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1.
Cell Physiol Biochem ; 54(1): 126-141, 2020 Feb 05.
Artículo en Inglés | MEDLINE | ID: mdl-32017483

RESUMEN

BACKGROUND/AIMS: Serotonin (5-hydroxytryptamine, 5-HT) is a neurotransmitter and hormone with important physiological functions in many organs, including the intestine. We have previously shown that 5-HT activates the aryl hydrocarbon receptor (AhR) in intestinal epithelial cells (IECs) via a serotonin transporter (SERT)-dependent mechanism. AhR is a nuclear receptor that binds a variety of molecules including tryptophan (TRP) metabolites to regulate physiological processes in the intestine including xenobiotic detoxification and immune modulation. We hypothesized that 5-HT activates AhR indirectly by interfering with metabolic clearance of AhR ligands by cytochrome P450 1A1 (CYP1A1). METHODS: Inhibition of CYP1A1 activity by 5-HT was assessed in the human intestinal epithelial cell line Caco-2 and recombinant CYP1A1 microsomes using both luciferase and LC-MS/MS. Degradation of 5-HT by recombinant CYP1A1 was measured by LC-MS/MS. For in vitro studies, CYP1A1 and CYP1B1 mRNA expression levels were measured by RT-PCR and CYP1A1 activity was measured by ethoxyresorufin-O-deethylase (EROD) assays. For in vivo studies, AhR ligands were administered to SERT KO mice and WT littermates and intestinal mucosa CYP1A1 mRNA was measured. RESULTS: We show that 5-HT inhibits metabolism of both the pro-luciferin CYP1A1 substrate Luc-CEE as well as the high affinity AhR ligand 6-formylindolo[3,2-b] carbazole (FICZ). Recombinant CYP1A1 assays revealed that 5-HT is metabolized by CYP1A1 in an NADPH dependent manner. Treatment with 5-HT in TRP-free medium, which is devoid of trace AhR ligands, showed that 5-HT requires the presence of AhR ligands to activate AhR. Cotreatment with 5-HT and FICZ confirmed that 5-HT potentiates induction of AhR target genes by AhR ligands. However, this was only true for ligands which are CYP1A1 substrates such as FICZ. Administration of ß-napthoflavone by gavage or indole-3-carbinol via diet to SERT KO mice revealed that lack of SERT impairs intestinal AhR activation. CONCLUSION: Our studies provide novel evidence of crosstalk between serotonergic and AhR signaling where 5-HT can influence the ability of AhR ligands to activate the receptor in the intestine.


Asunto(s)
Citocromo P-450 CYP1A1/metabolismo , Receptores de Hidrocarburo de Aril/metabolismo , Serotonina/farmacología , Transcripción Genética/efectos de los fármacos , Animales , Células CACO-2 , Carbazoles/farmacología , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1B1/genética , Citocromo P-450 CYP1B1/metabolismo , Humanos , Ligandos , Masculino , Ratones , Ratones Noqueados , ARN Mensajero/metabolismo , Receptores de Hidrocarburo de Aril/química , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas de Transporte de Serotonina en la Membrana Plasmática/deficiencia , Proteínas de Transporte de Serotonina en la Membrana Plasmática/genética , Transducción de Señal/efectos de los fármacos , Especificidad por Sustrato , beta-naftoflavona/administración & dosificación
2.
Life Sci ; 242: 117189, 2020 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-31891724

RESUMEN

AIMS: Neointimal hyperplasia contributes to arterial restenosis after percutaneous transluminal coronary angioplasty or vascular surgery. Neointimal thickening after arterial injury is determined by inflammatory processes. We investigated the role of the innate immune receptor toll-like receptor 2 (TLR2) in neointima formation after arterial injury in mice. MATERIALS AND METHODS: Carotid artery injury was induced by 10% ferric chloride in C57Bl/6J wild type (WT), TLR2 deficient (B6.129-Tlr2tm1Kir/J, TLR2-/-) and WT mice treated with a TLR2 blocking antibody. 21 days after injury, carotid arteries were assessed histomorphometrically and for smooth muscle cell (SMC) content. To identify the contribution of circulating cells in mediating the effects of TLR2-deficiency, arterial injury was induced in WT/TLR2-/--chimeric mice and the paracrine modulation of bone marrow-derived cells from WT and TLR2-/- on SMC migration compared in vitro. KEY FINDINGS: TLR2-/- mice and WT mice treated with TLR2 blocking antibodies exhibited reduced neointimal thickening (23.7 ± 4.2 and 6.5 ± 3.0 vs. 43.1 ± 5.9 µm, P < 0.05 and P < 0.01), neointimal area (5491 ± 1152 and 315 ± 76.7 vs. 13,756 ± 2627 µm2, P < 0.05 and P < 0.01) and less luminal stenosis compared to WT mice (8.5 ± 1.6 and 5.0 ± 1.3 vs. 22.4 ± 2.2%, both P < 0.001n = 4-8 mice/group). The phenotypes of TLR2-/- vs. WT mice were completely reverted in WT/TLR2-/- bone marrow chimeric mice (5.9 ± 1.5 µm neointimal thickness, 874.2 ± 290.2 µm2 neointima area and 2.7 ± 0.6% luminal stenoses in WT mice transplanted with TLR2-/- bone marrow vs. 23.6 ± 5.1 µm, 3555 ± 511 µm2 and 12.0 ± 1.3% in WT mice receiving WT bone marrow, all P < 0.05, n = 6/group). Neointimal lesions of WT and WT mice transplanted with TLR2-/- bone marrow chimeric mice showed increased numbers of SMC (10.8 ± 1.4 and 12.6 ± 1.4 vs. 3.8 ± 0.9 in TLR2-/- and 3.5 ± 1.1 cells in WT mice transplanted with TLR2-/- bone marrow, all P < 0.05, n = 6). WT bone marrow cells stimulated SMC migration more than TLR2-deficient bone marrow cells (1.7 ± 0.05 vs. 1.3 ± 0.06-fold, P < 0.05, n = 7) and this effect was aggravated by TLR2 stimulation and diminished by TLR2 blockade (1.1 ± 0.03-fold after stimulation with TLR2 agonists and 0.8 ± 0.02-fold after TLR2 blockade vs. control treated cells defined as 1.0, P < 0.05, n = 7). SIGNIFICANCE: TLR2-deficiency on hematopoietic but not vessel wall resident cells augments vascular healing after arterial injury. Pharmacological blockade of TLR2 may thus be a promising therapeutic option to improve vessel patency after iatrogenic arterial injury.


Asunto(s)
Células de la Médula Ósea/metabolismo , Receptor Toll-Like 2/deficiencia , Cicatrización de Heridas , Animales , Arterias/lesiones , Trasplante de Médula Ósea , Traumatismos de las Arterias Carótidas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Liso Vascular/lesiones , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Neointima/metabolismo , Neointima/patología , Receptor Toll-Like 2/metabolismo
3.
Hum Genet ; 139(2): 257-271, 2020 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-31942643

RESUMEN

Severe asthenozoospermia is a common cause of male infertility. Recent studies have revealed that SPEF2 mutations lead to multiple morphological abnormalities of the sperm flagella (MMAF) without primary ciliary dyskinesia (PCD) symptoms in males, but PCD phenotype was also found in one female individual. Therefore, whether there is a phenotypic continuum ranging from infertile patients with PCD to MMAF patients with no or low noise PCD manifestations remains elusive. Here, we performed whole-exome sequencing in 47 patients with severe asthenozoospermia from 45 unrelated Chinese families. We identified four novel biallelic mutations in SPEF2 (8.9%, 4/45) in six affected individuals (12.8%, 6/47), while no deleterious biallelic variants in SPEF2 were detected in 637 controls, including 219 with oligoasthenospermia, 195 with non-obstructive azoospermia, and 223 fertile controls. Notably, all six patients exhibited PCD-like symptoms, including recurrent airway infections, bronchitis, and rhinosinusitis. Ultrastructural analysis revealed normal 9 + 2 axonemes of respiratory cilia but consistently abnormal 9 + 0 axoneme or disordered accessory structures of sperm flagella, indicating different roles of SPEF2 in sperm flagella and respiratory cilia. Subsequently, a Spef2 knockout mouse model was used to validate the PCD-like phenotype and male infertility, where the subfertility of female Spef2-/- mice was found unexpectedly. Overall, our data bridge the link between MMAF and PCD based on the association of SPEF2 mutations with both infertility and PCD in males and provide basis for further exploring the molecular mechanism of SPEF2 during spermiogenesis and ciliogenesis.


Asunto(s)
Anomalías Múltiples/patología , Proteínas de Ciclo Celular/genética , Cilios/patología , Trastornos de la Motilidad Ciliar/patología , Infertilidad Masculina/patología , Proteínas/fisiología , Cola del Espermatozoide/patología , Anomalías Múltiples/genética , Animales , Cilios/genética , Trastornos de la Motilidad Ciliar/genética , Femenino , Humanos , Infertilidad Masculina/genética , Masculino , Ratones , Ratones Noqueados , Fenotipo , Motilidad Espermática , Cola del Espermatozoide/metabolismo
4.
Invest Ophthalmol Vis Sci ; 61(1): 3, 2020 Jan 23.
Artículo en Inglés | MEDLINE | ID: mdl-31995154

RESUMEN

Purpose: The purpose of this study was to investigate the expression of death ligands in the lacrimal glands (LGs), identify upstream factors that regulate their expression, and determine the functional roles of these factors in the pathogenesis of dry eye disease (DED). Methods: For DED experiment, ex vivo coculture system with LG and in vivo murine model using a controlled environment chamber were utilized. C57BL/6 mice and hypoxia-inducible factor (HIF)-1α conditional knockout (CKO) mice were used. Immunohistochemical staining, polymerase chain reaction, and immunoblotting were performed to determine levels of death ligands including tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in DED-induced LGs. Additionally, acinar cell and CD45+ cell apoptosis was determined with neutralizing TRAIL treatment. Results: Desiccating stress significantly increased HIF-1α expression in LG-acinar cells. Furthermore, HIF-1α deficiency significantly enhanced the infiltration of CD45+ inflammatory cells in LG and induced LG-acinar cell death. Meanwhile, only TRAIL expression was increased in DED-LG, but abrogated in HIF-1α CKO. Interestingly, the main source of TRAIL was the CD45- LG-acinar cells, but not CD45+ immune cells after DED induction. Using ex vivo coculture system, we confirmed LG-induced apoptosis of immune cells via HIF-1α-mediated TRAIL secretion following DED. Consistent with ex vivo, the insufficiency of HIF-1α and TRAIL enhanced recruitment of inflammatory cells to the LG and subsequently exacerbated ocular surface damage in DED mice. Conclusions: Our findings offer novel insight into the regulatory function of acinar cell-derived TRAIL in limiting inflammatory damage and could be implicated in the development of potential therapeutic strategies for DED.


