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Sialyllactose is an acidic oligosaccharide that has an immune-protective effect against pathogens and contributes to developing the immune system and intestinal microbes. In this study, a method for the determination of 3'-sialyllactose by high-performance liquid chromatography tandem mass spectrometry was established. The sample was treated with 0.1% formic acid methanol solution, and the gradient elution was performed with 0.05% formic acid water and 0.1% formic acid acetonitrile. The hydrophilic liquid chromatographic column was used for separation. The results showed that the linearity was good in the concentration range of 1~160 µg/L. The limit of detection (LOD) and the limit of quantification (LOQ) of the method were 0.3 µg/kg and 1.0 µg/kg, the recovery range was 91.6%~98.4%, and the relative standard deviation (RSD) was 1.5%~2.2%. This method is fast and sensitive. In addition, the 3'-sialyllactose content in edible bird's nest products produced by different processes was studied. It was found that within the tested range, 3'-sialyllactose in edible bird's nest products increased with the intensity of stewing and increased with the addition of sugar. In short, the results provided a new method for detecting the nutritional value of edible bird's nests, as well as a new direction for improving the nutritional value of edible bird's nest products.
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Aves , Oligosacáridos , Animales , Espectrometría de MasasRESUMEN
OBJECTIVES: Systemic sclerosis (SSc) is characterised by aberrant hedgehog signalling in fibrotic tissues. The hedgehog acyltransferase (HHAT) skinny hedgehog catalyses the attachment of palmitate onto sonic hedgehog (SHH). Palmitoylation of SHH is required for multimerisation of SHH proteins, which is thought to promote long-range, endocrine hedgehog signalling. The aim of this study was to evaluate the role of HHAT in the pathogenesis of SSc. METHODS: Expression of HHAT was analysed by real-time polymerase chain reaction(RT-PCR), immunofluorescence and histomorphometry. The effects of HHAT knockdown were analysed by reporter assays, target gene studies and quantification of collagen release and myofibroblast differentiation in cultured human fibroblasts and in two mouse models. RESULTS: The expression of HHAT was upregulated in dermal fibroblasts of patients with SSc in a transforming growth factor-ß (TGFß)/SMAD-dependent manner. Knockdown of HHAT reduced TGFß-induced hedgehog signalling as well as myofibroblast differentiation and collagen release in human dermal fibroblasts. Knockdown of HHAT in the skin of mice ameliorated bleomycin-induced and topoisomerase-induced skin fibrosis. CONCLUSION: HHAT is regulated in SSc in a TGFß-dependent manner and in turn stimulates TGFß-induced long-range hedgehog signalling to promote fibroblast activation and tissue fibrosis. Targeting of HHAT might be a novel approach to more selectively interfere with the profibrotic effects of long-range hedgehog signalling.
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Aciltransferasas/genética , Regulación de la Expresión Génica , ARN/genética , Esclerodermia Sistémica/genética , Piel/patología , Factor de Crecimiento Transformador beta/metabolismo , Aciltransferasas/biosíntesis , Adulto , Anciano , Animales , Western Blotting , Diferenciación Celular , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Masculino , Ratones , Persona de Mediana Edad , Esclerodermia Sistémica/metabolismo , Esclerodermia Sistémica/patología , Transducción de Señal , Piel/metabolismo , Adulto JovenRESUMEN
OBJECTIVES: The enzyme poly(ADP-ribose) polymerase-1 (PARP-1) transfers negatively charged ADP-ribose units to target proteins. This modification can have pronounced regulatory effects on target proteins. Recent studies showed that PARP-1 can poly(ADP-ribosyl)ate (PARylate) Smad proteins. However, the role of PARP-1 in the pathogenesis of systemic sclerosis (SSc) has not been investigated. METHODS: The expression of PARP-1 was determined by quantitative PCR and immunohistochemistry. DNA methylation was analysed by methylated DNA immunoprecipitation assays. Transforming growth factor-ß (TGFß) signalling was assessed using reporter assays, chromatin immunoprecipitation assays and target gene analysis. The effect of PARP-1 inactivation was investigated in bleomycin-induced and topoisomerase-induced fibrosis as well as in tight-skin-1 (Tsk-1) mice. RESULTS: The expression of PARP-1 was decreased in patients with SSc, particularly in fibroblasts. The promoter of PARP-1 was hypermethylated in SSc fibroblasts and in TGFß-stimulated normal fibroblasts. Inhibition of DNA methyltransferases (DNMTs) reduced the promoter methylation and reactivated the expression of PARP-1. Inactivation of PARP-1 promoted accumulation of phosphorylated Smad3, enhanced Smad-dependent transcription and upregulated the expression of TGFß/Smad target genes. Inhibition of PARP-1 enhanced the effect of TGFß on collagen release and myofibroblast differentiation in vitro and exacerbated experimental fibrosis in vivo. PARP-1 deficiency induced a more severe fibrotic response to bleomycin with increased dermal thickening, hydroxyproline content and myofibroblast counts. Inhibition of PARylation also exacerbated fibrosis in Tsk-1 mice and in mice with topoisomerase-induced fibrosis. CONCLUSION: PARP-1 negatively regulates canonical TGFß signalling in experimental skin fibrosis. The downregulation of PARP-1 in SSc fibroblasts may thus directly contribute to hyperactive TGFß signalling and to persistent fibroblast activation in SSc.
