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1.
Elife ; 82019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31535973

RESUMO

In most vertebrates, the upper digestive tract is composed of muscularized jaws linked to the esophagus that permits food ingestion and swallowing. Masticatory and esophagus striated muscles (ESM) share a common cardiopharyngeal mesoderm (CPM) origin, however ESM are unusual among striated muscles as they are established in the absence of a primary skeletal muscle scaffold. Using mouse chimeras, we show that the transcription factors Tbx1 and Isl1 are required cell-autonomously for myogenic specification of ESM progenitors. Further, genetic loss-of-function and pharmacological studies point to MET/HGF signaling for antero-posterior migration of esophagus muscle progenitors, where Hgf ligand is expressed in adjacent smooth muscle cells. These observations highlight the functional relevance of a smooth and striated muscle progenitor dialogue for ESM patterning. Our findings establish a Tbx1-Isl1-Met genetic hierarchy that uniquely regulates esophagus myogenesis and identify distinct genetic signatures that can be used as framework to interpret pathologies arising within CPM derivatives.

4.
J Cell Sci ; 131(14)2018 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-30054310

RESUMO

During growth, homeostasis and regeneration, stem cells are exposed to different energy demands. Here, we characterise the metabolic pathways that mediate the commitment and differentiation of mouse skeletal muscle stem cells, and how their modulation can influence the cell state. We show that quiescent satellite stem cells have low energetic demands and perturbed oxidative phosphorylation during ageing, which is also the case for cells from post-mortem tissues. We show also that myogenic fetal cells have distinct metabolic requirements compared to those proliferating during regeneration, with the former displaying a low respiration demand relying mostly on glycolysis. Furthermore, we show distinct requirements for peroxisomal and mitochondrial fatty acid oxidation (FAO) in myogenic cells. Compromising peroxisomal but not mitochondrial FAO promotes early differentiation of myogenic cells. Acute muscle injury and pharmacological block of peroxisomal and mitochondrial FAO expose differential requirements for these organelles during muscle regeneration. Taken together, these observations indicate that changes in myogenic cell state lead to significant alterations in metabolic requirements. In addition, perturbing specific metabolic pathways impacts on myogenic cell fates and the regeneration process.


Assuntos
Desenvolvimento Muscular , Músculo Esquelético/crescimento & desenvolvimento , Células-Tronco/citologia , Células-Tronco/metabolismo , Animais , Proliferação de Células , Ácidos Graxos/metabolismo , Camundongos , Mitocôndrias/metabolismo , Músculo Esquelético/metabolismo , Oxirredução , Peroxissomos/metabolismo , Células Satélites de Músculo Esquelético/citologia , Células Satélites de Músculo Esquelético/metabolismo
5.
Skelet Muscle ; 8(1): 19, 2018 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-29875011

RESUMO

After publication of this article [1], the authors noted that the legends for supplementary files Figures S3 and S4 were truncated in the production process, therefore lacking some information concerning these Figures. The complete legends are included in this Correction. The authors apologize for any inconvenience that this might have caused.

6.
Skelet Muscle ; 7(1): 28, 2017 12 22.
Artigo em Inglês | MEDLINE | ID: mdl-29273087

RESUMO

BACKGROUND: Skeletal muscle satellite (stem) cells are quiescent in adult mice and can undergo multiple rounds of proliferation and self-renewal following muscle injury. Several labs have profiled transcripts of myogenic cells during the developmental and adult myogenesis with the aim of identifying quiescent markers. Here, we focused on the quiescent cell state and generated new transcriptome profiles that include subfractionations of adult satellite cell populations, and an artificially induced prenatal quiescent state, to identify core signatures for quiescent and proliferating. METHODS: Comparison of available data offered challenges related to the inherent diversity of datasets and biological conditions. We developed a standardized workflow to homogenize the normalization, filtering, and quality control steps for the analysis of gene expression profiles allowing the identification up- and down-regulated genes and the subsequent gene set enrichment analysis. To share the analytical pipeline of this work, we developed Sherpa, an interactive Shiny server that allows multi-scale comparisons for extraction of desired gene sets from the analyzed datasets. This tool is adaptable to cell populations in other contexts and tissues. RESULTS: A multi-scale analysis comprising eight datasets of quiescent satellite cells had 207 and 542 genes commonly up- and down-regulated, respectively. Shared up-regulated gene sets include an over-representation of the TNFα pathway via NFKß signaling, Il6-Jak-Stat3 signaling, and the apical surface processes, while shared down-regulated gene sets exhibited an over-representation of Myc and E2F targets and genes associated to the G2M checkpoint and oxidative phosphorylation. However, virtually all datasets contained genes that are associated with activation or cell cycle entry, such as the immediate early stress response genes Fos and Jun. An empirical examination of fixed and isolated satellite cells showed that these and other genes were absent in vivo, but activated during procedural isolation of cells. CONCLUSIONS: Through the systematic comparison and individual analysis of diverse transcriptomic profiles, we identified genes that were consistently differentially expressed among the different datasets and shared underlying biological processes key to the quiescent cell state. Our findings provide impetus to define and distinguish transcripts associated with true in vivo quiescence from those that are first responding genes due to disruption of the stem cell niche.


Assuntos
Diferenciação Celular , Células Satélites de Músculo Esquelético/metabolismo , Transcriptoma , Animais , Bases de Dados Factuais , Feminino , Perfilação da Expressão Gênica , Masculino , Camundongos
7.
Front Immunol ; 8: 490, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28512459

RESUMO

Wiskott-Aldrich syndrome (WAS) is a primary immunodeficiency caused by mutations in the gene encoding the hematopoietic-specific WAS protein (WASp). WAS is frequently associated with autoimmunity, indicating a critical role of WASp in maintenance of tolerance. The role of B cells in the induction of autoreactive immune responses in WAS has been investigated in several settings, but the mechanisms leading to the development of autoimmune manifestations have been difficult to evaluate in the mouse models of the disease that do not spontaneously develop autoimmunity. We performed an extensive characterization of Was-/- mice that provided evidence of the potential alteration in B cell selection, because of the presence of autoantibodies against double-stranded DNA, platelets, and tissue antigens. To uncover the mechanisms leading to the activation of the potentially autoreactive B cells in Was-/- mice, we performed in vivo chronic stimulations with toll-like receptors agonists (LPS and CpG) and apoptotic cells or infection with lymphocytic choriomeningitis virus. All treatments led to increased production of autoantibodies, increased proteinuria, and kidney tissue damage in Was-/- mice. These findings demonstrate that a lower clearance of pathogens and/or self-antigens and the resulting chronic inflammatory state could cause B cell tolerance breakdown leading to autoimmunity in WAS.

8.
Methods Mol Biol ; 1556: 23-39, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28247343

RESUMO

Isolation of muscle stem cells from skeletal muscle is a critical step for the study of skeletal myogenesis and regeneration. Although stem cell isolation has been performed for decades, the emergence of flow cytometry with defined cell surface markers, or transgenic mouse models, has allowed the efficient isolation of highly enriched stem cell populations. Here, we describe the isolation of mouse muscle stem cells using two different combinations of enzyme treatments allowing the release of mononucleated muscle stem cells from their niche. Mouse muscle stem cells can be further isolated as a highly enriched population by flow cytometry using fluorescent reporters or cell surface markers. We will present advantages and drawbacks of these different approaches.


Assuntos
Separação Celular/métodos , Músculo Esquelético/citologia , Células-Tronco/citologia , Células-Tronco/metabolismo , Animais , Biomarcadores , Citometria de Fluxo/métodos , Imunofenotipagem/métodos , Camundongos , Fenótipo , Células Satélites de Músculo Esquelético/citologia , Células Satélites de Músculo Esquelético/metabolismo
9.
Sci Rep ; 6: 31453, 2016 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-27509848

RESUMO

Emotional deficits are part of the non-motor features of Parkinson's disease but few attention has been paid to specific aspects such as subjective emotional experience and autonomic responses. This study aimed to investigate the mechanisms of emotional recognition in Parkinson's Disease (PD) using the following levels: explicit evaluation of emotions (Self-Assessment Manikin) and implicit reactivity (Skin Conductance Response; electromyographic measure of facial feedback of the zygomaticus and corrugator muscles). 20 PD Patients and 34 healthy controls were required to observe and evaluate affective pictures during physiological parameters recording. In PD, the appraisal process on both valence and arousal features of emotional cues were preserved, but we found significant impairment in autonomic responses. Specifically, in comparison to healthy controls, PD patients revealed lower Skin Conductance Response values to negative and high arousing emotional stimuli. In addition, the electromyographic measures showed defective responses exclusively limited to negative and high arousing emotional category: PD did not show increasing of corrugator activity in response to negative emotions as happened in heathy controls. PD subjects inadequately respond to the emotional categories which were considered more "salient": they had preserved appraisal process, but impaired automatic ability to distinguish between different emotional contexts.


Assuntos
Sistema Nervoso Autônomo/fisiologia , Emoções/fisiologia , Músculos Faciais/fisiologia , Doença de Parkinson/psicologia , Idoso , Nível de Alerta/fisiologia , Eletromiografia , Expressão Facial , Reconhecimento Facial , Feminino , Resposta Galvânica da Pele , Humanos , Masculino , Pessoa de Meia-Idade
10.
J Clin Invest ; 125(10): 3941-51, 2015 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-26368308

RESUMO

Wiskott-Aldrich syndrome (WAS) is an X-linked immunodeficiency characterized by microthrombocytopenia, eczema, and high susceptibility to developing tumors and autoimmunity. Recent evidence suggests that B cells may be key players in the pathogenesis of autoimmunity in WAS. Here, we assessed whether WAS protein deficiency (WASp deficiency) affects the establishment of B cell tolerance by testing the reactivity of recombinant antibodies isolated from single B cells from 4 WAS patients before and after gene therapy (GT). We found that pre-GT WASp-deficient B cells were hyperreactive to B cell receptor stimulation (BCR stimulation). This hyperreactivity correlated with decreased frequency of autoreactive new emigrant/transitional B cells exiting the BM, indicating that the BCR signaling threshold plays a major role in the regulation of central B cell tolerance. In contrast, mature naive B cells from WAS patients were enriched in self-reactive clones, revealing that peripheral B cell tolerance checkpoint dysfunction is associated with impaired suppressive function of WAS regulatory T cells. The introduction of functional WASp by GT corrected the alterations of both central and peripheral B cell tolerance checkpoints. We conclude that WASp plays an important role in the establishment and maintenance of B cell tolerance in humans and that restoration of WASp by GT is able to restore B cell tolerance in WAS patients.


Assuntos
Linfócitos B/imunologia , Terapia Genética , Vetores Genéticos/uso terapêutico , Tolerância Imunológica , Proteína da Síndrome de Wiskott-Aldrich/uso terapêutico , Síndrome de Wiskott-Aldrich/terapia , Adulto , Sequência de Aminoácidos , Medula Óssea/patologia , Criança , Pré-Escolar , Deleção Clonal , Células Clonais/imunologia , Humanos , Lentivirus/genética , Masculino , Dados de Sequência Molecular , Receptores de Antígenos de Linfócitos B/imunologia , Proteínas Recombinantes de Fusão , Linfócitos T Reguladores/imunologia , Síndrome de Wiskott-Aldrich/imunologia , Proteína da Síndrome de Wiskott-Aldrich/deficiência , Proteína da Síndrome de Wiskott-Aldrich/genética
11.
J Allergy Clin Immunol ; 136(3): 692-702.e2, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25792466

RESUMO

BACKGROUND: Wiskott-Aldrich syndrome (WAS) is a severe X-linked immunodeficiency characterized by microthrombocytopenia, eczema, recurrent infections, and susceptibility to autoimmunity and lymphomas. Hematopoietic stem cell transplantation is the treatment of choice; however, administration of WAS gene-corrected autologous hematopoietic stem cells has been demonstrated as a feasible alternative therapeutic approach. OBJECTIVE: Because B-cell homeostasis is perturbed in patients with WAS and restoration of immune competence is one of the main therapeutic goals, we have evaluated reconstitution of the B-cell compartment in 4 patients who received autologous hematopoietic stem cells transduced with lentiviral vector after a reduced-intensity conditioning regimen combined with anti-CD20 administration. METHODS: We evaluated B-cell counts, B-cell subset distribution, B cell-activating factor and immunoglobulin levels, and autoantibody production before and after gene therapy (GT). WAS gene transfer in B cells was assessed by measuring vector copy numbers and expression of Wiskott-Aldrich syndrome protein. RESULTS: After lentiviral vector-mediated GT, the number of transduced B cells progressively increased in the peripheral blood of all patients. Lentiviral vector-transduced progenitor cells were able to repopulate the B-cell compartment with a normal distribution of B-cell subsets both in bone marrow and the periphery, showing a WAS protein expression profile similar to that of healthy donors. In addition, after GT, we observed a normalized frequency of autoimmune-associated CD19(+)CD21(-)CD35(-) and CD21(low) B cells and a reduction in B cell-activating factor levels. Immunoglobulin serum levels and autoantibody production improved in all treated patients. CONCLUSIONS: We provide evidence that lentiviral vector-mediated GT induces transgene expression in the B-cell compartment, resulting in ameliorated B-cell development and functionality and contributing to immunologic improvement in patients with WAS.


Assuntos
Subpopulações de Linfócitos B/metabolismo , Terapia Genética/métodos , Transplante de Células-Tronco Hematopoéticas , Proteína da Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/terapia , Autoanticorpos/biossíntese , Fator Ativador de Células B/genética , Fator Ativador de Células B/metabolismo , Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/patologia , Medula Óssea/imunologia , Medula Óssea/metabolismo , Medula Óssea/patologia , Criança , Pré-Escolar , Expressão Gênica , Perfilação da Expressão Gênica , Vetores Genéticos , Células-Tronco Hematopoéticas/imunologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Imunoglobulinas/biossíntese , Imunofenotipagem , Lactente , Lentivirus/genética , Masculino , Proteínas Recombinantes de Fusão/uso terapêutico , Transdução Genética , Condicionamento Pré-Transplante , Transplante Autólogo , Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/imunologia , Síndrome de Wiskott-Aldrich/patologia , Proteína da Síndrome de Wiskott-Aldrich/imunologia
12.
J Autoimmun ; 50: 42-50, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24369837

RESUMO

Wiskott-Aldrich Syndrome protein (WASp) regulates the cytoskeleton in hematopoietic cells and mutations in its gene cause the Wiskott-Aldrich Syndrome (WAS), a primary immunodeficiency with microthrombocytopenia, eczema and a higher susceptibility to develop tumors. Autoimmune manifestations, frequently observed in WAS patients, are associated with an increased risk of mortality and still represent an unsolved aspect of the disease. B cells play a crucial role both in immune competence and self-tolerance and defects in their development and function result in immunodeficiency and/or autoimmunity. We performed a phenotypical and molecular analysis of central and peripheral B-cell compartments in WAS pediatric patients. We found a decreased proportion of immature B cells in the bone marrow correlating with an increased presence of transitional B cells in the periphery. These results could be explained by the defective migratory response of WAS B cells to SDF-1α, essential for the retention of immature B cells in the BM. In the periphery, we observed an unusual expansion of CD21(low) B-cell population and increased plasma BAFF levels that may contribute to the high susceptibility to develop autoimmune manifestations in WAS patients. WAS memory B cells were characterized by a reduced in vivo proliferation, decreased somatic hypermutation and preferential usage of IGHV4-34, an immunoglobulin gene commonly found in autoreactive B cells. In conclusion, our findings demonstrate that WASp-deficiency perturbs B-cell homeostasis thus adding a new layer of immune dysregulation concurring to the increased susceptibility to develop autoimmunity in WAS patients.


Assuntos
Autoimunidade , Linfócitos B/imunologia , Suscetibilidade a Doenças/imunologia , Proteína da Síndrome de Wiskott-Aldrich/deficiência , Síndrome de Wiskott-Aldrich/imunologia , Fator Ativador de Células B/sangue , Fator Ativador de Células B/genética , Fator Ativador de Células B/imunologia , Linfócitos B/patologia , Medula Óssea/imunologia , Medula Óssea/patologia , Diferenciação Celular , Movimento Celular , Quimiocina CXCL12/genética , Quimiocina CXCL12/imunologia , Expressão Gênica , Homeostase/imunologia , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Memória Imunológica , Receptores de Complemento 3d/genética , Receptores de Complemento 3d/imunologia , Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/patologia , Proteína da Síndrome de Wiskott-Aldrich/genética , Proteína da Síndrome de Wiskott-Aldrich/imunologia
13.
Front Immunol ; 3: 209, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22826711

RESUMO

Wiskott-Aldrich Syndrome (WAS) is a severe X-linked Primary Immunodeficiency that affects 1-10 out of 1 million male individuals. WAS is caused by mutations in the WAS Protein (WASP) expressing gene that leads to the absent or reduced expression of the protein. WASP is a cytoplasmic protein that regulates the formation of actin filaments in hematopoietic cells. WASP deficiency causes many immune cell defects both in humans and in the WAS murine model, the Was(-/-) mouse. Both cellular and humoral immune defects in WAS patients contribute to the onset of severe clinical manifestations, in particular microthrombocytopenia, eczema, recurrent infections, and a high susceptibility to develop autoimmunity and malignancies. Autoimmune diseases affect from 22 to 72% of WAS patients and the most common manifestation is autoimmune hemolytic anemia, followed by vasculitis, arthritis, neutropenia, inflammatory bowel disease, and IgA nephropathy. Many groups have widely explored immune cell functionality in WAS partially explaining how cellular defects may lead to pathology. However, the mechanisms underlying the occurrence of autoimmune manifestations have not been clearly described yet. In the present review, we report the most recent progresses in the study of immune cell function in WAS that have started to unveil the mechanisms contributing to autoimmune complications in WAS patients.

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