Asunto(s)
Dacriocistitis/metabolismo , Síndromes de Ojo Seco/metabolismo , Regulación de la Expresión Génica/fisiología , Subunidad alfa del Factor 1 Inducible por Hipoxia/fisiología , Aparato Lagrimal/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/genética , Animales , Apoptosis , Técnicas de Cocultivo , Dacriocistitis/patología , Modelos Animales de Enfermedad , Síndromes de Ojo Seco/patología , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Immunoblotting , Etiquetado Corte-Fin in Situ , Aparato Lagrimal/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Técnicas de Cultivo de Órganos , Reacción en Cadena en Tiempo Real de la Polimerasa
5.
Immunity ; 52(1): 151-166.e6, 2020 01 14.
Artículo en Inglés | MEDLINE | ID: mdl-31924474

RESUMEN

In addition to helper and regulatory potential, CD4+ T cells also acquire cytotoxic activity marked by granzyme B (GzmB) expression and the ability to promote rejection of established tumors. Here, we examined the molecular and cellular mechanisms underpinning the differentiation of cytotoxic CD4+ T cells following immunotherapy. CD4+ transfer into lymphodepleted animals or regulatory T (Treg) cell depletion promoted GzmB expression by tumor-infiltrating CD4+, and this was prevented by interleukin-2 (IL-2) neutralization. Transcriptional analysis revealed a polyfunctional helper and cytotoxic phenotype characterized by the expression of the transcription factors T-bet and Blimp-1. While T-bet ablation restricted interferon-γ (IFN-γ) production, loss of Blimp-1 prevented GzmB expression in response to IL-2, suggesting two independent programs required for polyfunctionality of tumor-reactive CD4+ T cells. Our findings underscore the role of Treg cells, IL-2, and Blimp-1 in controlling the differentiation of cytotoxic CD4+ T cells and offer a pathway to enhancement of anti-tumor activity through their manipulation.


Asunto(s)
Granzimas/inmunología , Neoplasias/inmunología , Factor 1 de Unión al Dominio 1 de Regulación Positiva/metabolismo , Proteínas de Dominio T Box/metabolismo , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/trasplante , Traslado Adoptivo , Animales , Línea Celular Tumoral , Humanos , Interferón gamma/inmunología , Interleucina-2/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Linfocitos T Reguladores/citología , Microambiente Tumoral/inmunología
6.
Immunity ; 52(1): 136-150.e6, 2020 01 14.
Artículo en Inglés | MEDLINE | ID: mdl-31940267

RESUMEN

Effector CD8+ T cells are important mediators of adaptive immunity, and receptor-ligand interactions that regulate their survival may have therapeutic potential. Here, we identified a subset of effector CD8+ T cells that expressed the inhibitory fragment crystallizable (Fc) receptor FcγRIIB following activation and multiple rounds of division. CD8+ T cell-intrinsic genetic deletion of Fcgr2b increased CD8+ effector T cell accumulation, resulting in accelerated graft rejection and decreased tumor volume in mouse models. Immunoglobulin G (IgG) antibody was not required for FcγRIIB-mediated control of CD8+ T cell immunity, and instead, the immunosuppressive cytokine fibrinogen-like 2 (Fgl2) was a functional ligand for FcγRIIB on CD8+ T cells. Fgl2 induced caspase-3/7-mediated apoptosis in Fcgr2b+, but not Fcgr2b-/-, CD8+ T cells. Increased expression of FcγRIIB correlated with freedom from rejection following withdrawal from immunosuppression in a clinical trial of kidney transplant recipients. Together, these findings demonstrate a cell-intrinsic coinhibitory function of FcγRIIB in regulating CD8+ T cell immunity.


Asunto(s)
Apoptosis/inmunología , Linfocitos T CD8-positivos/inmunología , Fibrinógeno/inmunología , Receptores de IgG/inmunología , Adulto , Anciano , Animales , Caspasa 3/inmunología , Caspasa 7/inmunología , Línea Celular Tumoral , Femenino , Fibrinógeno/genética , Rechazo de Injerto/inmunología , Humanos , Inmunoglobulina G/inmunología , Inmunosupresión , Masculino , Melanoma Experimental , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Receptores de IgG/genética , Adulto Joven
7.
Clin Sci (Lond) ; 134(2): 239-259, 2020 Jan 31.
Artículo en Inglés | MEDLINE | ID: mdl-31943002

RESUMEN

Mitochondrial stress has been widely observed in diabetic kidney disease (DKD). Cyclophilin D (CypD) is a functional component of the mitochondrial permeability transition pore (mPTP) which allows the exchange of ions and solutes between the mitochondrial matrix to induce mitochondrial swelling and activation of cell death pathways. CypD has been successfully targeted in other disease contexts to improve mitochondrial function and reduced pathology. Two approaches were used to elucidate the role of CypD and the mPTP in DKD. Firstly, mice with a deletion of the gene encoding CypD (Ppif-/-) were rendered diabetic with streptozotocin (STZ) and followed for 24 weeks. Secondly, Alisporivir, a CypD inhibitor was administered to the db/db mouse model (5 mg/kg/day oral gavage for 16 weeks). Ppif-/- mice were not protected against diabetes-induced albuminuria and had greater glomerulosclerosis than their WT diabetic littermates. Renal hyperfiltration was lower in diabetic Ppif-/- as compared with WT mice. Similarly, Alisporivir did not improve renal function nor pathology in db/db mice as assessed by no change in albuminuria, KIM-1 excretion and glomerulosclerosis. Db/db mice exhibited changes in mitochondrial function, including elevated respiratory control ratio (RCR), reduced mitochondrial H2O2 generation and increased proximal tubular mitochondrial volume, but these were unaffected by Alisporivir treatment. Taken together, these studies indicate that CypD has a complex role in DKD and direct targeting of this component of the mPTP will likely not improve renal outcomes.


Asunto(s)
/metabolismo , Diabetes Mellitus Experimental/metabolismo , Enfermedades Renales/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Albuminuria/genética , Albuminuria/metabolismo , Animales , /genética , Ciclosporina/farmacología , Diabetes Mellitus Experimental/genética , Nefropatías Diabéticas/genética , Nefropatías Diabéticas/metabolismo , Peróxido de Hidrógeno/metabolismo , Riñón/efectos de los fármacos , Riñón/metabolismo , Riñón/patología , Enfermedades Renales/genética , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/genética , ATPasas de Translocación de Protón/metabolismo
8.
Toxicol Lett ; 319: 213-224, 2020 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-31783120

RESUMEN

The upregulated α-synuclein (α-syn) and Tau co-occur in methamphetamine (METH) abusers' brains. Here, we designed experiments mainly to investigate whether α-syn and Tau interact in METH exposure. We detected the expression of α-syn, total Tau, and phosphorylation of Tau at Serine 396 (pSer396 Tau) under in vitro and in vivo conditions after METH exposure to determine the co-occurrence of α-syn and Tau. We also explored the effect of α-syn or Tau on one another by silencing and knocking-out one of them in METH treatment. We found that METH increased the α-syn, total Tau, and pSer396 Tau protein level in SH-SY5Y cells, primary cultured neurons, and in mice brains. In additional, reducing α-syn level can relieve and even normalize the pSer396 Tau and total Tau overexpression after treatment of METH. Furthermore, knocking out Tau can effectively inhibit METH induced overexpression of α-syn in mice brains. Finally, knocking out α-syn or Tau can effectively reduce METH-induced neurotoxicity in mice brains. This research could provide potential therapeutic approaches targeting the vicious circle between α-syn and Tau in METH abusers and patients with neurodegenerative disorders.


Asunto(s)
Estimulantes del Sistema Nervioso Central/toxicidad , Metanfetamina/toxicidad , Síndromes de Neurotoxicidad/genética , Síndromes de Neurotoxicidad/metabolismo , alfa-Sinucleína/biosíntesis , Proteínas tau/biosíntesis , Animales , Conducta Animal/efectos de los fármacos , Línea Celular , Silenciador del Gen , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Síndromes de Neurotoxicidad/psicología , Cultivo Primario de Células , ARN Interferente Pequeño , alfa-Sinucleína/genética , Proteínas tau/genética , Proteínas tau/metabolismo
9.
Life Sci ; 240: 117107, 2020 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-31785241

RESUMEN

BACKGROUND: Toll-like receptor 4 (Tlr4) is recognized due to its role in the immune response. Also, this protein can participate in the signaling pathway of events triggered by physical exercise such as apoptosis, inflammation, and endoplasmic reticulum (ER) stress. The main objective of this study was to evaluate the role of Tlr4 in the markers of these events in the myocardium of mice submitted to acute physical exercise (APE) protocols at different intensities. METHODS: Echocardiogram, RT-qPCR, and immunoblotting technique were used to evaluate the left ventricle of wild-type (WT) and Tlr4 knockout (Tlr4 KO) submitted to APE protocols at 45, 60, and 75% of their maximal velocity. Also, we performed the bioinformatics analysis to establish the connection of heart mRNA levels of Tlr4 with heart genes of inflammation and ER stress of several isogenic strains of BXD mice. RESULTS: Under basal conditions, the Tlr4 deletion diminished the performance, and expression of inflammation and ER stress genes in the left ventricle, but increased the serum levels of CK, Il-17, and Tnf-alpha. Under the same exercise conditions, the Tlr4 deletion reduced the glycemia, serum levels of CK, Il-17, and Tnf-alpha, as well as genes and/or proteins related to apoptosis, inflammation and ER stress in the left ventricle, but increased the levels of CK-mb and LDH, as well as other genes related to apoptosis, inflammation, and ER stress in the left ventricle. CONCLUSION: Altogether, the current findings highlighted the effects of different acute exercise intensities were attenuated in the heart of Tlr4 KO mice.


Asunto(s)
Apoptosis/fisiología , Estrés del Retículo Endoplásmico/fisiología , Corazón/fisiología , Inflamación , Esfuerzo Físico/fisiología , Receptor Toll-Like 4/fisiología , Animales , Apoptosis/genética , Biología Computacional , Creatina Quinasa/sangre , Ecocardiografía , Estrés del Retículo Endoplásmico/genética , Corazón/diagnóstico por imagen , Interleucina-17/sangre , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Esfuerzo Físico/genética , Transducción de Señal/fisiología , Receptor Toll-Like 4/genética , Factor de Necrosis Tumoral alfa/sangre
10.
Life Sci ; 241: 117120, 2020 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-31825792

RESUMEN

AIMS: The present study explored the function and regulatory mechanism of High mobility group box 1 (HMGB1) in asthma. MAIN METHODS: OVA (ovalbumin)-induced asthmatic mice model and LPS-treated cellular model were established in this study. Airway inflammation was measured through detecting the expression of IL-4, IL-5, IL-13 and Interferon-γ (IFN-γ) in serum and BALF (bronchoalveolar lavage fluid) by ELISA kits. Bioinformatics predictive analysis, ChIP assays, Luciferase reporter assay and Western blotting were used to explore the relation between HMGB1 and HSF1 (Heat shock factor 1). KEY FINDINGS: HMGB1 expression was increased in OVA-induced asthmatic mice. Silencing HMGB1 attenuated the increasing of IgE, inflammatory factors (IL-4, IL-5 and IL-13), and airway hyperresponsiveness that induced by OVA. In addition, our study found that HSF1 directly bind with the HMGB1 promoter and negatively regulation of HMGB1. HSF-1 were upregulated in OVA-induced asthmatic mice, and knockdown of HSF1 aggravated the OVA-induced airway inflammation and airway hyperreactivity in mice may through promoting the expression of HMGB1 and the activation of the Toll-like receptor 4 (TLR4)/Myeloid differentiation primary response 88 (MyD88)/Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signal pathway. SIGNIFICANCE: The expression of HMGB1 could be negatively regulated by HSF1, and the TLR4/MyD88/NF-κB signal pathway was involved in HSF1/HMGB1-mediated regulation of asthma.