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Fibroblastos/fisiología , Fibrosis/genética , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Esclerodermia Sistémica/genética , Enfermedades de la Piel/genética , Adulto , Anciano , Animales , Metilación de ADN/genética , Modelos Animales de Enfermedad , Regulación hacia Abajo/genética , Femenino , Fibrosis/inducido químicamente , Fibrosis/enzimología , Humanos , Masculino , Ratones , Persona de Mediana Edad , Proteínas Serina-Treonina Quinasas , Esclerodermia Sistémica/enzimología , Transducción de Señal , Piel/metabolismo , Piel/patología , Enfermedades de la Piel/enzimología , Proteína smad3/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Adulto JovenRESUMEN
OBJECTIVES: Systemic sclerosis (SSc) fibroblasts remain activated even in the absence of exogenous stimuli. Epigenetic alterations are thought to play a role for this endogenous activation. Trimethylation of histone H3 on lysine 27 (H3K27me3) is regulated by Jumonji domain-containing protein 3 (JMJD3) and ubiquitously transcribed tetratricopeptide repeat on chromosome X (UTX) in a therapeutically targetable manner. The aim of this study was to explore H3K27me3 demethylases as potential targets for the treatment of fibrosis. METHODS: JMJD3 was inactivated by small interfering RNA-mediated knockdown and by pharmacological inhibition with GSKJ4. The effects of targeted inactivation of JMJD3 were analysed in cultured fibroblasts and in the murine models of bleomycin-induced and topoisomerase-I (topoI)-induced fibrosis. H3K27me3 at the FRA2 promoter was analysed by ChIP. RESULTS: The expression of JMJD3, but not of UTX, was increased in fibroblasts in SSc skin and in experimental fibrosis in a transforming growth factor beta (TGFß)-dependent manner. Inactivation of JMJD3 reversed the activated fibroblast phenotype in SSc fibroblasts and prevented the activation of healthy dermal fibroblasts by TGFß. Pharmacological inhibition of JMJD3 ameliorated bleomycin-induced and topoI-induced fibrosis in well-tolerated doses. JMJD3 regulated fibroblast activation in a FRA2-dependent manner: Inactivation of JMJD3 reduced the expression of FRA2 by inducing accumulation of H3K27me3 at the FRA2 promoter. Moreover, the antifibrotic effects of JMJD3 inhibition were reduced on knockdown of FRA2. CONCLUSION: We present first evidence for a deregulation of JMJD3 in SSc. JMJD3 modulates fibroblast activation by regulating the levels of H3K27me3 at the promoter of FRA2. Targeted inhibition of JMJD3 limits the aberrant activation of SSc fibroblasts and exerts antifibrotic effects in two murine models.
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Fibroblastos/enzimología , Histona Demetilasas con Dominio de Jumonji/metabolismo , Esclerodermia Sistémica/enzimología , Adulto , Anciano , Animales , Bleomicina , Estudios de Casos y Controles , Células Cultivadas , Activación Enzimática , Femenino , Fibrosis/inducido químicamente , Fibrosis/enzimología , Humanos , Masculino , Ratones , Persona de Mediana Edad , Adulto JovenRESUMEN
OBJECTIVES: Hedgehog signalling plays a critical role during the pathogenesis of fibrosis in systemic sclerosis (SSc). Besides canonical hedgehog signalling with smoothened (SMO)-dependent activation of GLI transcription factors, GLI can be activated independently of classical hedgehog ligands and receptors (so-called non-canonical pathways). Here, we aimed to evaluate the role of non-canonical hedgehog signalling in SSc and to test the efficacy of direct GLI inhibitors that target simultaneously canonical and non-canonical hedgehog pathways. METHODS: The GLI inhibitor GANT-61 was used to inhibit canonical as well as non-canonical hedgehog signalling, while the SMO inhibitor vismodegib was used to selectively target canonical hedgehog signalling. Furthermore, GLI2 was selectively depleted in fibroblasts using the Cre-LoxP system. The effects of pharmacological or genetic of GLI2 on transforming growth factor-ß (TGF-ß) signalling were analysed in cultured fibroblasts, in bleomycin-induced pulmonary fibrosis and in mice with overexpression of a constitutively active TGF-ß receptor I. RESULTS: TGF-ß upregulated GLI2 in a Smad3-dependent manner and induced nuclear accumulation and DNA binding of GLI2. Fibroblast-specific knockout of GLI2 protected mice from TBRact-induced fibrosis. Combined targeting of canonical and non-canonical hedgehog signalling with direct GLI inhibitors exerted more potent antifibrotic effects than selective targeting of canonical hedgehog signalling with SMO inhibitors in experimental dermal and pulmonary fibrosis. CONCLUSIONS: Our data demonstrate that hedgehog pathways and TGF-ß signalling both converge to GLI2 and that GLI2 integrates those signalling to promote tissue fibrosis. These findings may have translational implications as non-selective inhibitors of GLI2 are in clinical use and selective molecules are currently in development.
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Proteínas Hedgehog/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , Fibrosis Pulmonar/metabolismo , Esclerodermia Sistémica/genética , Esclerodermia Sistémica/metabolismo , Piel/patología , Factor de Crecimiento Transformador beta/metabolismo , Adulto , Anciano , Anilidas/farmacología , Animales , Células Cultivadas , Colágeno Tipo I/genética , Factor de Crecimiento del Tejido Conjuntivo/genética , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibrosis , Técnicas de Inactivación de Genes , Humanos , Factores de Transcripción de Tipo Kruppel/antagonistas & inhibidores , Factores de Transcripción de Tipo Kruppel/genética , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Persona de Mediana Edad , Inhibidor 1 de Activador Plasminogénico/genética , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Pteridinas/farmacología , Fibrosis Pulmonar/inducido químicamente , Piridinas/farmacología , Pirimidinas/farmacología , ARN Mensajero/metabolismo , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/antagonistas & inhibidores , Receptores de Factores de Crecimiento Transformadores beta/genética , Proteínas Recombinantes/farmacología , Transducción de Señal/efectos de los fármacos , Piel/efectos de los fármacos , Proteína smad3/metabolismo , Receptor Smoothened/antagonistas & inhibidores , Factor de Crecimiento Transformador beta/farmacología , Adulto Joven , Proteína Gli2 con Dedos de ZincRESUMEN
OBJECTIVES: Janus kinase 2 (JAK2) has recently been described as a novel downstream mediator of the pro-fibrotic effects of transforming growth factor-ß. Although JAK2 inhibitors are in clinical use for myelodysplastic syndromes, patients often rapidly develop resistance. Tumour cells can escape the therapeutic effects of selective JAK2 inhibitors by mutation-independent transactivation of JAK2 by JAK1. Here, we used selective JAK2 inhibition as a model to test the hypothesis that chronic treatment may provoke resistance by facilitating non-physiological signalling pathways in fibroblasts. METHODS: The antifibrotic effects of long-term treatment with selective JAK2 inhibitors and reactivation of JAK2 signalling by JAK1-dependent transphosphorylation was analysed in cultured fibroblasts and experimental dermal and pulmonary fibrosis. Combined JAK1/JAK2 inhibition and co-treatment with an HSP90 inhibitor were evaluated as strategies to overcome resistance. RESULTS: The antifibrotic effects of selective JAK2 inhibitors on fibroblasts decreased with prolonged treatment as JAK2 signalling was reactivated by JAK1-dependent transphosphorylation of JAK2. This reactivation could be prevented by HSP90 inhibition, which destabilised JAK2 protein, or with combined JAK1/JAK2 inhibitors. Treatment with combined JAK1/JAK2 inhibitors or with JAK2 inhibitors in combination with HSP90 inhibitors was more effective than monotherapy with JAK2 inhibitors in bleomycin-induced pulmonary fibrosis and in adTBR-induced dermal fibrosis. CONCLUSION: Fibroblasts can develop resistance to chronic treatment with JAK2 inhibitors by induction of non-physiological JAK1-dependent transactivation of JAK2 and that inhibition of this compensatory signalling pathway, for example, by co-inhibition of JAK1 or HSP90 is important to maintain the antifibrotic effects of JAK2 inhibition with long-term treatment.
