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1.
Cell ; 171(5): 1057-1071.e11, 2017 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-29033131

RESUMO

Type I interferon restrains interleukin-1ß (IL-1ß)-driven inflammation in macrophages by upregulating cholesterol-25-hydroxylase (Ch25h) and repressing SREBP transcription factors. However, the molecular links between lipid metabolism and IL-1ß production remain obscure. Here, we demonstrate that production of 25-hydroxycholesterol (25-HC) by macrophages is required to prevent inflammasome activation by the DNA sensor protein absent in melanoma 2 (AIM2). We find that in response to bacterial infection or lipopolysaccharide (LPS) stimulation, macrophages upregulate Ch25h to maintain repression of SREBP2 activation and cholesterol synthesis. Increasing macrophage cholesterol content is sufficient to trigger IL-1ß release in a crystal-independent but AIM2-dependent manner. Ch25h deficiency results in cholesterol-dependent reduced mitochondrial respiratory capacity and release of mitochondrial DNA into the cytosol. AIM2 deficiency rescues the increased inflammasome activity observed in Ch25h-/-. Therefore, activated macrophages utilize 25-HC in an anti-inflammatory circuit that maintains mitochondrial integrity and prevents spurious AIM2 inflammasome activation.


Assuntos
Colesterol/metabolismo , Inflamassomos/metabolismo , Macrófagos/metabolismo , Animais , Colesterol/biossíntese , DNA Mitocondrial/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Hidroxicolesteróis/metabolismo , Inflamassomos/imunologia , Inflamação/imunologia , Inflamação/patologia , Interleucina-1beta/imunologia , Interleucina-1beta/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Listeria monocytogenes/fisiologia , Listeriose/imunologia , Macrófagos/citologia , Macrófagos/imunologia , Proteínas de Membrana/metabolismo , Camundongos , Mitocôndrias/metabolismo , Oxisteróis/metabolismo
2.
Annu Rev Biochem ; 82: 1-24, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23472605

RESUMO

Chris Raetz passed away on August 16, 2011, still at the height of his productive years. His seminal contributions to biomedical research were in the genetics, biochemistry, and structural biology of phospholipid and lipid A biosynthesis in Escherichia coli and other gram-negative bacteria. He defined the catalytic properties and structures of many of the enzymes responsible for the "Raetz pathway for lipid A biosynthesis." His deep understanding of chemistry, coupled with knowledge of medicine, biochemistry, genetics, and structural biology, formed the underpinnings for his contributions to the lipid field. He displayed an intense passion for science and a broad interest that came from a strong commitment to curiosity-driven research, a commitment he imparted to his mentees and colleagues. What follows is a testament to both Chris's science and humanity from his friends and colleagues.


Assuntos
Pesquisa Biomédica/história , Biologia Molecular/história , Idoso , Alemanha , História do Século XX , História do Século XXI , Humanos , Masculino , Estados Unidos
3.
Cell ; 151(1): 138-52, 2012 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-23021221

RESUMO

Inflammation and macrophage foam cells are characteristic features of atherosclerotic lesions, but the mechanisms linking cholesterol accumulation to inflammation and LXR-dependent response pathways are poorly understood. To investigate this relationship, we utilized lipidomic and transcriptomic methods to evaluate the effect of diet and LDL receptor genotype on macrophage foam cell formation within the peritoneal cavities of mice. Foam cell formation was associated with significant changes in hundreds of lipid species and unexpected suppression, rather than activation, of inflammatory gene expression. We provide evidence that regulated accumulation of desmosterol underlies many of the homeostatic responses, including activation of LXR target genes, inhibition of SREBP target genes, selective reprogramming of fatty acid metabolism, and suppression of inflammatory-response genes, observed in macrophage foam cells. These observations suggest that macrophage activation in atherosclerotic lesions results from extrinsic, proinflammatory signals generated within the artery wall that suppress homeostatic and anti-inflammatory functions of desmosterol.


Assuntos
Aterosclerose/imunologia , Colesterol/biossíntese , Desmosterol/metabolismo , Células Espumosas/metabolismo , Metabolismo dos Lipídeos , Transcriptoma , Animais , Aterosclerose/metabolismo , Colesterol/análogos & derivados , Colesterol/metabolismo , Ácidos Graxos/metabolismo , Células Espumosas/imunologia , Técnicas de Silenciamento de Genes , Leucócitos Mononucleares/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptores de LDL/genética , Receptores de LDL/metabolismo , Proteínas de Ligação a Elemento Regulador de Esterol/metabolismo
4.
Cell ; 142(2): 196-8, 2010 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-20655462

RESUMO

The steroid 5alpha-reductase (SRD5A) family of enzymes produces steroid hormones that regulate male sexual development. Now, Cantagrel et al. (2010) identify a member of this family, SRD5A3, as a polyprenol reductase with a crucial role in N-linked protein glycosylation and pinpoint SRD5A3 mutations as the cause of a rare Mendelian disease.

5.
Annu Rev Biochem ; 78: 1017-40, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19489738

RESUMO

Cholesterol 24-hydroxylase is a highly conserved cytochrome P450 that is responsible for the majority of cholesterol turnover in the vertebrate central nervous system. The enzyme is expressed in neurons, including hippocampal and cortical neurons that are important for learning and memory formation. Disruption of the cholesterol 24-hydroxylase gene in the mouse reduces both cholesterol turnover and synthesis in the brain but does not alter steady-state levels of cholesterol in the tissue. The decline in synthesis reduces the flow of metabolites through the cholesterol biosynthetic pathway, of which one, geranylgeraniol diphosphate, is required for learning in the whole animal and for synaptic plasticity in vitro. This review focuses on how the link between cholesterol metabolism and higher-order brain function was experimentally established.


Assuntos
Encéfalo/metabolismo , Colesterol/metabolismo , Esteroide Hidroxilases/metabolismo , Animais , Encéfalo/citologia , Colesterol 24-Hidroxilase , Regulação Enzimológica da Expressão Gênica , Hipocampo/metabolismo , Humanos , Aprendizagem , Neurônios/metabolismo , Esteroide Hidroxilases/química , Esteroide Hidroxilases/genética
6.
Immunity ; 43(6): 1125-36, 2015 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-26682985

RESUMO

The interleukin-1 receptor I (IL-1RI) is critical for host resistance to Mycobacterium tuberculosis (Mtb), yet the mechanisms of IL-1RI-mediated pathogen control remain unclear. Here, we show that without IL-1RI, Mtb-infected newly recruited Ly6G(hi) myeloid cells failed to upregulate tumor necrosis factor receptor I (TNF-RI) and to produce reactive oxygen species, resulting in compromised pathogen control. Furthermore, simultaneous ablation of IL-1RI and TNF-RI signaling on either stroma or hematopoietic cells led to early lethality, indicating non-redundant and synergistic roles of IL-1 and TNF in mediating macrophage-stroma cross-talk that was critical for optimal control of Mtb infection. Finally, we show that even in the presence of functional Mtb-specific adaptive immunity, the lack of IL-1α and not IL-1ß led to an exuberant intracellular pathogen replication and progressive non-resolving inflammation. Our study reveals functional interdependence between IL-1 and TNF in enabling Mtb control mechanisms that are critical for host survival.


Assuntos
Interleucina-1alfa/imunologia , Tuberculose/imunologia , Fator de Necrose Tumoral alfa/imunologia , Animais , Separação Celular , Modelos Animais de Doenças , Citometria de Fluxo , Imunofluorescência , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mycobacterium tuberculosis , Receptores Tipo I de Interleucina-1/imunologia
7.
Mol Ther ; 29(2): 680-690, 2021 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-33554867

RESUMO

Adeno-associated virus (AAV) integrates into host genomes at low frequency, but when integration occurs in oncogenic hotspots it can cause hepatocellular carcinoma (HCC). Given the possibility of recombinant AAV (rAAV) integration leading to HCC, common causes of liver inflammation like non-alcoholic fatty liver disease (NAFLD) may increase the risk of rAAV-induced HCC. A rAAV targeting the oncogenic mouse Rian locus was used, and as expected led to HCC in all mice infected as neonates, likely due to growth-related hepatocyte proliferation in young mice. Mice infected with rAAV as adults did not develop HCC unless they were fed a diet leading to NAFLD, with increased inflammation and hepatocyte proliferation. Female mice were less susceptible to rAAV-induced HCC, and male mice with NAFLD treated with estrogen exhibited less inflammation and immune exhaustion associated with oncogenesis compared to those without estrogen. Adult NAFLD mice infected with a non-targeted control rAAV also developed HCC, though only half as frequently as those exposed to the Rian targeted rAAV. This study shows that adult mice exposed to rAAV gene therapy in the context of chronic liver disease developed HCC at high frequency, and thus warrants further study in humans given the high prevalence of NAFLD in the population.


Assuntos
Carcinoma Hepatocelular/etiologia , Dependovirus/genética , Terapia Genética/efeitos adversos , Vetores Genéticos/genética , Hepatopatias/complicações , Hepatopatias/etiologia , Neoplasias Hepáticas/etiologia , Animais , Carcinoma Hepatocelular/diagnóstico , Modelos Animais de Doenças , Terapia Genética/métodos , Incidência , Hepatopatias/patologia , Neoplasias Hepáticas/diagnóstico , Camundongos
8.
Immunity ; 37(3): 535-48, 2012 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-22999953

RESUMO

7α,25-dihydroxycholesterol (7α,25-OHC) is a ligand for the G protein-coupled receptor EBI2; however, the cellular sources of this oxysterol are undefined. 7α,25-OHC is synthesized from cholesterol by the stepwise actions of two enzymes, CH25H and CYP7B1, and is metabolized to a 3-oxo derivative by HSD3B7. We showed that all three enzymes control EBI2 ligand concentration in lymphoid tissues. Lymphoid stromal cells were the main CH25H- and CYP7B1-expressing cells required for positioning of B cells, and they also mediated 7α,25-OHC inactivation. CH25H and CYP7B1 were abundant at the follicle perimeter, whereas CH25H expression by follicular dendritic cells was repressed. CYP7B1, CH25H, and HSD3B7 deficiencies each resulted in defective T cell-dependent plasma cell responses. These findings establish that CYP7B1 and HSD3B7, as well as CH25H, have essential roles in controlling oxysterol production in lymphoid tissues, and they suggest that differential enzyme expression in stromal cell subsets establishes 7α,25-OHC gradients required for B cell responses.


Assuntos
Linfócitos B/imunologia , Movimento Celular/imunologia , Hidroxicolesteróis/imunologia , Imunidade Humoral/imunologia , Células Estromais/imunologia , 3-Hidroxiesteroide Desidrogenases/genética , 3-Hidroxiesteroide Desidrogenases/imunologia , 3-Hidroxiesteroide Desidrogenases/metabolismo , Animais , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Células Cultivadas , Família 7 do Citocromo P450 , Feminino , Citometria de Fluxo , Expressão Gênica , Células HEK293 , Humanos , Hidroxicolesteróis/metabolismo , Ativação Linfocitária/imunologia , Tecido Linfoide/imunologia , Tecido Linfoide/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/imunologia , Receptores Acoplados a Proteínas G/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Esteroide Hidroxilases/genética , Esteroide Hidroxilases/imunologia , Esteroide Hidroxilases/metabolismo , Células Estromais/metabolismo
9.
J Biol Chem ; 293(49): 18804-18827, 2018 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-30530852

RESUMO

On January 21, 2017, I received an E-mail from Herb Tabor that I had been simultaneously hoping for and dreading for several years: an invitation to write a "Reflections" article for the Journal of Biological Chemistry On the one hand, I was honored to receive an invitation from Herb, a man I have admired for over 40 years, known for 24 years, and worked with as a member of the Editorial Board and Associate Editor of the Journal of Biological Chemistry for 17 years. On the other hand, the invitation marked the waning of my career as an academic scientist. With these conflicting emotions, I wrote this article with the goals of recording my career history and recognizing the many mentors, trainees, and colleagues who have contributed to it and, perhaps with pretension, with the desire that students who are beginning a career in research will find inspiration in the path I have taken and appreciate the importance of luck.


Assuntos
Biologia Molecular/história , Animais , História do Século XX , História do Século XXI , Humanos , Texas
10.
Mol Ther ; 26(5): 1255-1265, 2018 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-29606506

RESUMO

X-linked severe combined immunodeficiency (X-SCID) has been successfully treated by hematopoietic stem cell (HSC) transduction with retroviral vectors expressing the interleukin-2 receptor subunit gamma gene (IL2RG), but several patients developed malignancies due to vector integration near cellular oncogenes. This adverse side effect could in principle be avoided by accurate IL2RG gene editing with a vector that does not contain a functional promoter or IL2RG gene. Here, we show that adeno-associated virus (AAV) gene editing vectors can insert a partial Il2rg cDNA at the endogenous Il2rg locus in X-SCID murine bone marrow cells and that these ex vivo-edited cells repopulate transplant recipients and produce CD4+ and CD8+ T cells. Circulating, edited lymphocytes increased over time and appeared in secondary transplant recipients, demonstrating successful editing in long-term repopulating cells. Random vector integration events were nearly undetectable, and malignant transformation of the transplanted cells was not observed. Similar editing frequencies were observed in human hematopoietic cells. Our results demonstrate that therapeutically relevant HSC gene editing can be achieved by AAV vectors in the absence of site-specific nucleases and suggest that this may be a safe and effective therapy for hematopoietic diseases where in vivo selection can increase edited cell numbers.


Assuntos
Dependovirus/genética , Edição de Genes , Vetores Genéticos/genética , Subunidade gama Comum de Receptores de Interleucina/genética , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/genética , Alelos , Animais , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Ordem dos Genes , Terapia Genética , Células-Tronco Hematopoéticas/metabolismo , Humanos , Imunoterapia Adotiva , Camundongos , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/imunologia , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/terapia
12.
Mol Ther ; 24(3): 582-91, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26444081

RESUMO

Many applications of pluripotent stem cells (PSCs) require efficient editing of silent chromosomal genes. Here, we show that a major limitation in isolating edited clones is silencing of the selectable marker cassette after homologous recombination and that this can be overcome by using a ubiquitous chromatin opening element (UCOE) promoter-driven transgene. We use this strategy to edit the silent IL2RG locus in human PSCs with a recombinant adeno-associated virus (rAAV)-targeting vector in the absence of potentially genotoxic, site-specific nucleases and show that IL2RG is required for natural killer and T-cell differentiation of human PSCs. Insertion of an active UCOE promoter into a silent locus altered the histone modification and cytosine methylation pattern of surrounding chromatin, but these changes resolved when the UCOE promoter was removed. This same approach could be used to correct IL2RG mutations in X-linked severe combined immunodeficiency patient-derived induced PSCs (iPSCs), to prevent graft versus host disease in regenerative medicine applications, or to edit other silent genes.


Assuntos
Edição de Genes , Inativação Gênica , Subunidade gama Comum de Receptores de Interleucina/genética , Células-Tronco Pluripotentes/metabolismo , Diferenciação Celular , Sobrevivência Celular/genética , Colágeno Tipo I/genética , Cadeia alfa 1 do Colágeno Tipo I , Epigênese Genética , Técnicas de Inativação de Genes , Marcação de Genes , Loci Gênicos , Humanos , Células Matadoras Naturais/citologia , Células-Tronco Pluripotentes/citologia , Regiões Promotoras Genéticas , Subpopulações de Linfócitos T/citologia , Transgenes , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/genética
13.
Proc Natl Acad Sci U S A ; 111(38): E4006-14, 2014 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-25201972

RESUMO

An unknown fraction of the genome participates in the metabolism of sterols and vitamin D, two classes of lipids with diverse physiological and pathophysiological roles. Here, we used mass spectrometry to measure the abundance of >60 sterol and vitamin D derivatives in 3,230 serum samples from a well-phenotyped patient population. Twenty-nine of these lipids were detected in a majority of samples at levels that varied over thousands of fold in different individuals. Pairwise correlations between sterol and vitamin D levels revealed evidence for shared metabolic pathways, additional substrates for known enzymes, and transcriptional regulatory networks. Serum levels of multiple sterols and vitamin D metabolites varied significantly by sex, ethnicity, and age. A genome-wide association study identified 16 loci that were associated with levels of 19 sterols and 25-hydroxylated derivatives of vitamin D (P < 10(-7)). Resequencing, expression analysis, and biochemical experiments focused on one such locus (CYP39A1), revealed multiple loss-of-function alleles with additive effects on serum levels of the oxysterol, 24S-hydroxycholesterol, a substrate of the encoded enzyme. Body mass index, serum lipid levels, and hematocrit were strong phenotypic correlates of interindividual variation in multiple sterols and vitamin D metabolites. We conclude that correlating population-based analytical measurements with genotype and phenotype provides productive insight into human intermediary metabolism.


Assuntos
Índice de Massa Corporal , