the patients and Sanger sequencing was performed in patients and family members. The in silico analysis tools such as Fathmm, M-CAP, Mutation Taster, PolyPhen 2, PROVEAN, REVEL, SIFT, MaxEntScan, Spliceailookup, Human Splicing Finder, NetGene2, and Fruitfly were used to predict the influence of the novel mutations. The pathogenicity of the mutation was interpreted according to the ACMG guidelines. Results: In this study, six patients from four different Vietnamese families were collected for genetic analysis at The Center for Gene and Protein Research and The Department of Molecular Pathology Faculty of Medical Technology, Hanoi Medical University, Hanoi, Vietnam. Based on clinical symptoms and serum creatine kinase (CK) levels, the patients were diagnosed with limb-girdle muscular dystrophies. Five mutations, including four (c.229C>T, p.Arg77Cys; exon one to three deletion; c.983 + 5G>C; and c.257_258insTGGCT, p.Phe88Leufs*125) in the SGCA gene and one (c.946-4_946-1delACAG) in the CAPN3 gene, were detected in six LGMD patients from four unrelated Vietnamese families. Two homozygous mutations (c.983 + 5G>C and c.257_258insTGGCT) in the SGCA gene were novel. These mutations were identified as the cause of the disease in the patients. Conclusion: Our results contribute to the general understanding of the etiology of the disease and provide the basis for definitive diagnosis and support genetic counseling and prenatal screening.
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Acute myeloid leukemia (AML) is the most aggressive hematopoietic malignancy characterized by uncontrolled proliferation of myeloid progenitor cells within the bone marrow. Tumor suppressor cylindromatosis (CYLD) is a deubiquitinating enzyme, which suppresses inflammatory response in macrophages. Macrophages have a central role in the defense against foreign substances and circulating cancer cells by their professional phagocytic capacity. Little is known about contributions of CYLD to changes in biological properties of human macrophages and its involvement in AML. The present study, therefore, explored whether macrophage functions in healthy individuals and AML patients are influenced by CYLD. To this end, ninety-two newly diagnosed AML patients and 80 healthy controls were recruited. The mRNA expression levels of inflammation-related genes were evaluated by real-time PCR, cell maturation, phagocytosis and apoptosis assays by flow cytometry and secretion of inflammatory cytokines by ELISA. As a result, AML patients with the low CYLD expression were significantly higher in M4/M5 than other subtypes according to the FAB type. The low CYLD expression was also closely associated with older patients and enhanced level of LDH in AML. Moreover, treatment of normal macrophages with CYLD siRNA enhanced activation of STAT-1, leading to increases in expressions of maturation markers and IL-6 production as well as suppression in cell apoptosis and phagocytosis, while macrophage phagocytosis from AML M4/M5b was higher than that from healthy controls upon CYLD siRNA transfection through STAT1 signalling. In conclusion, the inhibitory effects of CYLD on macrophage functions are expected to affect the immune response in AML.