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1.
Allergy ; 74(3): 549-559, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-29987849

RESUMO

BACKGROUND: Chronic rhinosinusitis (CRS) with nasal polyps (CRSwNP) is a chronic inflammatory disease often accompanied by impairment of sense of smell. This symptom has been somewhat overlooked, and its relationship to inflammatory cytokines, tissue compression, neuronal loss, and neurogenesis is still unclear. METHODS: In order to elucidate potential mechanisms leading to CRS in humans, we have established a type 2/T helper type 2 cell (Th2)-mediated allergic CRS mouse model, based on house dust mite (HDM) and Staphylococcus aureus enterotoxin B (SEB) sensitization. The inflammatory status of the olfactory epithelium (OE) was assessed using histology, biochemistry, and transcriptomics. The sense of smell was evaluated by studying olfactory behavior and recording electro-olfactograms (EOGs). RESULTS: After 22 weeks, a typical type 2/Th2-mediated inflammatory profile was obtained, as demonstrated by increased interleukin (IL)-4, IL-5, and IL-13 in the OE. The number of mast cells and eosinophils was increased, and infiltration of these cells into the olfactory mucosa was also observed. In parallel, transcriptomic and histology analyses indicated a decreased number of immature olfactory neurons, possibly due to decreased renewal. However, the number of mature sensory neurons was not affected and neither the EOG nor olfactory behavior was impaired. CONCLUSION: Our mouse model of CRS displayed an allergic response to HDM + SEB administration, including the type 2/Th2 inflammatory profile characteristic of human eosinophilic CRSwNP. Although the sense of smell did not appear to be altered in these conditions, the data reveal the influence of chronic inflammation on olfactory neurogenesis, suggesting that factors unique to humans may be involved in CRSwNP-associated anosmia.


Assuntos
Neurogênese , Mucosa Olfatória/metabolismo , Rinite/etiologia , Rinite/metabolismo , Sinusite/etiologia , Sinusite/metabolismo , Células Th2/imunologia , Células Th2/metabolismo , Animais , Biomarcadores , Doença Crônica , Modelos Animais de Doenças , Camundongos , Neurogênese/genética , Neurogênese/imunologia , Mucosa Olfatória/fisiopatologia , Neurônios Receptores Olfatórios/metabolismo , Mucosa Respiratória/imunologia , Mucosa Respiratória/metabolismo , Mucosa Respiratória/patologia , Rinite/fisiopatologia , Sinusite/fisiopatologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo
2.
Stem Cells ; 33(4): 1277-91, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25523907

RESUMO

Obesity-associated inflammation contributes to the development of metabolic diseases. Although brite adipocytes have been shown to ameliorate metabolic parameters in rodents, their origin and differentiation remain to be characterized in humans. Native CD45-/CD34+/CD31- cells have been previously described as human adipocyte progenitors. Using two additional cell surface markers, MSCA1 (tissue nonspecific alkaline phosphatase) and CD271 (nerve growth factor receptor), we are able to partition the CD45-/CD34+/CD31- cell population into three subsets. We establish serum-free culture conditions without cell expansion to promote either white/brite adipogenesis using rosiglitazone, or bone morphogenetic protein 7 (BMP7), or specifically brite adipogenesis using 3-isobuthyl-1-methylxanthine. We demonstrate that adipogenesis leads to an increase of MSCA1 activity, expression of white/brite adipocyte-related genes, and mitochondriogenesis. Using pharmacological inhibition and gene silencing approaches, we show that MSCA1 activity is required for triglyceride accumulation and for the expression of white/brite-related genes in human cells. Moreover, native immunoselected MSCA1+ cells exhibit brite precursor characteristics and the highest adipogenic potential of the three progenitor subsets. Finally, we provided evidence that MSCA1+ white/brite precursors accumulate with obesity in subcutaneous adipose tissue (sAT), and that local BMP7 and inflammation regulate brite adipogenesis by modulating MSCA1 in human sAT. The accumulation of MSCA1+ white/brite precursors in sAT with obesity may reveal a blockade of their differentiation by immune cells, suggesting that local inflammation contributes to metabolic disorders through impairment of white/brite adipogenesis. Stem Cells 2015;33:1277-1291.


Assuntos
Adipócitos Brancos/imunologia , Adipócitos Brancos/metabolismo , Adipogenia/fisiologia , Antígenos de Superfície/biossíntese , Imunidade Celular/fisiologia , Adulto , Idoso , Células Cultivadas , Feminino , Humanos , Pessoa de Meia-Idade
3.
Aging Cell ; 22(3): e13776, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36617688

RESUMO

Senescence is a key event in the impairment of adipose tissue (AT) function with obesity and aging but the underlying molecular and cellular players remain to be fully defined, particularly with respect to the human AT progenitors. We have found distinct profiles of senescent progenitors based on AT location between stroma from visceral versus subcutaneous AT. In addition to flow cytometry, we characterized the location differences with transcriptomic and proteomic approaches, uncovering the genes and developmental pathways that are underlying replicative senescence. We identified key components to include INBHA as well as SFRP4 and GREM1, antagonists for the WNT and BMP pathways, in the senescence-associated secretory phenotype and NOTCH3 in the senescence-associated intrinsic phenotype. Notch activation in AT progenitors inhibits adipogenesis and promotes myofibrogenesis independently of TGFß. In addition, we demonstrate that NOTCH3 is enriched in the premyofibroblast progenitor subset, which preferentially accumulates in the visceral AT of patients with an early obesity trajectory. Herein, we reveal that NOTCH3 plays a role in the balance of progenitor fate determination preferring myofibrogenesis at the expense of adipogenesis. Progenitor NOTCH3 may constitute a tool to monitor replicative senescence and to limit AT dysfunction in obesity and aging.


Assuntos
Senescência Celular , Proteômica , Humanos , Senescência Celular/genética , Tecido Adiposo/metabolismo , Envelhecimento/metabolismo , Obesidade/metabolismo
4.
Cell Rep Med ; 4(12): 101333, 2023 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-38118407

RESUMO

Gain-of-function mutations in stimulator of interferon gene 1 (STING1) result in STING-associated vasculopathy with onset in infancy (SAVI), a severe autoinflammatory disease. Although elevated type I interferon (IFN) production is thought to be the leading cause of the symptoms observed in patients, STING can induce a set of pathways, which have roles in the onset and severity of SAVI and remain to be elucidated. To this end, we performed a multi-omics comparative analysis of peripheral blood mononuclear cells (PBMCs) and plasma from SAVI patients and healthy controls, combined with a dataset of healthy PBMCs treated with IFN-ß. Our data reveal a subset of disease-associated monocyte, expressing elevated CCL3, CCL4, and IL-6, as well as a strong integrated stress response, which we suggest is the result of direct PERK activation by STING. Cell-to-cell communication inference indicates that these monocytes lead to T cell early activation, resulting in their senescence and apoptosis. Last, we propose a transcriptomic signature of STING activation, independent of type I IFN response.


Assuntos
Interferon Tipo I , Doenças Vasculares , Humanos , Monócitos/metabolismo , Leucócitos Mononucleares/metabolismo , Doenças Vasculares/genética , Doenças Vasculares/metabolismo , Interferon Tipo I/metabolismo , RNA
5.
Curr Protoc Mol Biol ; 129(1): e109, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31763778

RESUMO

Over the past decade, transcriptomic studies using next-generation sequencing (NGS)-based RNA sequencing (RNA-Seq) have greatly contributed to characterizing biochemical and physiological changes in cells and tissues across organisms and experimental conditions. Critical steps in RNA-Seq include the preparation of the sequencing library from extracted RNA. Currently, a large panoply of RNA-Seq kits are commercially available. In these kits, conversion of RNA into a sequencing library involves multiple steps, which are labor-intensive, and cost per sample for library preparation may limit routine use of RNA-Seq. Here we describe a simple method for RNA-Seq library construction, referred to as RNA Fragmentation and Sequencing (RF-Seq). RF-Seq requires as little as 10 ng of total RNA and facilitates the sequencing of the 3' end of mRNAs. RF-Seq involves the fragmentation of total RNA followed by reverse transcription in presence of the oligo(dT) primer/template switch oligonucleotide and a sample barcoding/enrichment within a single PCR tube/well. The sample barcoding/enrichment step provides more flexibility for individual sample handling. The use of just twenty orthogonal Illumina TruSeq HT barcoding primers facilitates the preparation of 96 uniquely labeled RF-Seq libraries in a single 96-well PCR plate. Twelve RF-Seq libraries can be prepared within 4 hr, with an approximate cost of $10/sample. We provide an example of using RF-Seq to measure gene expression upon activation of an innate immune pathway using STING activator in human blood cells, highlighting the potential usefulness of the proposed method in routine transcriptomic applications such as high-throughput drug screening and/or preclinical toxicity assays. © 2019 by John Wiley & Sons, Inc. Basic Protocol: RNA fragmentation and sequencing (RF-Seq): Cost-effective, time-efficient, and high-throughput 3' mRNA sequencing library construction in a single tube.


Assuntos
Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Reação em Cadeia da Polimerase/métodos , RNA Mensageiro/genética , Análise de Sequência de RNA/métodos , Humanos , Proteínas de Membrana/sangue , Proteínas de Membrana/genética , Transcriptoma
6.
Brain Res ; 953(1-2): 63-72, 2002 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-12384239

RESUMO

Three subtypes of neurotensin receptor have been described, two members of the heptahelical transmembrane domain G protein-coupled receptor superfamily NT-1R and NT-2R, and NT-3R unrelated to this family. We have generated NT-1R deficient (NT-1R(-/-)) mice. NT-1R(-/-) mice were born at the expected Mendelian frequency without obvious abnormalities and they were fertile. The NT-induced analgesia on the writhing induced by phenyl-p-benzoquinone administration remained at wild-type levels in the NT-1R(-/-) mice demonstrating that the NT-1R is not implicated in the analgesic effect of NT in this test. The NT-1R(-/-) mice were hyperthermic; their body temperature was not affected by intracerebroventricular (i.c.v.) administration of NT, contrasting with the hypothermia induced in NT-1R(+/+) mice. NT-1R(-/-) mice showed a small significant increase in body weight compared to the NT-1R(+/+) congeners as early as 10 weeks after birth, correlated with a higher food intake. NT-1R(-/-) mice showed similar spontaneous locomotion to the control littermates, but did not respond to i.c.v. NT-induced hypolocomotion. I.c.v. injection of NT inhibited feeding in fasted wild-type mice, but had no effect on feeding of the NT-1R(-/-) mice. I.c.v. administration of the orexigenic neuropeptide Y (NPY) stimulated feeding to the same extent in both wild-type and NT-1R(-/-) mice. This analysis of NT-1R-deficient mice shows that the NT-1R does not play a role in NT-induced analgesia, but that it is clearly implicated in thermal and feeding regulation, weight control, and NT-induced hypolocomotion.


Assuntos
Regulação da Temperatura Corporal/fisiologia , Comportamento Alimentar/fisiologia , Receptores de Neurotensina/genética , Receptores de Neurotensina/metabolismo , Analgésicos/farmacologia , Animais , Feminino , Deleção de Genes , Hipotermia/induzido quimicamente , Hipotermia/fisiopatologia , Injeções Intraventriculares , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atividade Motora/fisiologia , Neuropeptídeo Y/farmacologia , Neurotensina/farmacologia , Dor/fisiopatologia
7.
Proc Natl Acad Sci U S A ; 99(12): 8384-8, 2002 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-12060781

RESUMO

Do endocannabinoids (eCBs) participate in long-term synaptic plasticity in the brain? Using pharmacological approaches and genetically altered mice, we show that stimulation of prelimbic cortex afferents at naturally occurring frequencies causes a long-term depression of nucleus accumbens glutamatergic synapses mediated by eCB release and presynaptic CB1 receptors. Translation of glutamate synaptic transmission into eCB retrograde signaling involved metabotropic glutamate receptors and postsynaptic intracellular Ca(2+) stores. These findings unveil the role of the eCB system in activity-dependent long-term synaptic plasticity and identify a mechanism by which marijuana can alter synaptic functions in the endogenous brain reward system.


Assuntos
Canabinoides/farmacologia , Potenciais Pós-Sinápticos Excitadores/fisiologia , Núcleo Accumbens/fisiologia , Receptores de Droga/fisiologia , Animais , Moduladores de Receptores de Canabinoides , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Ácido Glutâmico/fisiologia , Técnicas In Vitro , Camundongos , Camundongos Knockout , Núcleo Accumbens/efeitos dos fármacos , Técnicas de Patch-Clamp , Reação em Cadeia da Polimerase , Receptores de Canabinoides , Receptores de Droga/deficiência , Receptores de Droga/genética
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