Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 3.564
Filtrar
1.
Zhonghua Xue Ye Xue Za Zhi ; 40(8): 662-666, 2019 Aug 14.
Artigo em Chinês | MEDLINE | ID: mdl-31495133

RESUMO

Objective: To establish a new method for chimerism analysis after allogeneic hematopoietic stem cell transplantation by using multiple nucleotide polymorphism sequencing (MNPseq) , and to explore its feasibility and superiority. Methods: One hundred MNP fragments were screened and chimeric analysis was performed by high-throughput sequencing technology. The accuracy and sensitivity of the method were verified by simulating chimeric samples and post-transplant samples and comparing them with short tandem repeat (STR) analysis, fusion gene quantitative detection and flow cytometry for minimal residual disease. Results: The accuracy and sensitivity of MNPseq were better than those of STR, in which the sensitivity could reach 0.01%, about 100 times more sensitive than STR. MNPseq could further distinguish 42 STR fully chimeric samples, and after corrected by cutoff value, it was correlated with the quantitative detection of fusion gene. MNPseq could correct false positive of STR caused by the shadow peak, and could be used to detect chimeric samples lacking pre-transplant information from donors and recipients. Conclusion: MNPseq analysis based on high-throughput sequencing is a more accurate and sensitive chimerism detection method, and it solves the problem that chimerism cannot be detected due to the lack of pre-transplant information, which has extremely high clinical application value.


Assuntos
Transplante de Células-Tronco Hematopoéticas , Humanos , Polimorfismo Genético , Doadores de Tecidos , Quimeras de Transplante
2.
Nat Commun ; 10(1): 3027, 2019 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-31289275

RESUMO

Fibrosis accompanying wound healing can drive the failure of many different organs. Activated fibroblasts are the principal determinants of post-injury pathological fibrosis along with physiological repair, making them a difficult therapeutic target. Although activated fibroblasts are phenotypically heterogeneous, they are not recognized as distinct functional entities. Using mice that express GFP under the FSP1 or αSMA promoter, we characterized two non-overlapping fibroblast subtypes from mouse hearts after myocardial infarction. Here, we report the identification of FSP1-GFP+ cells as a non-pericyte, non-hematopoietic fibroblast subpopulation with a predominant pro-angiogenic role, characterized by in vitro phenotypic/cellular/ultrastructural studies and in vivo granulation tissue formation assays combined with transcriptomics and proteomics. This work identifies a fibroblast subtype that is functionally distinct from the pro-fibrotic αSMA-expressing myofibroblast subtype. Our study has the potential to shift our focus towards viewing fibroblasts as molecularly and functionally heterogeneous and provides a paradigm to approach treatment for organ fibrosis.


Assuntos
Fibroblastos/metabolismo , Infarto do Miocárdio/patologia , Miocárdio/patologia , Neovascularização Fisiológica/fisiologia , Cicatrização/fisiologia , Actinas/genética , Actinas/metabolismo , Animais , Transplante de Medula Óssea , Proteínas de Ligação ao Cálcio/metabolismo , Diferenciação Celular , Modelos Animais de Doenças , Fibroblastos/patologia , Fibrose/etiologia , Fibrose/patologia , Proteínas de Fluorescência Verde/genética , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Infarto do Miocárdio/etiologia , Infarto do Miocárdio/fisiopatologia , Miocárdio/citologia , Proteína A4 de Ligação a Cálcio da Família S100/genética , Proteína A4 de Ligação a Cálcio da Família S100/metabolismo , Quimeras de Transplante
3.
Nat Commun ; 10(1): 2220, 2019 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-31101805

RESUMO

Both medullary thymic epithelial cells (mTEC) and dendritic cells (DC) present tissue-restricted antigens (TRA) to thymocytes to induce central tolerance, but the relative contributions of these antigen-presenting cell (APC) subsets remain unresolved. Here we developed a two-photon microscopy approach to observe thymocytes interacting with intact APCs presenting TRAs. We find that mTECs and DCs cooperate extensively to induce tolerance, with their relative contributions regulated by the cellular form of the TRA and the class of major histocompatibility complex (MHC) on which antigen is presented. Even when TRA expression is restricted to mTECs, DCs still present self-antigens at least as frequently as mTECs. Notably, the DC subset cDC2 efficiently acquires secreted mTEC-derived TRAs for cross-presentation on MHC-I. By directly imaging interactions between thymocytes and APCs, while monitoring intracellular signaling, this study reveals that distinct DC subsets and AIRE+ mTECs contribute substantially to presentation of diverse self-antigens for establishing central tolerance.


Assuntos
Tolerância Central/imunologia , Células Dendríticas/imunologia , Timócitos/imunologia , Timo/imunologia , Animais , Apresentação do Antígeno/imunologia , Autoantígenos/imunologia , Autoantígenos/metabolismo , Transplante de Medula Óssea , Separação Celular/métodos , Células Dendríticas/metabolismo , Células Epiteliais/imunologia , Feminino , Citometria de Fluxo/métodos , Microscopia Intravital/métodos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Linfócitos T Reguladores/imunologia , Timócitos/metabolismo , Timo/citologia , Fatores de Transcrição/imunologia , Fatores de Transcrição/metabolismo , Quimeras de Transplante/imunologia
4.
Nat Commun ; 10(1): 2011, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-31043609

RESUMO

TET enzymes oxidize 5-methylcytosine to 5-hydroxymethylcytosine and other oxidized methylcytosines in DNA. Here we examine the role of TET proteins in regulatory T (Treg) cells. Tet2/3fl/flFoxp3Cre mice lacking Tet2 and Tet3 in Treg cells develop inflammatory disease, and Treg cells from these mice show altered expression of Treg signature genes and upregulation of genes involved in cell cycle, DNA damage and cancer. In littermate mice with severe inflammation, both CD4+Foxp3+ and CD4+Foxp3- cells show strong skewing towards Tfh/Th17 phenotypes. Wild-type Treg cells in mixed bone marrow chimeras and in Tet2/3fl/flFoxp3WT/Cre heterozygous female mice are unable to rescue the aberrant properties of Tet2/3fl/flFoxp3Cre Treg cells. Treg cells from Tet2/3fl/flFoxp3Cre mice tend to lose Foxp3 expression, and transfer of total CD4+ T cells isolated from Tet2/3fl/flFoxp3Cre mice could elicit inflammatory disease in fully immunocompetent mice. Together, these data indicate that Tet2 and Tet3 are guardians of Treg cell stability and immune homeostasis.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Inflamação/imunologia , Proteínas Proto-Oncogênicas/metabolismo , Linfócitos T Reguladores/imunologia , Transferência Adotiva , Animais , Transplante de Medula Óssea , Colite , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/imunologia , Modelos Animais de Doenças , Feminino , Humanos , Inflamação/genética , Inflamação/patologia , Masculino , Camundongos , Camundongos Knockout , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/imunologia , Linfócitos T Reguladores/metabolismo , Linfócitos T Reguladores/transplante , Quimeras de Transplante
5.
Nat Commun ; 10(1): 2184, 2019 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-31097716

RESUMO

Chronic hepatitis B virus (HBV) infection can cause cirrhosis and hepatocellular carcinoma and is therefore a serious public health problem. Infected patients are currently treated with nucleoside/nucleotide analogs and interferon α, but this approach is not curative. Here, we screen 978 FDA-approved compounds for their ability to inhibit HBV replication in HBV-expressing HepG2.2.15 cells. We find that ciclopirox, a synthetic antifungal agent, strongly inhibits HBV replication in cells and in mice by blocking HBV capsid assembly. The crystal structure of the HBV core protein and ciclopirox complex reveals a unique binding mode at dimer-dimer interfaces. Ciclopirox synergizes with nucleoside/nucleotide analogs to prevent HBV replication in cells and in a humanized liver mouse model. Therefore, orally-administered ciclopirox may provide a novel opportunity to combat chronic HBV infection by blocking HBV capsid assembly.


Assuntos
Antivirais/farmacologia , Ciclopirox/farmacologia , Vírus da Hepatite B/fisiologia , Hepatite B Crônica/tratamento farmacológico , Montagem de Vírus/efeitos dos fármacos , Animais , Antivirais/uso terapêutico , Capsídeo/efeitos dos fármacos , Capsídeo/metabolismo , Ciclopirox/química , Ciclopirox/uso terapêutico , Cristalografia por Raios X , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Sinergismo Farmacológico , Células Hep G2 , Vírus da Hepatite B/efeitos dos fármacos , Hepatite B Crônica/patologia , Hepatite B Crônica/virologia , Hepatócitos/transplante , Hepatócitos/virologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , RNA Viral/metabolismo , Quimeras de Transplante , Resultado do Tratamento , Proteínas do Core Viral/química , Proteínas do Core Viral/metabolismo , Replicação Viral/efeitos dos fármacos
6.
Bull Cancer ; 106(6): 574-583, 2019 Jun.
Artigo em Francês | MEDLINE | ID: mdl-31060736

RESUMO

Allogeneic hematopoietic stem cell transplantation is the only curative therapy for numerous malignant and non-malignant haematological diseases. A sustained engraftment of the donor stem cells is essential for transplant success and overall outcome. Graft failure is a rare but severe event after allogeneic hematopoietic stem cell transplantation. While different risk factors such as underlying disease, graft source or HLA matching have been found to be consistently associated with graft failure, other factors such as ABO mismatch graft-versus-host disease prophylaxis or infections, particularly viral reactivations, are more controversial. In this article, we review the different factors associated with graft failure.


Assuntos
Rejeição de Enxerto , Transplante de Células-Tronco Hematopoéticas , Aloenxertos , Incompatibilidade de Grupos Sanguíneos , Gerenciamento Clínico , Rejeição de Enxerto/epidemiologia , Rejeição de Enxerto/etiologia , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/fisiopatologia , Doença Enxerto-Hospedeiro/complicações , Doença Enxerto-Hospedeiro/prevenção & controle , Doenças Hematológicas/terapia , Transplante de Células-Tronco Hematopoéticas/mortalidade , Histocompatibilidade , Humanos , Incidência , Neutropenia/etiologia , Pancitopenia/etiologia , Fatores de Risco , Quimeras de Transplante
7.
Gastroenterology ; 157(2): 552-568, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31029706

RESUMO

BACKGROUND & AIMS: Acetaminophen (APAP) overdose is a major cause of acute liver failure (ALF). Mitochondrial SH3BP5 (also called SAB) and phosphorylation of c-Jun N-terminal kinase (JNK) mediate the hepatotoxic effects of APAP. We investigated the involvement of steroidogenic acute regulatory protein (STARD1), a mitochondrial cholesterol transporter, in this process and sensitization by valproic acid (VPA), which depletes glutathione and stimulates steroidogenesis. METHODS: Nonfasted C57BL/6J mice (control) and mice with liver-specific deletion of STARD1 (Stard1ΔHep), SAB (SabΔHep), or JNK1 and JNK2 (Jnk1+2ΔHep) were given VPA with or without APAP. Liver tissues were collected and analyzed by histology and immunohistochemistry and for APAP metabolism, endoplasmic reticulum (ER) stress, and mitochondrial function. Adult human hepatocytes were transplanted into Fah-/-/Rag2-/-/Il2rg-/-/NOD (FRGN) mice to create mice with humanized livers. RESULTS: Administration of VPA before administration of APAP increased the severity of liver damage in control mice. The combination of VPA and APAP increased expression of CYP2E1, formation of NAPQI-protein adducts, and depletion of glutathione from liver tissues of control mice, resulting in ER stress and the upregulation of STARD1. Livers from control mice given VPA and APAP accumulated cholesterol in the mitochondria and had sustained mitochondrial depletion of glutathione and mitochondrial dysfunction. Inhibition of ER stress, by administration of tauroursodeoxycholic acid to control mice, prevented upregulation of STARD1 in liver and protected the mice from hepatoxicity following administration of VPA and APAP. Administration of N-acetylcysteine to control mice prevented VPA- and APAP-induced ER stress and liver injury. Stard1ΔHep mice were resistant to induction of ALF by VPA and APAP, despite increased mitochondrial levels of glutathione and phosphorylated JNK; we made similar observations in fasted Stard1ΔHep mice given APAP alone. SabΔHep mice or Jnk1+2ΔHep mice did not develop ALF following administration of VPA and APAP. The ability of VPA to increase the severity of APAP-induced liver damage was observed in FRGN mice with humanized liver. CONCLUSIONS: In studies of mice, we found that upregulation of STARD1 following ER stress mediates APAP hepatoxicity via SH3BP5 and phosphorylation of JNK1 and JNK2.


Assuntos
Acetaminofen/toxicidade , Doença Hepática Induzida por Substâncias e Drogas/patologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Hepatócitos/patologia , Fosfoproteínas/metabolismo , Adulto , Animais , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Modelos Animais de Doenças , Overdose de Drogas/complicações , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Hepatócitos/transplante , Humanos , Lipogênese/efeitos dos fármacos , Fígado/citologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fosfoproteínas/genética , Esteroides/metabolismo , Quimeras de Transplante , Regulação para Cima , Ácido Valproico/administração & dosagem
8.
In Vivo ; 33(3): 675-688, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31028184

RESUMO

Background/Aim: The cause of fatal neuromuscular amyotrophic lateral sclerosis (ALS) is not known. Materials and Methods: Ninety-day-old superoxide-dismutase-1 G93A (SOD1 G93A ) mice demonstrating level 1 paralysis, received 9.0 Gy total body irradiation (TBI) from a cesium source at 340 cGy per minute, and intravenous transplantation with 1×10 6 C57BL/6 green fluorescent protein (GFP)+ donor bone marrow cells. Results: Paralysis-free survival was prolonged in TBI and bone marrow-transplanted SOD1 G93A mice from 100 to over 250 days (p=0.0018). Other mice transplanted with SOD1 G93A marrow or marrow treated with the free-radical scavenger MMS350 showed no therapeutic effect. GFP+ macrophage-2 (M2) microglial cells of bone marrow origin, were seen at sites of degenerating anterior horn motor neurons. SOD1 G93A mice had a disruption in the blood-brain barrier permeability which was reversed by marrow transplant from C57BL/6 mice. SOD1 G93A marrow showed unexpected robust hematopoiesis in vitro, and radioresistance. Conclusion: After TBI, M2 microglial cells from transplanted donor marrow extended the paralysis-free interval in SOD1 G93A mice.


Assuntos
Esclerose Amiotrófica Lateral/metabolismo , Microglia/metabolismo , Mutação , Superóxido Dismutase-1/genética , Substituição de Aminoácidos , Esclerose Amiotrófica Lateral/etiologia , Esclerose Amiotrófica Lateral/fisiopatologia , Esclerose Amiotrófica Lateral/terapia , Animais , Barreira Hematoencefálica/metabolismo , Transplante de Medula Óssea , Diferenciação Celular/genética , Modelos Animais de Doenças , Expressão Gênica , Sobrevivência de Enxerto , Hematopoese/genética , Camundongos , Camundongos Transgênicos , Microglia/imunologia , Tolerância a Radiação/genética , Superóxido Dismutase-1/metabolismo , Quimeras de Transplante
9.
Gastroenterology ; 157(2): 537-551.e9, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30978357

RESUMO

BACKGROUND & AIMS: The mechanisms of hepatitis C virus (HCV) infection, liver disease progression, and hepatocarcinogenesis are only partially understood. We performed genomic, proteomic, and metabolomic analyses of HCV-infected cells and chimeric mice to learn more about these processes. METHODS: Huh7.5.1dif (hepatocyte-like cells) were infected with culture-derived HCV and used in RNA sequencing, proteomic, metabolomic, and integrative genomic analyses. uPA/SCID (urokinase-type plasminogen activator/severe combined immunodeficiency) mice were injected with serum from HCV-infected patients; 8 weeks later, liver tissues were collected and analyzed by RNA sequencing and proteomics. Using differential expression, gene set enrichment analyses, and protein interaction mapping, we identified pathways that changed in response to HCV infection. We validated our findings in studies of liver tissues from 216 patients with HCV infection and early-stage cirrhosis and paired biopsy specimens from 99 patients with hepatocellular carcinoma, including 17 patients with histologic features of steatohepatitis. Cirrhotic liver tissues from patients with HCV infection were classified into 2 groups based on relative peroxisome function; outcomes assessed included Child-Pugh class, development of hepatocellular carcinoma, survival, and steatohepatitis. Hepatocellular carcinomas were classified according to steatohepatitis; the outcome was relative peroxisomal function. RESULTS: We quantified 21,950 messenger RNAs (mRNAs) and 8297 proteins in HCV-infected cells. Upon HCV infection of hepatocyte-like cells and chimeric mice, we observed significant changes in levels of mRNAs and proteins involved in metabolism and hepatocarcinogenesis. HCV infection of hepatocyte-like cells significantly increased levels of the mRNAs, but not proteins, that regulate the innate immune response; we believe this was due to the inhibition of translation in these cells. HCV infection of hepatocyte-like cells increased glucose consumption and metabolism and the STAT3 signaling pathway and reduced peroxisome function. Peroxisomes mediate ß-oxidation of very long-chain fatty acids; we found intracellular accumulation of very long-chain fatty acids in HCV-infected cells, which is also observed in patients with fatty liver disease. Cells in livers from HCV-infected mice had significant reductions in levels of the mRNAs and proteins associated with peroxisome function, indicating perturbation of peroxisomes. We found that defects in peroxisome function were associated with outcomes and features of HCV-associated cirrhosis, fatty liver disease, and hepatocellular carcinoma in patients. CONCLUSIONS: We performed combined transcriptome, proteome, and metabolome analyses of liver tissues from HCV-infected hepatocyte-like cells and HCV-infected mice. We found that HCV infection increases glucose metabolism and the STAT3 signaling pathway and thereby reduces peroxisome function; alterations in the expression levels of peroxisome genes were associated with outcomes of patients with liver diseases. These findings provide insights into liver disease pathogenesis and might be used to identify new therapeutic targets.


Assuntos
Hepacivirus/patogenicidade , Hepatite C Crônica/patologia , Hepatócitos/patologia , Fígado/patologia , Animais , Linhagem Celular Tumoral , Conjuntos de Dados como Assunto , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Glucose/metabolismo , Hepatite C Crônica/metabolismo , Hepatite C Crônica/virologia , Hepatócitos/transplante , Hepatócitos/virologia , Humanos , Fígado/citologia , Fígado/virologia , Metabolômica , Camundongos , Peroxissomos/metabolismo , Peroxissomos/patologia , Proteômica , Fator de Transcrição STAT3/metabolismo , Quimeras de Transplante
10.
Nat Commun ; 10(1): 1668, 2019 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-30971695

RESUMO

Therapies that target the function of immune cells have significant clinical efficacy in diseases such as cancer and autoimmunity. Although functional genomics has accelerated therapeutic target discovery in cancer, its use in primary immune cells is limited because vector delivery is inefficient and can perturb cell states. Here we describe CHIME: CHimeric IMmune Editing, a CRISPR-Cas9 bone marrow delivery system to rapidly evaluate gene function in innate and adaptive immune cells in vivo without ex vivo manipulation of these mature lineages. This approach enables efficient deletion of genes of interest in major immune lineages without altering their development or function. We use this approach to perform an in vivo pooled genetic screen and identify Ptpn2 as a negative regulator of CD8+ T cell-mediated responses to LCMV Clone 13 viral infection. These findings indicate that this genetic platform can enable rapid target discovery through pooled screening in immune cells in vivo.


Assuntos
Imunidade Adaptativa/genética , Sistemas CRISPR-Cas/genética , Técnicas de Transferência de Genes , Testes Genéticos/métodos , Imunidade Inata/genética , Animais , Transplante de Medula Óssea , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Linhagem da Célula/genética , Linhagem da Célula/imunologia , Modelos Animais de Doenças , Estudos de Viabilidade , Feminino , Vetores Genéticos/genética , Genômica/métodos , Células HEK293 , Humanos , Coriomeningite Linfocítica/genética , Coriomeningite Linfocítica/imunologia , Coriomeningite Linfocítica/virologia , Vírus da Coriomeningite Linfocítica/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteína Tirosina Fosfatase não Receptora Tipo 2/genética , Proteína Tirosina Fosfatase não Receptora Tipo 2/imunologia , RNA Guia/genética , Quimeras de Transplante , Células Vero
11.
Nat Commun ; 10(1): 1634, 2019 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-30967552

RESUMO

Gene correction in human long-term hematopoietic stem cells (LT-HSCs) could be an effective therapy for monogenic diseases of the blood and immune system. Here we describe an approach for X-linked sSevere cCombined iImmunodeficiency (SCID-X1) using targeted integration of a cDNA into the endogenous start codon to functionally correct disease-causing mutations throughout the gene. Using a CRISPR-Cas9/AAV6 based strategy, we achieve up to 20% targeted integration frequencies in LT-HSCs. As measures of the lack of toxicity we observe no evidence of abnormal hematopoiesis following transplantation and no evidence of off-target mutations using a high-fidelity Cas9 as a ribonucleoprotein complex. We achieve high levels of targeting frequencies (median 45%) in CD34+ HSPCs from six SCID-X1 patients and demonstrate rescue of lymphopoietic defect in a patient derived HSPC population in vitro and in vivo. In sum, our study provides specificity, toxicity and efficacy data supportive of clinical development of genome editing to treat SCID-Xl.


Assuntos
DNA Complementar/genética , Edição de Genes/métodos , Transplante de Células-Tronco Hematopoéticas , Subunidade gama Comum de Receptores de Interleucina/genética , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/terapia , Animais , Antígenos CD34/metabolismo , Sistemas CRISPR-Cas/genética , Linhagem Celular , Códon de Iniciação/genética , Éxons/genética , Sangue Fetal/citologia , Vetores Genéticos/genética , Voluntários Saudáveis , Células-Tronco Hematopoéticas/metabolismo , Humanos , Masculino , Camundongos , Mutação , Parvovirinae/genética , Cultura Primária de Células , Fatores de Tempo , Transdução Genética/métodos , Quimeras de Transplante/genética , Transplante Heterólogo/métodos , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/genética
12.
Nat Commun ; 10(1): 1183, 2019 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-30862784

RESUMO

T regulatory (Treg) cells maintain immunological tolerance and organ homeostasis. Activated Treg cells differentiate into effector Treg subsets that acquire tissue-specific functions. Ca2+ influx via Ca2+ release-activated Ca2+ (CRAC) channels formed by STIM and ORAI proteins is required for the thymic development of Treg cells, but its function in mature Treg cells remains unclear. Here we show that deletion of Stim1 and Stim2 genes in mature Treg cells abolishes Ca2+ signaling and prevents their differentiation into follicular Treg and tissue-resident Treg cells. Transcriptional profiling of STIM1/STIM2-deficient Treg cells reveals that Ca2+ signaling regulates transcription factors and signaling pathways that control the identity and effector differentiation of Treg cells. In the absence of STIM1/STIM2 in Treg cells, mice develop a broad spectrum of autoantibodies and fatal multiorgan inflammation. Our findings establish a critical role of CRAC channels in controlling lineage identity and effector functions of Treg cells.


Assuntos
Doenças Autoimunes/imunologia , Canais de Cálcio Ativados pela Liberação de Cálcio/fisiologia , Cálcio/metabolismo , Diferenciação Celular/imunologia , Linfócitos T Reguladores/fisiologia , Animais , Autoanticorpos/imunologia , Doenças Autoimunes/genética , Transplante de Medula Óssea , Cátions Bivalentes/metabolismo , Modelos Animais de Doenças , Feminino , Fatores de Transcrição Forkhead/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Molécula 1 de Interação Estromal/genética , Molécula 1 de Interação Estromal/metabolismo , Molécula 2 de Interação Estromal/genética , Molécula 2 de Interação Estromal/metabolismo , Quimeras de Transplante
13.
Nat Commun ; 10(1): 1076, 2019 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-30842418

RESUMO

Phagocytes, including neutrophils and macrophages, have been suggested to function in a cooperative way in the initial phase of inflammatory responses, but their interaction and integration in the resolution of inflammation and tissue repair remain unclear. Here we show that neutrophils have crucial functions in liver repair by promoting the phenotypic conversion of pro-inflammatory Ly6ChiCX3CR1lo monocytes/macrophages to pro-resolving Ly6CloCX3CR1hi macrophages. Intriguingly, reactive oxygen species (ROS), expressed predominantly by neutrophils, are important mediators that trigger this phenotypic conversion to promote liver repair. Moreover, this conversion is prevented by the depletion of neutrophils via anti-Ly6G antibody, genetic deficiency of granulocyte colony-stimulating factor, or genetic deficiency of NADPH oxidase 2 (Nox2). By contrast, adoptive transfer of WT rather than Nox2-/- neutrophils rescues the impaired phenotypic conversion of macrophages in neutrophil-depleted mice. Our findings thus identify an intricate cooperation between neutrophils and macrophages that orchestrate resolution of inflammation and tissue repair.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas/imunologia , Regeneração Hepática/imunologia , Macrófagos/imunologia , Neutrófilos/imunologia , Espécies Reativas de Oxigênio/metabolismo , Acetaminofen/toxicidade , Transferência Adotiva/métodos , Animais , Transplante de Medula Óssea , Células Cultivadas , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Doença Hepática Induzida por Substâncias e Drogas/patologia , Modelos Animais de Doenças , Humanos , Fígado/imunologia , Fígado/patologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Knockout , NADPH Oxidase 2/genética , NADPH Oxidase 2/imunologia , Neutrófilos/metabolismo , Neutrófilos/transplante , Cultura Primária de Células , Quimeras de Transplante
14.
Nat Commun ; 10(1): 1072, 2019 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-30842549

RESUMO

The balance between self-renewal and differentiation of hematopoietic stem and progenitor cells (HSPCs) maintains hematopoietic homeostasis, failure of which can lead to hematopoietic disorder. HSPC fate is controlled by signals from the bone marrow niche resulting in alteration of the stem cell transcription network. Regnase-1, a member of the CCCH zinc finger protein family possessing RNAse activity, mediates post-transcriptional regulatory activity through degradation of target mRNAs. The precise function of Regnase-1 has been explored in inflammation-related cytokine expression but its function in hematopoiesis has not been elucidated. Here, we show that Regnase-1 regulates self-renewal of HSPCs through modulating the stability of Gata2 and Tal1 mRNA. In addition, we found that dysfunction of Regnase-1 leads to the rapid onset of abnormal hematopoiesis. Thus, our data reveal that Regnase-1-mediated post-transcriptional regulation is required for HSPC maintenance and suggest that it represents a leukemia tumor suppressor.


Assuntos
Células-Tronco Hematopoéticas/fisiologia , Leucemia Mieloide Aguda/genética , Processamento Pós-Transcricional do RNA/fisiologia , Ribonucleases/metabolismo , Fatores de Transcrição/metabolismo , Animais , Medula Óssea/patologia , Diferenciação Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Autorrenovação Celular/genética , Conjuntos de Dados como Assunto , Fator de Transcrição GATA2/genética , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Células HEK293 , Transplante de Células-Tronco Hematopoéticas , Humanos , Leucemia Mieloide Aguda/mortalidade , Leucemia Mieloide Aguda/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Prognóstico , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Ribonucleases/genética , Proteína 1 de Leucemia Linfocítica Aguda de Células T/genética , Fatores de Transcrição/genética , Quimeras de Transplante
15.
Transpl Infect Dis ; 21(3): e13070, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30864271

RESUMO

Recovery of cytomegalovirus (CMV)-specific immunity after hematopoietic cell transplantation (HCT) is essential in controlling CMV infection. We hypothesize that mixed donor engraftment as measured by chimerism at day 30 in CMV D(+) HCTs and full chimerism in CMV D(-) HCTs will be predictive of CMV reactivation. Prospectively collected data for 407 CMV R+ HCT recipients transplanted from 2006 to 2014 at the University of Minnesota were retrospectively analyzed. Full and mixed donor engraftment were defined as ≥95% or <95% donor cells at day 30, respectively. Source of engraftment determination included preferentially peripheral blood CD3 fraction, then myeloid cell fraction (CD15+), then bone marrow. In 407 CMV R+ subjects, 77% (n = 313) were CMV D(-) cells from umbilical cord blood (n = 209), peripheral blood (n = 58) or marrow (n = 46). Fifty three per cent received reduced intensity conditioning (RIC). At day +30, full donor engraftment was seen in 82% of myeloablative and 55% of RIC transplants. The cumulative incidence of CMV infection 1-year after transplant was not different in patients with full (54%, n = 276) or mixed (53%, n = 131) donor engraftment. Control of CMV did not significantly differ among the two groups. In multiple regression analysis, there was no significant association between donor engraftment (mixed or full) and incidence or control of CMV.


Assuntos
Infecções por Citomegalovirus/prevenção & controle , Doença Enxerto-Hospedeiro/complicações , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Quimeras de Transplante , Ativação Viral , Adolescente , Adulto , Antivirais/administração & dosagem , Criança , Citomegalovirus , Feminino , Humanos , Incidência , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Estudos Retrospectivos , Doadores de Tecidos , Transplante Homólogo , Adulto Jovem
16.
Cell Prolif ; 52(3): e12591, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30896067

RESUMO

OBJECTIVES: To date, many efforts have been made to establish porcine embryonic stem (pES) cells without success. Extraembryonic endoderm (XEN) cells can self-renew and differentiate into the visceral endoderm and parietal endoderm. XEN cells are derived from the primitive endoderm of the inner cell mass of blastocysts and may be an intermediate state in cell reprogramming. MATERIALS AND METHODS: Porcine XEN cells (pXENCs) were generated from porcine pluripotent stem cells (pPSCs) and were characterized by RNA sequencing and immunofluorescence analyses. The developmental potential of pXENCs was investigated in chimeric mouse embryos. RESULTS: Porcine XEN cells derived from porcine pPSCs were successfully expanded in N2B27 medium supplemented with bFGF for least 30 passages. RNA sequencing and immunofluorescence analyses showed that pXENCs expressed the murine and canine XEN markers Gata6, Gata4, Sox17 and Pdgfra but not the pluripotent markers Oct4, Sox2 and TE marker Cdx2. Moreover, these cells contributed to the XEN when injected into four-cell stage mouse embryos. Supplementation with Chir99021 and SB431542 promoted the pluripotency of the pXENCs. CONCLUSIONS: We successfully derived pXENCs and showed that supplementation with Chir99021 and SB431542 confer them with pluripotency. Our results provide a new resource for investigating the reprogramming mechanism of porcine-induced pluripotent stem cells.


Assuntos
Endoderma/citologia , Endoderma/embriologia , Suínos/embriologia , Animais , Técnicas de Cultura de Células , Diferenciação Celular , Linhagem Celular , Técnicas de Cocultura , Cães , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Endoderma/metabolismo , Expressão Gênica , Camundongos , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Análise de Sequência de RNA , Transdução de Sinais , Suínos/genética , Suínos/metabolismo , Quimeras de Transplante
17.
J Korean Med Sci ; 34(6): e46, 2019 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-30787679

RESUMO

Background: The impact of early peripheral blood chimerism on the outcome of allogeneic hematopoietic stem cell transplantation (allo-HSCT) is unclear. We aimed to determine whether day 14 peripheral blood chimerism after allo-HSCT predicts outcomes in patients with non-malignant diseases. Methods: Data from 56 patients who received allo-HSCT between April 2007 and March 2016 were retrospectively analyzed. Chimerism was evaluated using short-tandem repeat polymerase chain reaction, with mixed chimerism (MC) defined as greater than 1% recipient cells which was further categorized into low-level MC (> 1% and < 15% of recipient-derived cells) and high-level MC (≥ 15% of the recipient-derived cells). Results: Thirty-six patients showed complete donor chimerism (CC), 14 low-level MC, and 6 high-level MC at day 14 post-transplant. The estimated 5-year event-free survival (EFS) was higher in the CC or low-level MC groups than in the high-level MC group (86.1% vs. 71.4% vs. 33.3%; P = 0.001). In BM or peripheral blood stem cell (BM/PBSC) transplants, the 5-year EFS was higher in the CC or low-level MC group than in the high-level MC group (93.1% vs. 66.7% vs. 0%; P < 0.001). However, in cord blood transplants, the 5-year OS and EFS according to the day 14 peripheral blood chimerism did not reach statistical significance. Conclusion: Although CC is not always necessary after allo-HSCT for non-malignant diseases, our data suggest that day 14 peripheral blood chimerism may predict outcomes in patients with non-malignant diseases who underwent BM/PBSC transplants.


Assuntos
Transplante de Células-Tronco Hematopoéticas , Quimeras de Transplante/genética , Adolescente , Adulto , Anemia Aplástica/mortalidade , Anemia Aplástica/patologia , Anemia Aplástica/terapia , Criança , Pré-Escolar , Intervalo Livre de Doença , Feminino , Doença Enxerto-Hospedeiro , Humanos , Lactente , Recém-Nascido , Masculino , Aplasia Pura de Série Vermelha/mortalidade , Aplasia Pura de Série Vermelha/patologia , Aplasia Pura de Série Vermelha/terapia , Estudos Retrospectivos , Taxa de Sobrevida , Quimeras de Transplante/sangue , Transplante Homólogo , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/mortalidade , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/patologia , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/terapia , Adulto Jovem
18.
Ann Hematol ; 98(5): 1279-1291, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30783731

RESUMO

Short tandem repeat (STR) analysis is used in chimerism monitoring after allogeneic hematopoietic stem cell transplantation (HSCT) for patients with various hematologic malignancies. Commercial forensic STR kits often contain loci with huge differences in power of discrimination (PD) across populations, causing some loci to be less informative for chimerism analysis in certain populations. This study aimed to construct a new STR multiplex panel with highly informative loci for efficient chimerism analysis. Thirteen STR markers which exhibit high PD (> 0.9) in at least 80% of 50 populations globally were selected to form a new panel and used in STR analysis of 253 Malaysian subjects. Cumulative power of discrimination (CPD) and combined power of exclusion (CPE) were determined from 253 Malaysian individuals. Loci informativity was assessed and compared to the commercial AmpFLSTR Identifiler PCR Amplification kit in 14 donor-recipient pairs. The new panel had detected 202 unique alleles including five novel alleles from the 253 individuals with high CPD and CPE (> 0.99999999999999999 and > 0.999999997 respectively). All loci from the new panel in the donor-recipient pair analysis showed higher than 50% informativity, while five loci from the commercial kit demonstrated lower than 50% informativity. Four loci from the new panel ranked the highest informativity. A sequenced allelic ladder which consists of 202 unique alleles from the 253 subjects was also developed to ensure accurate allele designation. The new 13-loci STR panel, thus, could serve as an additional powerful, accurate, and highly informative panel for chimerism analysis for HSCT patients.


Assuntos
Loci Gênicos , Transplante de Células-Tronco Hematopoéticas , Repetições de Microssatélites , Reação em Cadeia da Polimerase Multiplex , Kit de Reagentes para Diagnóstico/normas , Quimeras de Transplante/genética , Aloenxertos , Feminino , Humanos , Malásia , Masculino , Reação em Cadeia da Polimerase Multiplex/métodos , Reação em Cadeia da Polimerase Multiplex/normas , Quimeras de Transplante/sangue
19.
PLoS One ; 14(2): e0212708, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30794643

RESUMO

BACKGROUND: The analysis of molecular haematopoietic chimerisms (HC) has become a well-established method to monitor the transplant evolution and to assess the risk of relapse after allogeneic stem cells transplantation (allo-STC). Different techniques and molecular markers are being used for chimerism surveillance after transplantation, including quantitative real-time PCR (qPCR) and the recently developed digital PCR (dPCR). This study aims to compare the sensitivity and accuracy of both methods to quantify HC and predict early relapse. METHODOLOGY: HC was evaluated using custom PCR systems for the specific detection of the Y-chromosome, null alleles and insertion-deletion polymorphisms. A total of 281 samples from 28 adult patients who underwent an allo-SCT were studied. Increasing mixed chimerism was detected prior to relapse in 100% of patients (18 relapses). RESULTS: Compared with conventional qPCR amplification, dPCR predicted relapse with a median anticipation period of 63 days versus 45.5 days by qPCR. Overall, 56% of the relapses were predicted earlier with dPCR whereas 38% of the relapses where detected simultaneously using both techniques and only in 1 case, relapse was predicted earlier with qPCR. CONCLUSIONS: In conclusion, chimerism determination by dPCR constitutes a suitable technique for the follow-up of patients with haematological pathologies after allo-STC, showing greater sensitivity to predict an early relapse.


Assuntos
Neoplasias Hematológicas/genética , Transplante de Células-Tronco Hematopoéticas , Mutação INDEL , Polimorfismo Genético , Reação em Cadeia da Polimerase em Tempo Real , Quimeras de Transplante/genética , Adulto , Feminino , Neoplasias Hematológicas/terapia , Humanos , Masculino , Pessoa de Meia-Idade , Recidiva , Transplante Homólogo
20.
Nat Commun ; 10(1): 616, 2019 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-30728353

RESUMO

Hematopoietic chimerism after allogeneic bone marrow transplantation may establish a state of donor antigen-specific tolerance. However, current allotransplantation protocols involve genotoxic conditioning which has harmful side-effects and predisposes to infection and cancer. Here we describe a non-genotoxic conditioning protocol for fully MHC-mismatched bone marrow allotransplantation in mice involving transient immunosuppression and selective depletion of recipient hematopoietic stem cells with a CD117-antibody-drug-conjugate (ADC). This protocol resulted in multilineage, high level (up to 50%), durable, donor-derived hematopoietic chimerism after transplantation of 20 million total bone marrow cells, compared with ≤ 2.1% hematopoietic chimerism from 50 million total bone marrow cells without conditioning. Moreover, long-term survival of bone marrow donor-type but not third party skin allografts is achieved in CD117-ADC-conditioned chimeric mice without chronic immunosuppression. The only observed adverse event is transient elevation of liver enzymes in the first week after conditioning. These results provide proof-of-principle for CD117-ADC as a non-genotoxic, highly-targeted conditioning agent in allotransplantation and tolerance protocols.


Assuntos
Transplante de Medula Óssea/métodos , Sobrevivência de Enxerto , Transplante de Células-Tronco Hematopoéticas/métodos , Imunoconjugados/farmacologia , Proteínas Proto-Oncogênicas c-kit/imunologia , Tolerância ao Transplante/efeitos dos fármacos , Animais , Células da Medula Óssea/efeitos dos fármacos , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/prevenção & controle , Células-Tronco Hematopoéticas , Tolerância Imunológica , Imunossupressão/métodos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Modelos Animais , Pele/patologia , Transplante de Pele/métodos , Quimeras de Transplante , Transplante Homólogo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA