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1.
EMBO J ; 43(15): 3141-3174, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38877304

RESUMO

Migrating cells preferentially breach and integrate epithelial and endothelial monolayers at multicellular vertices. These sites are amenable to forces produced by the migrating cell and subsequent opening of the junctions. However, the cues that guide migrating cells to these entry portals, and eventually drive the transmigration process, are poorly understood. Here, we show that lymphatic endothelium multicellular junctions are the preferred sites of dendritic cell transmigration in both primary cell co-cultures and in mouse dermal explants. Dendritic cell guidance to multicellular junctions was dependent on the dendritic cell receptor CCR7, whose ligand, lymphatic endothelial chemokine CCL21, was exocytosed at multicellular junctions. Characterization of lymphatic endothelial secretory routes indicated Golgi-derived RAB6+ vesicles and RAB3+/27+ dense core secretory granules as intracellular CCL21 storage vesicles. Of these, RAB6+ vesicles trafficked CCL21 to the multicellular junctions, which were enriched with RAB6 docking factor ELKS (ERC1). Importantly, inhibition of RAB6 vesicle exocytosis attenuated dendritic cell transmigration. These data exemplify how spatially-restricted exocytosis of guidance cues helps to determine where dendritic cells transmigrate.


Assuntos
Quimiocina CCL21 , Células Dendríticas , Exocitose , Receptores CCR7 , Proteínas rab de Ligação ao GTP , Animais , Camundongos , Quimiocina CCL21/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab de Ligação ao GTP/genética , Células Dendríticas/metabolismo , Receptores CCR7/metabolismo , Receptores CCR7/genética , Junções Intercelulares/metabolismo , Migração Transendotelial e Transepitelial , Endotélio Linfático/metabolismo , Endotélio Linfático/citologia , Células Endoteliais/metabolismo , Camundongos Endogâmicos C57BL , Humanos , Técnicas de Cocultura , Células Cultivadas , Movimento Celular
2.
Eur J Immunol ; 44(2): 480-8, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24307058

RESUMO

Although mesenchymal stromal cells (MSCs) possess the capacity to modulate immune responses, little is known about the mechanisms that underpin these processes. In this study, we show that immunosupression is mediated by activation of nuclear factor kappa B (NF-κB) in human MSCs. This pathway is activated by TNF-α that is generated following TCR stimulation of T cells. Inhibition of NF-κB through silencing of IκB kinase ß or the TNF-α receptor abolishes the immunosuppressive capacity of MSCs. Our data also indicate that MSC-associated NF-κB activation primarily leads to inhibition of T-cell proliferation with little effect on expression of the activation markers CD69 and CD25. Thus, our data support the hypothesis that the TNF-α/NF-κB signalling pathway is required for the initial priming of immunosuppressive function in human MSCs. Interestingly, drugs that interfere with NF-κB activation significantly antagonise the immunoregulatory effect of MSCs, which could have important implications for immunosuppression regimens in the clinic.


Assuntos
Ativação Linfocitária/imunologia , Células-Tronco Mesenquimais/imunologia , Células-Tronco Mesenquimais/metabolismo , NF-kappa B/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Antígenos CD/imunologia , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/imunologia , Antígenos de Diferenciação de Linfócitos T/metabolismo , Proliferação de Células , Células Cultivadas , Humanos , Quinase I-kappa B/imunologia , Quinase I-kappa B/metabolismo , Subunidade alfa de Receptor de Interleucina-2/imunologia , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Lectinas Tipo C/imunologia , Lectinas Tipo C/metabolismo , NF-kappa B/imunologia , Receptores do Fator de Necrose Tumoral/imunologia , Receptores do Fator de Necrose Tumoral/metabolismo , Transdução de Sinais/imunologia , Fator de Necrose Tumoral alfa/imunologia
3.
Nat Commun ; 14(1): 5878, 2023 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-37735168