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Fibroblastos/efectos de los fármacos , Janus Quinasa 1/efectos de los fármacos , Janus Quinasa 2/efectos de los fármacos , Pulmón/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Fibrosis Pulmonar/metabolismo , Pirazoles/farmacología , Pirimidinas/farmacología , Esclerodermia Sistémica , Sulfonamidas/farmacología , Adulto , Animales , Antibióticos Antineoplásicos/toxicidad , Benzoquinonas/farmacología , Bleomicina/toxicidad , Western Blotting , Modelos Animales de Enfermedad , Fibroblastos/metabolismo , Fibrosis , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Humanos , Inmunohistoquímica , Janus Quinasa 1/antagonistas & inhibidores , Janus Quinasa 1/metabolismo , Janus Quinasa 2/antagonistas & inhibidores , Janus Quinasa 2/metabolismo , Lactamas Macrocíclicas/farmacología , Pulmón/patología , Masculino , Ratones , Persona de Mediana Edad , Nitrilos , Fosforilación/efectos de los fármacos , Fibrosis Pulmonar/inducido químicamente , Reacción en Cadena en Tiempo Real de la Polimerasa , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
OBJECTIVES: Autophagy has recently been shown to regulate osteoclast activity and osteoclast differentiation. Here, we aim to investigate the impact of autophagy inhibition as a potential therapeutic approach for the treatment of osteoporosis in preclinical models. METHODS: Systemic bone loss was induced in mice by glucocorticoids and by ovariectomy (OVX). Autophagy was targeted by conditional inactivation of autophagy-related gene 7 (Atg7) and by treatment with chloroquine (CQ). Bone density was evaluated by microCT. The role of autophagy on osteoclastogenesis was analysed by osteoclastogenesis and bone resorption assays. The quantification of receptor activator of nuclear factor κ B ligand and osteoprotegerin proteins in cocultures was performed using ELISA whereas that of osteoclast and osteoblast differentiation markers was by qPCR. RESULTS: Selective deletion of Atg7 in monocytes from Atg7(fl/fl)_x_LysM-Cre mice mitigated glucocorticoid-induced and OVX-induced osteoclast differentiation and bone loss compared with Atg7(fl/fl) littermates. Pharmacological inhibition of autophagy by treatment with CQ suppressed glucocorticoid-induced osteoclastogenesis and protected mice from bone loss. Similarly, inactivation of autophagy shielded mice from OVX-induced bone loss. Inhibition of autophagy led to decreased osteoclast differentiation with lower expression of osteoclast markers such as NFATc1, tartrate-resistant acid phosphatase, OSCAR and cathepsin K and attenuated bone resorption in vitro. In contrast, osteoblast differentiation was not affected by inhibition of autophagy. CONCLUSIONS: Pharmacological or genetic inactivation of autophagy ameliorated glucocorticoid-induced and OVX-induced bone loss by inhibiting osteoclastogenesis. These findings may have direct translational implications for the treatment of osteoporosis, since inhibitors of autophagy such as CQ are already in clinical use.
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Autofagia/efectos de los fármacos , Osteoporosis/prevención & control , Animales , Proteína 7 Relacionada con la Autofagia/genética , Células Cultivadas , Cloroquina/farmacología , Cloroquina/uso terapéutico , Modelos Animales de Enfermedad , Femenino , Eliminación de Gen , Glucocorticoides , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Terapia Molecular Dirigida , Monocitos/metabolismo , Osteoclastos/efectos de los fármacos , Osteoclastos/fisiología , Osteogénesis/efectos de los fármacos , Osteoporosis/inducido químicamente , Osteoporosis/etiología , Osteoporosis/patología , OvariectomíaRESUMEN
UNLABELLED: Interleukin (IL)-27, a newly discovered IL-12 family cytokine, is composed of p28 and EBI3. In this study, CD11c-p28(f/f) conditional knockout mice were generated to delete p28 specifically in dendritic cells (DCs). We demonstrated that in the absence of DC-derived p28, these mice were highly susceptible to both low and higher concentrations of concanavalin A (ConA) (5 mg/kg or 10 mg/kg), with extremely early and steady high levels of interferon-γ (IFN-γ) in sera. Neutralizing IFN-γ prevented ConA-induced liver damage in these mice, indicating a critical role of IFN-γ in this pathological process. Interestingly, the main source of the increased IFN-γ in CD11c-p28(f/f) mice was CD4+ T cells, but not natural killer T (NKT) cells. Depletion of CD4+ , but not NK1.1+ , cells completely abolished liver damage, whereas transferring CD4+ T cells from CD11c-p28(f/f) mice, but not from wild-type mice or CD11c-p28(f/f) -IFN-γ(-/-) double knockout mice to CD4(-/-) mice, restored the increased liver damage. Further studies defined higher levels of IFN-γ and T-bet messenger RNA in naïve CD4+ T cells from CD11c-p28(f/f) mice, and these CD4+ T cells were highly responsive to both low and higher concentrations of anti-CD3, indicating a programmed functional alternation of CD4+ T cells. CONCLUSION: We provide a unique model for studying the pathology of CD4+ T cell-mediated liver injury and reveal a novel function of DC-derived p28 on ConA-induced fulminant hepatitis through regulation of the intrinsic ability for IFN-γ production by CD4+ T cells.
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Linfocitos T CD4-Positivos/patología , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Enfermedad Hepática Inducida por Sustancias y Drogas/fisiopatología , Concanavalina A/efectos adversos , Susceptibilidad a Enfermedades/patología , Interferón gamma/fisiología , Animales , Antígeno CD11c/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Femenino , Interferón gamma/genética , Interleucina-17/genética , Interleucina-17/fisiología , Células Asesinas Naturales/metabolismo , Células Asesinas Naturales/patología , Masculino , Ratones , Ratones NoqueadosRESUMEN
PB201 is an orally active, partial glucokinase activator targeting both pancreatic and hepatic glucokinase. As the second glucokinase activator studied beyond phase I, PB201 has demonstrated promising glycemic effects as well as favorable pharmacokinetic (PK) and safety profiles in patients with type 2 diabetes mellitus (T2DM). This study aims to develop a population PK/pharmacodynamic (PD) model for PB201 using the pooled data from nine phase I/II clinical trials conducted in non-Chinese healthy volunteers and a T2DM population and to predict the PK/PD profile of PB201 in a Chinese T2DM population. We developed the PK/PD model using the non-linear mixed-effects modeling approach. All runs were performed using the first-order conditional estimation method with interaction. The pharmacokinetics of PB201 were well fitted by a one-compartment model with saturable absorption and linear elimination. The PD effects of PB201 on reducing the fasting plasma glucose and glycosylated hemoglobin levels in the T2DM population were described by indirect response models as stimulating the elimination of fasting plasma glucose, where the production of glycosylated hemoglobin was assumed to be stimulated by fasting plasma glucose. Covariate analyses revealed enhanced absorption of PB201 by food and decreased systemic clearance with ketoconazole co-administration, while no significant covariate was identified for the pharmacodynamics. The population PK model established for non-Chinese populations was shown to be applicable to the Chinese T2DM population as verified by the PK data from the Chinese phase I study. The final population PK/PD model predicted persistent and dose-dependent reductions in fasting plasma glucose and glycosylated hemoglobin levels in the Chinese T2DM population receiving 50/50 mg, 100/50 mg, and 100/100 mg PB201 twice daily for 24 weeks independent of co-administration of metformin. Overall, the proposed population PK/PD model quantitatively characterized the PK/PD properties of PB201 and the impact of covariates on its target populations, which allows the leveraging of extensive data in non-Chinese populations with the limited data in the Chinese T2DM population to successfully supported the waiver of the clinical phase II trial and facilitate the optimal dose regimen design of a pivotal phase III study of PB201 in China.
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Diabetes Mellitus Tipo 2 , Humanos , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Glucoquinasa , Hemoglobina Glucada , Glucemia , Voluntarios Sanos , Modelos Biológicos , Relación Dosis-Respuesta a DrogaRESUMEN
Con A-induced fulminant hepatitis is a well-known animal model for acute liver failure. However, the role of γδ T cells in this model is undefined. In this report, using TCR δ(-/-) mice, we demonstrated a protective role of γδ T cells in Con A-induced hepatitis model. TCR δ(-/-) mice showed significantly decreased levels of IL-17A and IL-17F in the Con A-treated liver tissue, and reconstitution of TCR δ(-/-) mice with wild-type (Wt), but not IL-17A(-/-), γδ T cells significantly reduced hepatitis, strongly suggesting a critical role of IL-17A in mediating the protective effect of γδ T cells. Interestingly, only Vγ4, but not Vγ1, γδ T cells exerted such a protective effect. Furthermore, depletion of NKT cells in TCR δ(-/-) mice completely abolished hepatitis, and NKT cells from Con A-challenged liver tissues of TCR δ(-/-) mice expressed significantly higher amounts of proinflammatory cytokine IFN-γ than those from Wt mice, indicating that γδ T cells protected hepatitis through targeting NKT cells. Finally, abnormal capacity of IFN-γ production by NKT cells of TCR δ(-/-) mice could only be downregulated by transferring Wt, but not IL-17(-/-), Vγ4 γδ T cells, confirming an essential role of Vγ4-derived IL-17A in regulating the function of NKT cells. In summary, our report thus demonstrated a novel function of Vγ4 γδ T cells in mediating a protective effect against Con A-induced fulminant hepatitis through negatively regulating function of NKT cells in an IL-17A-dependent manner, and transferring Vγ4 γδ T cells may provide a novel therapeutic approach for this devastating liver disease.
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Concanavalina A/toxicidad , Regulación hacia Abajo/inmunología , Interleucina-17/fisiología , Fallo Hepático Agudo/inmunología , Fallo Hepático Agudo/prevención & control , Células T Asesinas Naturales/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/biosíntesis , Subgrupos de Linfocitos T/inmunología , Animales , Concanavalina A/antagonistas & inhibidores , Modelos Animales de Enfermedad , Regulación hacia Abajo/genética , Femenino , Interferón gamma/antagonistas & inhibidores , Interferón gamma/biosíntesis , Interleucina-17/deficiencia , Interleucina-17/genética , Fallo Hepático Agudo/patología , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Células T Asesinas Naturales/metabolismo , Células T Asesinas Naturales/patología , Receptores de Antígenos de Linfocitos T gamma-delta/clasificación , Receptores de Antígenos de Linfocitos T gamma-delta/deficiencia , Receptores de Antígenos de Linfocitos T gamma-delta/genética , Receptores de Antígenos de Linfocitos T gamma-delta/fisiología , Subgrupos de Linfocitos T/metabolismo , Subgrupos de Linfocitos T/patologíaRESUMEN
BACKGROUND: Ovarian cancer (OC) typically develops an immunosuppressive microenvironment by funtional changes of host immune cells. Dysregulated m6A level is associated with cancer progression via the intrinsic oncogenic pathways. However, the role of m6A in regulating host immune cell function during anti-tumor immunity needs comprehensive analysis. This study aimed to investigate the role of METTL3, a catalytic subunit of the methyltransferase complex, in regulating host immune cell response against OC. METHODS: In this study, myeloid-specific Mettl3 gene knockout (Mettl3-cKO) mice were bred using the Cre-LoxP system. Intraperitoneally injection of ID8 cells was used as a syngeneic OC model. Furthermore, the compositions of immune cell populations were analyzed by flow cytometry and single-cell sequencing. Moreover, chemokines and cytokines secretion were assessed using ELISA. Lastly, the role of METTL3 in regulating IL-1ß secretion and inflammasome activation in bone marrow-derived macrophages cocultured with ID8 cells was specified by ELISA and immunoblotting. RESULTS: It was revealed that OC cell growth was enhanced in Mettl3-cKO mice. Furthermore, a shift of decreased M1 to increased M2 macrophage polarization was observed during OC progression. Moreover, Mettl3 depletion in myeloid lineage cells increased secretion of CCL2 and CXCL2 in peritoneal lavage fluild. Interestingly, Mettl3 deficiency enhanced IL-1ß secretion induced by viable ID8 cells independent of inflammasome activation and cell death. Therefore, OC cells in tumor-bearing mice trigger a slight inflammatory response with a low-to-moderate secretion of pro-inflammatory cytokines and chemokines. CONCLUSION: This study provides new insights into METTL3-mediated m6A methylation, which regulates host immune response against OC.
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PURPOSE: Probing efficacy and safety of lusutrombopag in Chinese chronic liver disease (CLD) and severe thrombocytopenia (PLT < 50 × 109/L) patients undergoing elective invasive procedures. METHODS: In this double-blind, parallel-group phase 3 study, 66 patients with CLD and severe thrombocytopenia were randomized 2:1 to lusutrombopag or placebo arm treatment regimens for seven days at 9 centers in China. Responders (PLT ≥ 50 × 109/L that increased to ≥ 20 × 109/L from the baseline and not received rescue therapy for bleeding) on Day 8 (the day after seven-day treatment) were assessed. PLT ≥ 50 × 109/L on or after Day 8 and within 2 days before invasive procedure (alternative criteria for not requiring platelet transfusion) were also analyzed. Adverse events (AEs) were recorded. RESULTS: The proportion of responders on Day 8 was evidently higher (p = 0.0011) in the lusutrombopag group (43.2%, 19/44) versus placebo (4.5%, 1/22). And 72.7% (32/44) patients receiving lusutrombopag met the alternative criteria for not requiring platelet transfusion, while 18.2% (4/22) in the placebo group. The median maximum PLT in lusutrombopag group increased to 80.5 × 109/L, and median time to reach maximum was 14.5 days. Compared with placebo, the lusutrombopag group had a lower incidence of bleeding events (6.8% versus 13.6%), and only one patient had thrombotic-related AE. Overall, the incidence of treatment-emergent AEs was comparable between two groups. CONCLUSIONS: Lusutrombopag was effective in raising PLT, diminishing platelet transfusion requirement, and documented a safety profile like the placebo in CLD and severe thrombocytopenia patients in a Chinese cohort undergoing elective invasive procedures. Chinese clinical trial registration number: CTR20192384.
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Hepatopatías , Trombocitopenia , Humanos , Pueblos del Este de Asia , Hepatopatías/complicaciones , Hepatopatías/tratamiento farmacológico , Trombocitopenia/tratamiento farmacológico , Tiazoles/uso terapéuticoRESUMEN
Bone mass is maintained by the balance between osteoclast-induced bone resorption and osteoblast-triggered bone formation. In inflammatory arthritis such as rheumatoid arthritis (RA), however, increased osteoclast differentiation and activity skew this balance resulting in progressive bone loss. O-GlcNAcylation is a posttranslational modification with attachment of a single O-linked ß-D-N-acetylglucosamine (O-GlcNAc) residue to serine or threonine residues of target proteins. Although O-GlcNAcylation is one of the most common protein modifications, its role in bone homeostasis has not been systematically investigated. We demonstrate that dynamic changes in O-GlcNAcylation are required for osteoclastogenesis. Increased O-GlcNAcylation promotes osteoclast differentiation during the early stages, whereas its downregulation is required for osteoclast maturation. At the molecular level, O-GlcNAcylation affects several pathways including oxidative phosphorylation and cell-cell fusion. TNFα fosters the dynamic regulation of O-GlcNAcylation to promote osteoclastogenesis in inflammatory arthritis. Targeted pharmaceutical or genetic inhibition of O-GlcNAc transferase (OGT) or O-GlcNAcase (OGA) arrests osteoclast differentiation during early stages of differentiation and during later maturation, respectively, and ameliorates bone loss in experimental arthritis. Knockdown of NUP153, an O-GlcNAcylation target, has similar effects as OGT inhibition and inhibits osteoclastogenesis. These findings highlight an important role of O-GlcNAcylation in osteoclastogenesis and may offer the potential to therapeutically interfere with pathologic bone resorption.
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Psammochloa villosa is an ecologically important desert grass that occurs in the Inner Mongolian Plateau where it is frequently the dominant species and is involved in sand stabilization and wind breaking. We sought to generate a preliminary demographic framework for P. villosa to support the future studies of this species, its conservation, and sustainable utilization. To accomplish this, we characterized the genetic diversity and structure of 210 individuals from 43 natural populations of P. villosa using amplified fragment length polymorphism (AFLP) markers. We obtained 1,728 well-defined amplified bands from eight pairs of primers, of which 1,654 bands (95.7%) were polymorphic. Results obtained from the AFLPs suggested effective alleles among populations of 1.32, a Nei's standard genetic distance value of 0.206, a Shannon index of 0.332, a coefficient of gene differentiation (G ST) of 0.469, and a gene flow parameter (Nm) of 0.576. All these values indicate that there is abundant genetic diversity in P. villosa, but limited gene flow. An analysis of molecular variance (AMOVA) showed that genetic variation mainly exists within populations (64.2%), and we found that the most genetically similar populations were often not geographically adjacent. Thus, this suggests that the mechanisms of gene flow are surprisingly complex in this species and may occur over long distances. In addition, we predicted the distribution dynamics of P. villosa based on the spatial distribution modeling and found that its range has contracted continuously since the last interglacial period. We speculate that dry, cold climates have been critical in determining the geographic distribution of P. villosa during the Quaternary period. Our study provides new insights into the population genetics and evolutionary history of P. villosa in the Inner Mongolian Plateau and provides a resource that can be used to design in situ conservation actions and prioritize sustainable utilization.
RESUMEN
Bioactive glass nanoparticles containing copper (Cu-BGNs) were introduced into polycaprolactone (PCL) coating systems to improve the bioactivity, antibacterial properties, and corrosion resistance of vulnerable magnesium matrices under physiological conditions. The influence of different amounts of Cu-BGNs in PCL coatings was thoroughly investigated in determining the wettability, electrochemical properties, and antibacterial effects against Staphylococcus carnosus and Escherichia coli, as well as their cyto-compatibility. Cu-BGNs were observed randomly scattered in PCL coatings. Increasing the concentration of Cu-BGNs resulted in a slight decrease of the water contact angle, and a reduction in anticorrosion properties of the Cu-BGN composite coatings. Yet higher Cu-BGN content in coatings led to more calcium phosphate formation on the surface after 7 days of immersion in Dulbecco's modified Eagle's medium, which was confirmed by Fourier-transform infrared spectroscopy and x-ray photoelectron spectroscopy. The growth of S. carnosus and E. coli was inhibited by Cu2+ ions released from the Cu-BGN coatings. In addition, both direct and indirect cyto-compatibility experiments showed that the viability and proliferation of MG-63 cells on Cu-BGN coatings were highly increased compared to pure magnesium; however, an additional increase of Cu-BGN concentration showed a slight decrease of cell proliferation and cell activity. In summary, Cu-BGN/PCL composite coatings impart magnesium-based biomaterials with antibacterial and anticorrosive properties for clinical applications.
Asunto(s)
Antibacterianos/química , Cobre/química , Vidrio/química , Magnesio/química , Poliésteres/química , Ingeniería de Tejidos/métodos , Materiales Biocompatibles/química , Huesos , Fosfatos de Calcio/química , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Escherichia coli/efectos de los fármacos , Humanos , Pruebas de Sensibilidad Microbiana , Espectroscopía de Fotoelectrones , Espectroscopía Infrarroja por Transformada de Fourier , Staphylococcus/efectos de los fármacosRESUMEN
The development of drug delivery coating systems for local and long-term drug release is gaining increasing interest especially to functionalize bioinert implants with osseointegration and antibacterial properties. In this study, a biodegradable drug delivery coating platform consisting of drug-loaded PHBV microspheres embedded in an alginate-PVA matrix was fabricated by a one-step electrophoretic deposition (EPD) process. Layer by layer (LbL) deposition was exploited to generate chitosan-alginate multilayers on the EPD-produced coating to enlarge the diffusional barrier around the microspheres for controlled drug release. Daidzein, selected as a model drug due to its anti-osteoporosis properties, was pre-encapsulated in PHBV microspheres. The parameters for microsphere fabrication were optimized by an orthogonal design approach. The loading efficiency of daidzein in both the microspheres and in the deposited coatings was adjusted by varying the processing parameters during microsphere fabrication and the EPD process. The degradation of the deposited multilayers was investigated in PBS for up to 14 days. The degradation rate, surface roughness and wettability, as well as adhesion strength of the coatings during degradation were evaluated by applying a range of techniques. A controlled and sustained daidzein release was detected from both free microspheres and microsphere-containing coatings. Finally cytotoxicity and stimulatory effects of daidzein or daidzein-loaded coatings, on both MC3T3-E1 and RAW264.7 cell lines, were studied to validate the potential of the developed coatings for orthopedic applications.