Asunto(s)
Asma/patología , Proteína HMGB1/metabolismo , Factores de Transcripción del Choque Térmico/fisiología , Inflamación/patología , Factor 88 de Diferenciación Mieloide/metabolismo , FN-kappa B/metabolismo , Receptor Toll-Like 4/metabolismo , Animales , Apoptosis , Asma/inducido químicamente , Asma/genética , Asma/metabolismo , Secuencia de Bases , Hiperreactividad Bronquial/etiología , Hiperreactividad Bronquial/metabolismo , Hiperreactividad Bronquial/patología , Citocinas/metabolismo , Células HEK293 , Proteína HMGB1/genética , Humanos , Inflamación/etiología , Inflamación/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 88 de Diferenciación Mieloide/genética , FN-kappa B/genética , Ovalbúmina/toxicidad , Regiones Promotoras Genéticas , Transducción de Señal , Receptor Toll-Like 4/genética
11.
Toxicol Lett ; 320: 1-8, 2020 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-31756458

RESUMEN

With the spread of hexavalent chromium [Cr(VI)] contamination, risk of exposure in non-occupational populations is increasing. The liver is the main target organ for Cr(VI) accumulation; however, the effect of long-term Cr(VI) exposure on liver toxicity is largely unknown. In this study, we investigated the effect of chronic Cr(VI) exposure on liver fibrosis and its possible mechanism. Mice were injected with Cr(VI) for two months, and our results showed Cr(VI) treatment caused liver toxicity characterized by liver structure disorganization, liver dysfunction, and antioxidant enzyme system inhibition. The development of liver fibrosis was also found via the emergence of collagen fibril deposition, increased expression of extracellular matrix-related genes, activation of hepatic stellate cells (HSCs) and increase the expression levels of Hedgehog (Hh) signaling pathway-related molecules. To demonstrate the role of Hh signaling in the regulation of Cr(VI)-induced liver fibrosis, genetically modified mice with heterozygous deficiency of Shh (Shh+/-) were used. In the Shh+/- mice, Hh signaling, HSCs activation and liver fibrosis development were all ameliorated. In conclusion, we demonstrated that Cr(VI)-induced liver fibrosis development resulted from Hh pathway-mediated HSCs activation. Our findings strongly suggest that inhibition of Hh pathway may help in the development of new strategies for Cr(VI)-associated liver fibrosis.


Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Cromo , Proteínas Hedgehog/metabolismo , Células Estrelladas Hepáticas/metabolismo , Cirrosis Hepática Experimental/metabolismo , Hígado/metabolismo , Dicromato de Potasio , Transducción de Señal , Animales , Enfermedad Hepática Inducida por Sustancias y Drogas/genética , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Modelos Animales de Enfermedad , Proteínas Hedgehog/deficiencia , Proteínas Hedgehog/genética , Células Estrelladas Hepáticas/patología , Hígado/patología , Cirrosis Hepática Experimental/inducido químicamente , Cirrosis Hepática Experimental/genética , Cirrosis Hepática Experimental/patología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados
12.
Toxicol Lett ; 319: 160-167, 2020 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-31734271

RESUMEN

Overexposure to 1,2-dichloroethane (1,2-DCE) can induce brain edema, but the underlying mechanisms remain largely unknown. Aquaporin 4 (AQP4) is the most prevalent water channel in the brain, and the pool of AQP4 facilitates brain edema by controlling the inflow and clearance of brain water. MicroRNAs play an important role in the regulation of brain edema via RNA silencing and post-transcriptional regulation of gene expression. To explore the regulation role of AQP4 and microRNA in 1,2-DCE-induced brain edema, Sprague-Dawley (SD) rats and AQP4 knockout CD-1 mice were exposed to 1,2-DCE by inhalation for 7 days (0, 600, 1,800 mg/m3) and 28 days (0, 100, 350, 700 mg/m3), respectively. The results showed that 1,2-DCE induces brain edema, in both rats and mice, characterized by an increase in brain water content and vacuolations in the brain parenchyma and around the vessels of the cerebral cortex. Notably, 1,2-DCE exposure can down-regulate AQP4 expression, in both rats and mice. Also, deleting AQP4 intensifies 1,2-DCE-induced brain edema in mice. Meanwhile, microRNA-29b-3p (miR-29b) expression increases with 1,2-DCE exposure, in both rats and mice. A negative correlation was found between the expression of miR-29b and AQP4 in vivo. Moreover, the negative regulation of miR-29b by direct targeting to AQP4 was confirmed by dual luciferase reporter assay in vitro. Taken together, our findings indicate that AQP4 plays an important role in balancing water content in 1,2-DCE-induced brain edema. The dysregulation of miR-29b after 1,2-DCE exposure can aggravate brain edema by directly suppressing the expression of AQP4.


Asunto(s)
Acuaporina 4/efectos de los fármacos , Edema Encefálico/inducido químicamente , Dicloruros de Etileno/toxicidad , MicroARNs/genética , Administración por Inhalación , Animales , Acuaporina 4/genética , Agua Corporal/metabolismo , Química Encefálica/efectos de los fármacos , Edema Encefálico/patología , Femenino , Masculino , Ratones , Ratones Noqueados , MicroARNs/biosíntesis , Ratas , Ratas Sprague-Dawley
13.
Life Sci ; 240: 116985, 2020 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-31647949

RESUMEN

BACKGROUND: The infiltration and activation of macrophages play key roles in arterial restenosis, providing a promising strategy for the treatment of restenosis caused by intimal hyperplasia. Although miR-150 has been implicated in cardiovascular diseases, the individual effect of miR-150 on intimal hyperplasia remains unclear. METHODS AND RESULTS: We observed that the expression of miR-150 was robustly reduced in proinflammatory M1 macrophages and reversely induced in resolving M2 macrophages. An in vitro experiment demonstrated that miR-150 deficiency promoted extensive upregulation of the expression of M1 markers but attenuated the expression of M2 macrophage markers. MiR-150 enhanced the proliferation and migration of vascular smooth muscle cells (VSMCs) when co-cultured with conditioned medium from polarized macrophages upon LPS or IL-4 stimulation. Mechanistically, the bioinformatics analysis and luciferase assay results showed that miR-150 directly targeted STAT1 and STAT1 was required for the effect of miR-150 knockout on macrophage polarization. More importantly, we showed that knockout of miR-150 accelerated neointima formation, accompanied by the activation of M1 macrophages and the inactivation of M2 macrophages. Furthermore, miR-150 deficiency in marrow-derived cell accelerated neointima formation. CONCLUSION: Our research demonstrated that miR-150 deficiency promoted intimal hyperplasia with high ratios of M1 to M2 macrophages and subsequently increased VSMCs proliferation and migration, which were partially mediated by directly targeting to STAT1. Collectively, these results suggested that miR-150 may act as a novel therapeutic target for arterial restenosis.


Asunto(s)
Polaridad Celular/genética , Hiperplasia/genética , Macrófagos , MicroARNs/genética , Neointima/genética , Animales , Movimiento Celular/genética , Proliferación Celular , Biología Computacional , Hiperplasia/patología , Inflamación/genética , Inflamación/patología , Interleucina-4/farmacología , Lipopolisacáridos/farmacología , Masculino , Ratones , Ratones Noqueados , Músculo Liso Vascular , Neointima/patología , Factor de Transcripción STAT1/genética
14.
Cancer Sci ; 111(1): 137-147, 2020 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-31724799

RESUMEN

As a member of the epidermal growth factor receptor (EGFR) family, ERBB3 plays an essential role in development and disease independent of inherently inactive kinase domain. Recently, ERBB3 has been found to bind to ATP and has catalytic activity in vitro. However, the biological function of ERBB3 kinase activity remains elusive in vivo. Here we have identified the physiological function of inactivated ERBB3 kinase activity by creating Erbb3-K740M knockin mice in which ATP cannot bind to ERBB3. Unlike Erbb3 knockout mice, kinase-inactive Erbb3K740M homozygous mice were born in Mendelian ratios and showed normal development. After dextran sulfate sodium-induced colitis, the kinase-inactive Erbb3 mutant mice showed normal recovery. However, the outgrowth of ileal organoids by neuregulin-1 treatment was more attenuated in Erbb3 mutant mice than in WT mice. Moreover, in combination with the ApcMin mouse, the proportion of polyps less than 1 mm in diameter in mutant mice was higher than in control mice and an increase in the number of apoptotic cells was observed in polyps from mutant mice compared with polyps from control mice. Taken together, the ERBB3 kinase activity contributes to the outgrowth of ileal organoids and intestinal tumorigenesis, and the development of ERBB3 kinase inhibitors, including epidermal growth factor receptor family members, can be a potential way to target colorectal cancer.


Asunto(s)
Carcinogénesis/metabolismo , Carcinogénesis/patología , Intestinos/patología , Organoides/metabolismo , Organoides/patología , Receptor ErbB-3/metabolismo , Animales , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Carcinogénesis/efectos de los fármacos , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Intestinos/efectos de los fármacos , Ratones , Ratones Noqueados , Organoides/efectos de los fármacos , Fosforilación/efectos de los fármacos , Fosforilación/fisiología , Pólipos/tratamiento farmacológico , Pólipos/patología , Inhibidores de Proteínas Quinasas/farmacología
15.
Toxicol Lett ; 321: 146-154, 2020 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-31836503

RESUMEN

BACKGROUND: Exposure to particulate matters (PMs) can lead to an acute exacerbation of allergic airway diseases, increasing the severity of symptoms and mortality. However, little is known about the underlying molecular mechanism. This study aimed to investigate the effects of PMs on acute exacerbation of allergic airway inflammation and seek potential therapeutic targets. METHODS: Non-allergic control and ovalbumin (OVA)-allergic wide-type (WT) and Toll-like receptor 2 knockout (Tlr2-/-) mice were exposed to 100 µg of PM (diameter 5.85 µm) or saline by the oropharyngeal instillation. The responses were examined three days after exposure. In the RAW264.7 macrophage cell line, Tlr2 was knocked down by small-interfering RNA or the NF-κB inhibitor JSH-23 was used, and then the cells were stimulated with PMs for 12 h before comparison of the inflammatory responses. RESULTS: PM exposure led to increased inflammatory cell recruitment and airway intensity of PAS + staining in OVA-allergic WT mice, accompanied with an accumulation of inflammatory cells and elevated inflammatory cytokines, such as IL-6 and IL-18, in the bronchoalveolar lavage fluid (BALF). Furthermore, the protein levels of TLR2 and the NLRP3 inflammasome were elevated concomitantly with the airway inflammation post-OVA/PMs challenge. Tlr2 deficiency effectively inhibited the airway inflammation, including pulmonary inflammatory cell recruitment, mucus secretion, serum OVA-specific immunoglobulin E (IgE), and BALF inflammatory cytokine production. Additionally, the P-induced NLRP3 activation in the RAW 264.7 cell line was diminished by the knockdown of Tlr2 or JSH-23 treatment in vitro. CONCLUSION: Our results indicated that PMs exacerbate the allergic airway inflammation mediated by the TLR2/ NF-κB/NLRP3 signaling pathway. Inhibition of NF-κB seems to be a possible treatment.


Asunto(s)
Pulmón/efectos de los fármacos , Macrófagos/efectos de los fármacos , FN-kappa B/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Material Particulado/toxicidad , Hipersensibilidad Respiratoria/inducido químicamente , Receptor Toll-Like 2/metabolismo , Alérgenos , Animales , Citocinas/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Pulmón/metabolismo , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ovalbúmina , Tamaño de la Partícula , Células RAW 264.7 , Hipersensibilidad Respiratoria/genética , Hipersensibilidad Respiratoria/metabolismo , Transducción de Señal , Receptor Toll-Like 2/deficiencia , Receptor Toll-Like 2/genética
16.
Scand J Immunol ; 91(1): e12805, 2020 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-31267543

RESUMEN

Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency disease caused by a mutation in the WAS gene that encodes the WAS protein (WASp); up to 5-10% of these patients develop inflammatory bowel disease (IBD). The mechanisms by which WASp deficiency causes IBD are unclear. Intestinal microbial dysbiosis and imbalances in host immune responses play important roles in the pathogenesis of polygenetic IBD; however, few studies have conducted detailed examination of the microbial alterations and their relationship with IBD in WAS. Here, we collected faecal samples from 19 children (all less than 2 years old) with WAS and samples from WASp-KO mice with IBD and subjected them to 16S ribosomal RNA sequencing. We found that microbial community richness and structure in WAS children were different from those in controls; WAS children revealed reduced microbial community richness and diversity. Relative abundance of Bacteroidetes and Verrucomicrobiain in WAS children was significantly lower, while that of Proteobacteria was markedly higher. WASp-KO mice revealed a significantly decreased abundance of Firmicutes. Faecal microbial dysbiosis caused by WASp deficiency is similar to that observed for polygenetic IBD, suggesting that WASp may play crucial function in microbial homoeostasis and that microbial dysbiosis may contribute to IBD in WAS. These microbial alterations may be useful targets for monitoring and therapeutically managing intestinal inflammation in WAS.


Asunto(s)
Disbiosis , Heces/microbiología , Microbioma Gastrointestinal , Síndrome de Wiskott-Aldrich/etiología , Animales , Biodiversidad , Biomarcadores , Estudios de Casos y Controles , Preescolar , Modelos Animales de Enfermedad , Femenino , Humanos , Lactante , Enfermedades Inflamatorias del Intestino/etiología , Masculino , Metagenoma , Metagenómica/métodos , Ratones , Ratones Noqueados , Mutación , ARN Ribosómico 16S/genética , Síndrome de Wiskott-Aldrich/diagnóstico , Proteína del Síndrome de Wiskott-Aldrich/deficiencia
17.
Gut ; 69(1): 177-186, 2020 01.
Artículo en Inglés | MEDLINE | ID: mdl-30954949

RESUMEN

OBJECTIVE: Increased de novo fatty acid (FA) synthesis and cholesterol biosynthesis have been independently described in many tumour types, including hepatocellular carcinoma (HCC). DESIGN: We investigated the functional contribution of fatty acid synthase (Fasn)-mediated de novo FA synthesis in a murine HCC model induced by loss of Pten and overexpression of c-Met (sgPten/c-Met) using liver-specific Fasn knockout mice. Expression arrays and lipidomic analysis were performed to characterise the global gene expression and lipid profiles, respectively, of sgPten/c-Met HCC from wild-type and Fasn knockout mice. Human HCC cell lines were used for in vitro studies. RESULTS: Ablation of Fasn significantly delayed sgPten/c-Met-driven hepatocarcinogenesis in mice. However, eventually, HCC emerged in Fasn knockout mice. Comparative genomic and lipidomic analyses revealed the upregulation of genes involved in cholesterol biosynthesis, as well as decreased triglyceride levels and increased cholesterol esters, in HCC from these mice. Mechanistically, loss of Fasn promoted nuclear localisation and activation of sterol regulatory element binding protein 2 (Srebp2), which triggered cholesterogenesis. Blocking cholesterol synthesis via the dominant negative form of Srebp2 (dnSrebp2) completely prevented sgPten/c-Met-driven hepatocarcinogenesis in Fasn knockout mice. Similarly, silencing of FASN resulted in increased SREBP2 activation and hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase (HMGCR) expression in human HCC cell lines. Concomitant inhibition of FASN-mediated FA synthesis and HMGCR-driven cholesterol production was highly detrimental for HCC cell growth in culture. CONCLUSION: Our study uncovers a novel functional crosstalk between aberrant lipogenesis and cholesterol biosynthesis pathways in hepatocarcinogenesis, whose concomitant inhibition might represent a therapeutic option for HCC.


Asunto(s)
Carcinoma Hepatocelular/metabolismo , Colesterol/biosíntesis , Acido Graso Sintasa Tipo I/metabolismo , Ácidos Grasos/biosíntesis , Neoplasias Hepáticas/metabolismo , Animales , Vías Biosintéticas/efectos de los fármacos , Vías Biosintéticas/genética , Carcinogénesis/genética , Carcinoma Hepatocelular/genética , Línea Celular Tumoral , Acido Graso Sintasa Tipo I/genética , Femenino , Técnicas de Silenciamiento del Gen , Silenciador del Gen , Genómica , Humanos , Hidroximetilglutaril-CoA Reductasas/genética , Hidroximetilglutaril-CoA Reductasas/metabolismo , Neoplasias Hepáticas/genética , Masculino , Ratones , Ratones Noqueados , Fosfohidrolasa PTEN/metabolismo , Proteínas Proto-Oncogénicas c-met/metabolismo , Proteína 2 de Unión a Elementos Reguladores de Esteroles/genética , Proteína 2 de Unión a Elementos Reguladores de Esteroles/metabolismo , Transcriptoma
18.
Gut ; 69(1): 133-145, 2020 01.
Artículo en Inglés | MEDLINE | ID: mdl-31409605

RESUMEN

OBJECTIVE: The Fragile X mental retardation (FMR) syndrome is a frequently inherited intellectual disability caused by decreased or absent expression of the FMR protein (FMRP). Lack of FMRP is associated with neuronal degradation and cognitive dysfunction but its role outside the central nervous system is insufficiently studied. Here, we identify a role of FMRP in liver disease. DESIGN: Mice lacking Fmr1 gene expression were used to study the role of FMRP during tumour necrosis factor (TNF)-induced liver damage in disease model systems. Liver damage and mechanistic studies were performed using real-time PCR, Western Blot, staining of tissue sections and clinical chemistry. RESULTS: Fmr1null mice exhibited increased liver damage during virus-mediated hepatitis following infection with the lymphocytic choriomeningitis virus. Exposure to TNF resulted in severe liver damage due to increased hepatocyte cell death. Consistently, we found increased caspase-8 and caspase-3 activation following TNF stimulation. Furthermore, we demonstrate FMRP to be critically important for regulating key molecules in TNF receptor 1 (TNFR1)-dependent apoptosis and necroptosis including CYLD, c-FLIPS and JNK, which contribute to prolonged RIPK1 expression. Accordingly, the RIPK1 inhibitor Necrostatin-1s could reduce liver cell death and alleviate liver damage in Fmr1null mice following TNF exposure. Consistently, FMRP-deficient mice developed increased pathology during acute cholestasis following bile duct ligation, which coincided with increased hepatic expression of RIPK1, RIPK3 and phosphorylation of MLKL. CONCLUSIONS: We show that FMRP plays a central role in the inhibition of TNF-mediated cell death during infection and liver disease.


Asunto(s)
Proteína del Retraso Mental del Síndrome del Cromosoma X Frágil/fisiología , Hepatitis Viral Animal/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Animales , Infecciones por Arenaviridae/inmunología , Infecciones por Arenaviridae/patología , Linfocitos T CD8-positivos/inmunología , Muerte Celular/efectos de los fármacos , Muerte Celular/inmunología , Muerte Celular/fisiología , Células Cultivadas , Colestasis/inmunología , Colestasis/metabolismo , Colestasis/patología , Proteína del Retraso Mental del Síndrome del Cromosoma X Frágil/metabolismo , Hepatitis Viral Animal/patología , Hepatitis Viral Animal/prevención & control , Hepatocitos/patología , Imidazoles/farmacología , Imidazoles/uso terapéutico , Indoles/farmacología , Indoles/uso terapéutico , Virus de la Coriomeningitis Linfocítica , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína Serina-Treonina Quinasas de Interacción con Receptores/antagonistas & inhibidores , Proteína Serina-Treonina Quinasas de Interacción con Receptores/fisiología
19.
Immunology ; 159(1): 121-129, 2020 01.
Artículo en Inglés | MEDLINE | ID: mdl-31606895

RESUMEN

The transcription factor hypoxia-inducible factor-1 alpha (HIF-1α) is a key regulator of the response and function of myeloid cells in hypoxic and inflammatory microenvironments. To define the role of HIF-1α in tuberculosis, the progression of aerosol Mycobacterium tuberculosis infection was analysed in mice deficient in HIF-1α in the myeloid lineage (mHIF-1α-/- ). We show that myeloid HIF-1α is not required for the containment of the infection, as both wild-type (WT) and mHIF-1α-/- mice mounted normal Th1 responses and maintained control of bacterial growth throughout infection. However, during chronic infection mHIF-1α-/- mice developed extensive lymphocytic inflammatory involvement of the interstitial lung tissue and died earlier than WT mice. These data support the hypothesis that HIF-1α activity coordinates the response of myeloid cells during M. tuberculosis infection to prevent excessive leucocyte recruitment and immunopathological consequences to the host.


Asunto(s)
Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Pulmón/metabolismo , Mycobacterium tuberculosis/crecimiento & desarrollo , Células Mieloides/metabolismo , Neumonía/metabolismo , Tuberculosis Pulmonar/metabolismo , Animales , Carga Bacteriana , Células Cultivadas , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Interacciones Huésped-Patógeno , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Pulmón/inmunología , Pulmón/microbiología , Ratones Endogámicos C57BL , Ratones Noqueados , Mycobacterium tuberculosis/inmunología , Células Mieloides/inmunología , Células Mieloides/microbiología , Neumonía/genética , Neumonía/inmunología , Neumonía/microbiología , Transducción de Señal , Tuberculosis Pulmonar/genética , Tuberculosis Pulmonar/inmunología , Tuberculosis Pulmonar/microbiología
20.
Int J Cancer ; 146(1): 192-207, 2020 01 01.
Artículo en Inglés | MEDLINE | ID: mdl-31107974

RESUMEN

Malignant pleural mesothelioma (MPM) is a tumor with high chemoresistance and poor prognosis. MPM-initiating cells (ICs) are known to be drug resistant, but it is unknown if and how stemness-related pathways determine chemoresistance. Moreover, there are no predictive markers of IC-associated chemoresistance. Aim of this work is to clarify if and by which mechanisms the chemoresistant phenotype of MPM IC was due to specific stemness-related pathways. We generated MPM IC from primary MPM samples and compared the gene expression and chemo-sensitivity profile of IC and differentiated/adherent cells (AC) of the same patient. Compared to AC, IC had upregulated the drug efflux transporter ABCB5 that determined resistance to cisplatin and pemetrexed. ABCB5-knocked-out (KO) IC clones were resensitized to the drugs in vitro and in patient-derived xenografts. ABCB5 was transcriptionally activated by the Wnt/GSK3ß/ß-catenin/c-myc axis that also increased IL-8 and IL-1ß production. IL-8 and IL-1ß-KO IC clones reduced the c-myc-driven transcription of ABCB5 and reacquired chemosensitivity. ABCB5-KO clones had lower IL-8 and IL-1ß secretion, and c-myc transcriptional activity, suggesting that either Wnt/GSK3ß/ß-catenin and IL-8/IL-1ß signaling drive c-myc-mediated transcription of ABCB5. ABCB5 correlated with lower time-to-progression and overall survival in MPM patients treated with cisplatin and pemetrexed. Our work identified multiple autocrine loops linking stemness pathways and resistance to cisplatin and pemetrexed in MPM IC. ABCB5 may represent a new target to chemosensitize MPM IC and a potential biomarker to predict the response to the first-line chemotherapy in MPM patients.


Asunto(s)
Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Resistencia a Antineoplásicos/genética , Interleucina-1beta/metabolismo , Interleucina-8/metabolismo , Mesotelioma/tratamiento farmacológico , Neoplasias Pleurales/tratamiento farmacológico , Vía de Señalización Wnt , Animales , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Femenino , Humanos , Mesotelioma/metabolismo , Mesotelioma/patología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Neoplasias Pleurales/metabolismo , Neoplasias Pleurales/patología
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