RESUMO

Branching morphogenesis is a ubiquitous process that gives rise to high exchange surfaces in the vasculature and epithelial organs. Lymphatic capillaries form branched networks, which play a key role in the circulation of tissue fluid and immune cells. Although mouse models and correlative patient data indicate that the lymphatic capillary density directly correlates with functional output, i.e., tissue fluid drainage and trafficking efficiency of dendritic cells, the mechanisms ensuring efficient tissue coverage remain poorly understood. Here, we use the mouse ear pinna lymphatic vessel network as a model system and combine lineage-tracing, genetic perturbations, whole-organ reconstructions and theoretical modeling to show that the dermal lymphatic capillaries tile space in an optimal, space-filling manner. This coverage is achieved by two complementary mechanisms: initial tissue invasion provides a non-optimal global scaffold via self-organized branching morphogenesis, while VEGF-C dependent side-branching from existing capillaries rapidly optimizes local coverage by directionally targeting low-density regions. With these two ingredients, we show that a minimal biophysical model can reproduce quantitatively whole-network reconstructions, across development and perturbations. Our results show that lymphatic capillary networks can exploit local self-organizing mechanisms to achieve tissue-scale optimization.


Assuntos
Pavilhão Auricular , Vasos Linfáticos , Animais , Camundongos , Humanos , Biofísica , Modelos Animais de Doenças , Líquido Extracelular
4.
Carbohydr Polym ; 277: 118771, 2022 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-34893216

RESUMO

The enzymatic hydrolysis of barley beta-glucan, konjac glucomannan and carboxymethyl cellulose by a ß-1,4-D-endoglucanase MeCel45A from blue mussel, Mytilus edulis, which belongs to subfamily B of glycoside hydrolase family 45 (GH45), was compared with GH45 members of subfamilies A (Humicola insolens HiCel45A), B (Trichoderma reesei TrCel45A) and C (Phanerochaete chrysosporium PcCel45A). Furthermore, the crystal structure of MeCel45A is reported. Initial rates and hydrolysis yields were determined by reducing sugar assays and product formation was characterized using NMR spectroscopy. The subfamily B and C enzymes exhibited mannanase activity, whereas the subfamily A member was uniquely able to produce monomeric glucose. All enzymes were confirmed to be inverting glycoside hydrolases. MeCel45A appears to be cold adapted by evolution, as it maintained 70% activity on cellohexaose at 4 °C relative to 30 °C, compared to 35% for TrCel45A. Both enzymes produced cellobiose and cellotetraose from cellohexaose, but TrCel45A additionally produced cellotriose.


Assuntos
Glicosídeo Hidrolases/metabolismo , Mananas/metabolismo , Mytilus edulis/enzimologia , beta-Glucanas/metabolismo , Animais , Gênero de Fungos Humicola/enzimologia , Glicosídeo Hidrolases/química , Hypocreales/enzimologia , Isoenzimas/química , Isoenzimas/metabolismo , Phanerochaete/enzimologia
5.
Acta Crystallogr D Biol Crystallogr ; 65(Pt 8): 796-803, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19622863

RESUMO

The Modoc virus (MODV) is a flavivirus with no known vector (NKV). Evolutionary studies have shown that the viruses in the MODV group have evolved in association with mammals (bats, rodents) without transmission by an arthropod vector. MODV methyltransferase is the first enzyme from this evolutionary branch to be structurally characterized. The high-resolution structure of the methyltransferase domain of the MODV NS5 protein (MTase(MODV)) was determined. The protein structure was solved in the apo form and in complex with its cofactor S-adenosyl-L-methionine (SAM). Although it belongs to a separate evolutionary branch, MTase(MODV) shares structural characteristics with flaviviral MTases from the other branches. Its capping machinery is a relatively new target in flaviviral drug development and the observed structural conservation between the three flaviviral branches indicates that it may be possible to identify a drug that targets a range of flaviviruses. The structural conservation also supports the choice of MODV as a possible model for flavivirus studies.


