Suchergebnisse

Lipopolysaccharides (LPS), the major components of the wall of gram-negative bacteria, trigger powerful defensive responses in the airways via mechanisms thought to rely solely on the Toll-like receptor 4 (TLR4) immune pathway. Here we show that airway epithelial cells display an increase in intracellular Ca2+ concentration within seconds of LPS application. This response occurs in a TLR4-independent manner, via activation of the transient receptor potential vanilloid 4 cation channel (TRPV4). We found that TRPV4 mediates immediate LPS-induced increases in ciliary beat frequency and the production of bactericidal nitric oxide. Upon LPS challenge TRPV4-deficient mice display exacerbated ventilatory changes and recruitment of polymorphonuclear leukocytes into the airways. We conclude that LPS-induced activation of TRPV4 triggers signaling mechanisms that operate faster and independently from the canonical TLR4 immune pathway, leading to immediate protective responses such as direct antimicrobial action, increase in airway clearance, and the regulation of the inflammatory innate immune reaction. ; B.B. was funded by a Ph.D. grant of the Agency for Innovation by Science and Technology (IWT). Research was supported by grants from the Belgian Federal Government (IUAP P7/13), the FWO (G.0702.12, 1.5.068.16 N) and the Research Council of the KU Leuven (Grants GOA/14/011 and PF-TRPLe), the Spanish Ministry of Economy and Competitiveness (SAF2015-69762-R and María de Maeztu Programme for Units of Excellence in R&D MDM-2014-0370), Fondo de Investigación Sanitaria (RD12/0042/0014), and the FEDER Funds

Allergic Contact Dermatitis (ACD) resulting from skin sensitization is a frequent inflammatory skin disease linked to the use of chemicals, called haptens. At this time, the sensitizing potential of a new chemical is evaluated on animal models. However, new European legislation requires alternative methods for skin sensitization. In this context, a better knowledge of ACD and the capacity to reproduce in vitro its mechanisms lead to the development of new alternative methods. The aim of this study performed with human dendritic cells (DCs) and the human cell line U937 was to determine the early events involved in dendritic cell activation induced by contact sensitizers and especially by the mercury compound thimerosal. Data show that oxidative stress induced by sensitizers is an early signaling event leading to DC activation (expression of CD86 and IL-8 release) and to apoptosis. Using antioxidants, our data show that oxidative stress, characterized by ROS production in correlation with the depletion of the mitochondrial membrane potential and of intracellular glutathione, is a key player in signal transduction induced by sensitizers, especially in the response of DCs towards thimerosal and DNCB. More specifically, these studies demonstrate that thiol groups play a direct role in the initiating events leading to DC activation. In addition, calcium influx was detected in DCs exposed to sensitizers, in correlation with oxidative stress. These data highlight the great interest in the development of a hapten-protein binding assay based on thiol groups and the necessity to better understand the redox status of chemicals as well as cell metabolism for predicting skin sensitization. ; L'eczéma allergique de contact (EAC) est une pathologie inflammatoire cutanée de plus en plus fréquente. Il s'agit d'une sensibilisation vis-à-vis de substances chimiques, appelées haptènes, qui sont en contact répété avec la peau. A l'heure actuelle, le pouvoir sensibilisant d'une molécule est évalué principalement grâce à des modèles ...

Regulation of lipid metabolism is essential for treatment and prevention of several chronic diseases such as obesity, diabetes, and cardiovascular diseases, which are responsible for most deaths worldwide. It has been demonstrated that the AMP-activated protein kinase (AMPK) has a direct impact on lipid metabolism by modulating several downstream-signaling components. The main objective of the present work was to evaluate the in vitro effect of a methanolic strawberry extract on AMPK and its possible repercussion on lipid metabolism in human hepatocellular carcinoma cells (HepG2). For such purpose, the lipid profile and the expression of proteins metabolically related to AMPK were determined on cells lysates. The results demonstrated that strawberry methanolic extract decreased total cholesterol, low-density lipoprotein (LDL)-cholesterol, and triglycerides levels (up to 0.50-, 0.30-, and 0.40-fold, respectively) while it stimulated the p-AMPK/AMPK expression (up to 3.06-fold), compared to the control. AMPK stimulation led to the phosphorylation and consequent inactivation of acetyl coenzyme A carboxylase (ACC) and inhibition of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), the major regulators of fatty acids and cholesterol synthesis, respectively. Strawberry treatment also entailed a 4.34-, 2.37-, and 2.47-fold overexpression of LDL receptor, sirtuin 1 (Sirt1), and the peroxisome proliferator activated receptor gamma coactivator 1-alpha (PGC-1α), respectively, compared to control. The observed results were counteracted by treatment with compound C, an AMPK pharmacological inhibitor, confirming that multiple effects of strawberries on lipid metabolism are mediated by the activation of this protein. ; European Union (EU) 679303

BACKGROUND: Endogenous retroviruses (ERVs), which are responsible for 10% of spontaneous mouse mutations, are kept under control via several epigenetic mechanisms. The H3K9 histone methyltransferase SETDB1 is essential for ERV repression in embryonic stem cells (ESCs), with DNA methylation also playing an important role. It has been suggested that SETDB1 protects ERVs from TET-dependent DNA demethylation, but the relevance of this mechanism for ERV expression remains unclear. Moreover, previous studies have been performed in primed ESCs, which are not epigenetically or transcriptionally representative of preimplantation embryos. RESULTS: We use naïve ESCs to investigate the role of SETDB1 in ERV regulation and its relationship with TET-mediated DNA demethylation. Naïve ESCs show an increased dependency on SETDB1 for ERV silencing when compared to primed ESCs, including at the highly mutagenic intracisternal A particles (IAPs). We find that in the absence of SETDB1, TET2 activates IAP elements in a catalytic-dependent manner. Surprisingly, TET2 does not drive changes in DNA methylation levels at IAPs, suggesting that it regulates these retrotransposons indirectly. Instead, SETDB1 depletion leads to a TET2-dependent loss of H4R3me2s, which is indispensable for IAP silencing during epigenetic reprogramming. CONCLUSIONS: Our results demonstrate a novel and unexpected role for SETDB1 in protecting IAPs from TET2-dependent histone arginine demethylation. ; M.R.B. is a Sir Henry Dale Fellow (101225/Z/13/Z), jointly funded by the Wellcome Trust and the Royal Society. Ö.D. and L.R. have received funding from the People Programme (Marie Curie Actions) of the European Union's Seventh Framework Programme (FP7/2007-2013) under REA grant agreement number 608765.

Allergic Contact Dermatitis (ACD) resulting from skin sensitization is a frequent inflammatory skin disease linked to the use of chemicals, called haptens. At this time, the sensitizing potential of a new chemical is evaluated on animal models. However, new European legislation requires alternative methods for skin sensitization. In this context, a better knowledge of ACD and the capacity to reproduce in vitro its mechanisms lead to the development of new alternative methods. The aim of this study performed with human dendritic cells (DCs) and the human cell line U937 was to determine the early events involved in dendritic cell activation induced by contact sensitizers and especially by the mercury compound thimerosal. Data show that oxidative stress induced by sensitizers is an early signaling event leading to DC activation (expression of CD86 and IL-8 release) and to apoptosis. Using antioxidants, our data show that oxidative stress, characterized by ROS production in correlation with the depletion of the mitochondrial membrane potential and of intracellular glutathione, is a key player in signal transduction induced by sensitizers, especially in the response of DCs towards thimerosal and DNCB. More specifically, these studies demonstrate that thiol groups play a direct role in the initiating events leading to DC activation. In addition, calcium influx was detected in DCs exposed to sensitizers, in correlation with oxidative stress. These data highlight the great interest in the development of a hapten-protein binding assay based on thiol groups and the necessity to better understand the redox status of chemicals as well as cell metabolism for predicting skin sensitization. ; L'eczéma allergique de contact (EAC) est une pathologie inflammatoire cutanée de plus en plus fréquente. Il s'agit d'une sensibilisation vis-à-vis de substances chimiques, appelées haptènes, qui sont en contact répété avec la peau. A l'heure actuelle, le pouvoir sensibilisant d'une molécule est évalué principalement grâce à des modèles animaux. Cependant, dans un contexte européen exigeant le développement de méthodes alternatives, la compréhension et la reproduction in vitro des mécanismes de l'EAC permettent le développement de nouvelles stratégies. Le but de ce travail, réalisé sur des cellules dendritiques (DCs) et la lignée humaine U937, est de mettre en évidence les événements précoces de la signalisation intracellulaire à l'origine de l'activation des DCs induite par des allergènes, et notamment par le composé mercurique thimerosal. Les résultats obtenus démontrent que l'induction d'un stress oxydant par les allergènes est un mécanisme induit précocement. L'utilisation d'antioxydants montre que ce mécanisme participe de manière directe à l'initiation du processus d'activation des DCs (expression du CD86 et sécrétion d'IL-8) et de l'apoptose. Le stress oxydant, caractérisé par une production d'ERO associée à une chute du potentiel membranaire mitochondrial et une déplétion en glutathion intracellulaire, est un acteur majeur de la signalisation induite par les allergènes et notamment dans la réponse induite par le thimerosal et le DNCB. Plus particulièrement, ce travail démontre le rôle des groupements thiols dans l'initiation de la transduction du signal aboutissant à l'activation des DCs. Par ailleurs, une signalisation calcique, dépendante du stress oxydant, a également été mise en évidence en réponse aux allergènes. Ces résultats mettent en valeur l'importance de l'étude de la réactivité des allergènes vis-à-vis des groupements thiols et de la nécessité de tenir compte du potentiel oxydant (rédox) des haptènes ainsi que du métabolisme cellulaire dans la mise en place de modèles prédictifs in vitro.

Allergic Contact Dermatitis (ACD) resulting from skin sensitization is a frequent inflammatory skin disease linked to the use of chemicals, called haptens. At this time, the sensitizing potential of a new chemical is evaluated on animal models. However, new European legislation requires alternative methods for skin sensitization. In this context, a better knowledge of ACD and the capacity to reproduce in vitro its mechanisms lead to the development of new alternative methods. The aim of this study performed with human dendritic cells (DCs) and the human cell line U937 was to determine the early events involved in dendritic cell activation induced by contact sensitizers and especially by the mercury compound thimerosal. Data show that oxidative stress induced by sensitizers is an early signaling event leading to DC activation (expression of CD86 and IL-8 release) and to apoptosis. Using antioxidants, our data show that oxidative stress, characterized by ROS production in correlation with the depletion of the mitochondrial membrane potential and of intracellular glutathione, is a key player in signal transduction induced by sensitizers, especially in the response of DCs towards thimerosal and DNCB. More specifically, these studies demonstrate that thiol groups play a direct role in the initiating events leading to DC activation. In addition, calcium influx was detected in DCs exposed to sensitizers, in correlation with oxidative stress. These data highlight the great interest in the development of a hapten-protein binding assay based on thiol groups and the necessity to better understand the redox status of chemicals as well as cell metabolism for predicting skin sensitization. ; L'eczéma allergique de contact (EAC) est une pathologie inflammatoire cutanée de plus en plus fréquente. Il s'agit d'une sensibilisation vis-à-vis de substances chimiques, appelées haptènes, qui sont en contact répété avec la peau. A l'heure actuelle, le pouvoir sensibilisant d'une molécule est évalué principalement grâce à des modèles animaux. Cependant, dans un contexte européen exigeant le développement de méthodes alternatives, la compréhension et la reproduction in vitro des mécanismes de l'EAC permettent le développement de nouvelles stratégies. Le but de ce travail, réalisé sur des cellules dendritiques (DCs) et la lignée humaine U937, est de mettre en évidence les événements précoces de la signalisation intracellulaire à l'origine de l'activation des DCs induite par des allergènes, et notamment par le composé mercurique thimerosal. Les résultats obtenus démontrent que l'induction d'un stress oxydant par les allergènes est un mécanisme induit précocement. L'utilisation d'antioxydants montre que ce mécanisme participe de manière directe à l'initiation du processus d'activation des DCs (expression du CD86 et sécrétion d'IL-8) et de l'apoptose. Le stress oxydant, caractérisé par une production d'ERO associée à une chute du potentiel membranaire mitochondrial et une déplétion en glutathion intracellulaire, est un acteur majeur de la signalisation induite par les allergènes et notamment dans la réponse induite par le thimerosal et le DNCB. Plus particulièrement, ce travail démontre le rôle des groupements thiols dans l'initiation de la transduction du signal aboutissant à l'activation des DCs. Par ailleurs, une signalisation calcique, dépendante du stress oxydant, a également été mise en évidence en réponse aux allergènes. Ces résultats mettent en valeur l'importance de l'étude de la réactivité des allergènes vis-à-vis des groupements thiols et de la nécessité de tenir compte du potentiel oxydant (rédox) des haptènes ainsi que du métabolisme cellulaire dans la mise en place de modèles prédictifs in vitro.

Zhongyang Liu,1,* Liangliang Huang,1,* Liang Liu,1,* Beier Luo,2,* Miaomiao Liang,3 Zhen Sun,1 Shu Zhu,1 Xin Quan,1 Yafeng Yang,1 Teng Ma,1 Jinghui Huang,1 Zhuojing Luo1 1Department of Orthopaedics, Xijing Hospital, Fourth Military Medical University, Xi'an, 2Department of Orthopaedics, Changhai Hospital, Second Military Medical University, Shanghai, 3Department of Neurosurgery, Xijing Hospital, Fourth Military Medical University, Xi'an, People's Republic of China *These authors contributed equally to this work Abstract: Schwann cells (SCs) are attractive seed cells in neural tissue engineering, but their application is limited by attenuated biological activities and impaired functions with aging. Therefore, it is important to explore an approach to enhance the viability and biological properties of SCs. In the present study, a magnetic composite made of magnetically responsive magnetic nanoparticles (MNPs) and a biodegradable chitosan–glycerophosphate polymer were prepared and characterized. It was further explored whether such magnetic nanocomposites via applied magnetic fields would regulate SC biological activities. The magnetization of the magnetic nanocomposite was measured by a vibrating sample magnetometer. The compositional characterization of the magnetic nanocomposite was examined by Fourier-transform infrared and X-ray diffraction. The tolerance of SCs to the magnetic fields was tested by flow-cytometry assay. The proliferation of cells was examined by a 5-ethynyl-2-deoxyuridine-labeling assay, a PrestoBlue assay, and a Live/Dead assay. Messenger ribonucleic acid of BDNF, GDNF, NT-3, and VEGF in SCs was assayed by quantitative real-time polymerase chain reaction. The amount of BDNF, GDNF, NT-3, and VEGF secreted from SCs was determined by enzyme-linked immunosorbent assay. It was found that magnetic nanocomposites containing 10% MNPs showed a cross-section diameter of 32.33±1.81 µm, porosity of 80.41%±0.72%, and magnetization of 5.691 emu/g at 8 kOe. The 10% MNP magnetic nanocomposites were able to support cell adhesion and spreading and further promote proliferation of SCs under magnetic field exposure. Interestingly, a magnetic field applied through the 10% MNP magnetic scaffold significantly increased the gene expression and protein secretion of BDNF, GDNF, NT-3, and VEGF. This work is the first stage in our understanding of how to precisely regulate the viability and biological properties of SCs in tissue-engineering grafts, which combined with additional molecular factors may lead to the development of new nerve grafts. Keywords: Schwann cell, magnetic field, nanocomposite, cell proliferation

Members of the tumour necrosis factor (TNF) superfamily OX40L and CD70 and their receptors are costimulating signalling axes critical for adequate T cell activation in humans and mice but characterisation of these molecules in other species including ruminants is lacking. Here we cloned and expressed the predicted ovine orthologues of the receptors OX40 and CD27, as well as soluble recombinant forms of their potential ovine ligands, OaOX40L and OaCD70. Using biochemical and immunofluorescence analyses, we show that both signalling axes are functional in sheep. We show that oligomeric recombinant ligand constructs are able to induce signalling through their receptors on transfected cells. Recombinant defective human adenoviruses were constructed to express the soluble forms of OaOX40L and OaCD70. Both proteins were detected in the supernatant of adenovirus-infected cells and shown to activate NF-B signalling pathway through their cognate receptor. These adenovirus-secreted OaOX40L and OaCD70 forms could also activate ovine T cell proliferation and enhance IFN- production in CD4+ and CD8+ T cells. Altogether, this study provides the first characterisation of the ovine costimulatory OX40L-OX40 and CD70-CD27 signalling axes, and indicates that their activation in vivo may be useful to enhance vaccination-induced immune responses in sheep and other ruminants. ; Ministerio de Economía y Competitividad. This work was funded by grants AGL2015-64290R, RTI2018-094616-B-I00, ADEDONET-Redes de Excelencia from the Spanish Ministerio de Economía y Competitividad; grant S2013/ABI-2906-PLATESA and S2018/BAA-4370-PLATESA2 from the Comunidad de Madrid and VetBionet INFRAIA-731014 from the European Union

Abstract
Androgen deficiency is a common medical conditions that affects males of all ages. Transplantation of testosterone-producing cells is a promising treatment for male hypogonadism. However, getting a cell source with the characteristics of Leydig cells (LCs) is still a challenge. Here, a high-efficiency reprogramming of skin-derived fibroblasts into functional Leydig-like cells (LLCs) based on epigenetic mechanism was described. By performing an integrated analysis of genome-wide DNA methylation and transcriptome profiling in LCs and fibroblasts, the potentially epigenetic-regulating steroidogenic genes and signaling pathways were identified. Then by using CRISPR/dCas9 activation system and signaling pathway regulators, the male- or female-derived fibroblasts were reprogrammed into LLCs with main LC-specific traits. Transcriptomic analysis further indicated that the correlation coefficients of global genes and transcription factors between LLCs and LCs were higher than 0.81 and 0.96, respectively. After transplantation in the testes of hypogonadal rodent models, LLCs increased serum testosterone concentration significantly. In type 2 diabetic rats model, LLCs which were transplanted in armpit, have the capability to restore the serum testosterone level and improve the hyperglycemia status. In conclusion, our approach enables skin-derived fibroblasts reprogramming into LLCs with high fidelity, providing a potential cell source for the therapeutics of male hypogonadism and metabolic-related comorbidities.