Assuntos
Infecções por Flavivirus/enzimologia , Flavivirus/enzimologia , Metiltransferases/química , Proteínas não Estruturais Virais/química , Animais , Vetores Artrópodes , Quirópteros , Cristalização , Cristalografia por Raios X , Evolução Molecular , Infecções por Flavivirus/tratamento farmacológico , Infecções por Flavivirus/genética , Infecções por Flavivirus/transmissão , Metiltransferases/genética , Metiltransferases/metabolismo , Camundongos , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína/genética , Análogos de Capuz de RNA/uso terapêutico , Capuzes de RNA/metabolismo , Ratos , S-Adenosilmetionina/metabolismo , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo
6.
PLoS One ; 12(1): e0169362, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28076364

RESUMO

γδ T cells play a role in a wide range of diseases such as autoimmunity and cancer. The majority of circulating human γδ T lymphocytes express a Vγ9Vδ2+ (Vδ2+) T cell receptor (TCR) and following activation release pro-inflammatory cytokines. In this study, we show that IFNγ, produced by Vδ2+ cells, activates mesenchymal stem cell (MSC)-mediated immunosupression, which in turn exerts a negative feedback mechanism on γδ T cell function ranging from cytokine production to proliferation. Importantly, this modulatory effect is limited to a short period of time (<24 hours) post-T cell activation, after which MSCs can no longer exert their immunoregulatory capacity. Using genetically modified MSCs with the IFNγ receptor 1 constitutively silenced, we demonstrate that IFNγ is essential to this process. Activated γδ T cells induce expression of several factors by MSCs that participate in the depletion of amino acids. In particular, we show that indolamine 2,3-dioxygenase (IDO), an enzyme involved in L-tryptophan degradation, is responsible for MSC-mediated immunosuppression of Vδ2+ T cells. Thus, our data demonstrate that γδ T cell responses can be immuno-modulated by different signals derived from MSC.


Assuntos
Interferon gama/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Células-Tronco Mesenquimais/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/fisiologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Retroalimentação Fisiológica/efeitos dos fármacos , Humanos , Tolerância Imunológica/efeitos dos fármacos , Células-Tronco Mesenquimais/fisiologia , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo
7.
Biochim Biophys Acta ; 1649(1): 40-50, 2003 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-12818189

RESUMO

We describe the 1.6 A crystal structure of the fatty-acid-binding protein EgFABP1 from the parasitic platyhelminth Echinococcus granulosus. E. granulosus causes hydatid disease, which is a major zoonosis. EgFABP1 has been implicated in the acquisition, storage, and transport of lipids, and may be important to the organism since it is incapable of synthesising most of its lipids de novo. Moreover, EgFABP1 is a promising candidate for a vaccine against hydatid disease. The crystal structure reveals that EgFABP1 has the expected 10-stranded beta-barrel fold typical of the family of intracellular lipid-binding proteins, and that it is structurally most similar to P2 myelin protein. We describe the comparison of the crystal structure of EgFABP1 with these proteins and with an older homology model for EgFABP1. The electron density reveals the presence of a bound ligand inside the cavity, which we have interpreted as palmitic acid. The carboxylate group of the fatty acid interacts with the protein's P2 motif, consisting of a conserved triad R em leader R-x-Y. The hydrophobic tail of the ligand assumes a fairly flat, U-shaped conformation and has relatively few interactions with the protein.We discuss some of the structural implications of the crystal structure of EgFABP1 for related platyhelminthic FABPs.


Assuntos
Proteínas de Transporte/química , Echinococcus/química , Proteínas de Helminto/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Proteínas de Transporte/metabolismo , Cristalografia por Raios X , Echinococcus/metabolismo , Elétrons , Proteínas de Ligação a Ácido Graxo , Proteínas de Helminto/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Metionina/metabolismo , Metilação , Modelos Moleculares , Dados de Sequência Molecular , Ácido Palmítico/metabolismo , Conformação Proteica , Homologia de Sequência de Aminoácidos , Serina/química , Homologia Estrutural de Proteína
8.
Artigo em Inglês | MEDLINE | ID: mdl-16511016

RESUMO

Human semicarbazide-sensitive amine oxidase (SSAO) is a homodimeric copper-containing monoamine oxidase that occurs in both a membrane-bound and a soluble form. SSAO is also known as vascular adhesion protein-1 (VAP-1). A truncated soluble form of human SSAO (comprising residues 29-763) was expressed in human embryonic kidney 293 cells and purified to homogeneity. Tetragonal crystals were obtained and a data set extending to 2.5 A was collected. The crystals are merohedrally twinned and the estimation of the twinning fraction was complicated by pseudo-symmetry and the anisotropic character of the crystals. Using a recently developed method for twinning detection that is insensitive to phenomena such as anisotropy or pseudo-symmetry [Padilla & Yeates (2003), Acta Cryst. D59, 1124-1130], the twinning fraction was estimated to be 0.3. The structure was eventually solved by molecular replacement in space group P4(3).


Assuntos
Amina Oxidase (contendo Cobre)/química , Amina Oxidase (contendo Cobre)/genética , Amina Oxidase (contendo Cobre)/isolamento & purificação , Animais , Linhagem Celular , Cristalização , Dimerização , Vetores Genéticos , Humanos , Rim/embriologia , Mamíferos , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Deleção de Sequência , Transfecção , Difração de Raios X
9.
Bone Marrow Res ; 2013: 203643, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24187625

RESUMO

Mesenchymal stromal cells (MSCs) are multipotent cells found in connective tissues that can differentiate into bone, cartilage, and adipose tissue. Interestingly, they can regulate immune responses in a paracrine way and allogeneic MSCs do not elicit immune response. These properties have encouraged a number of clinical trials in a broad range of regenerative therapies. Although these trials were first focused on their differentiation properties, in the last years, the immunosuppressive features have gained most of the attention. In this review, we will summarize the up-to-date knowledge about the immunosuppressive mechanisms of MSCs in vivo and in vitro and the most promising approaches in clinical investigation.

10.
Stem Cells Dev ; 21(14): 2581-91, 2012 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-22455388

RESUMO

Epigenetic changes are regarded as emerging major players for hematopoietic stem cell (HSC) biology. Although some histone deacetylase (HDAC) inhibitors, such as valproic acid (VA), induce differentiation and apoptosis in a variety of leukemic cells in vitro, they produce a favorable effect on the expansion of normal HSCs. In this study, we have identified the VA target HDAC3 as a negative regulator of umbilical cord blood HSC expansion. We demonstrate that knockdown of the transcript dramatically improves CD34+ cell expansion, which correlates with a higher potential to generate colony-forming units in functional assays. We show that this effect is mediated at the level of primitive hematopoietic cells and that it is not due to negative effects on specific cell commitment or alterations in the cell cycle. HDAC3 inhibition does not block commitment to the monocytic lineage and the maturation of monocyte precursors, which are the main inhibited pathways in the presence of VA. Therefore, our results identify HDAC3 as a promising target for therapies aiming to expand HSCs.


Assuntos
Proliferação de Células , Células-Tronco Hematopoéticas/enzimologia , Histona Desacetilases/metabolismo , Antígenos CD34/metabolismo , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem da Célula , Proliferação de Células/efeitos dos fármacos , Sangue Fetal/citologia , Citometria de Fluxo , Técnicas de Silenciamento de Genes , Células HEK293 , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/genética , Humanos , Lentivirus/genética , Monócitos/citologia , Monócitos/efeitos dos fármacos , Monócitos/enzimologia , RNA Interferente Pequeno/genética , Ácido Valproico/farmacologia
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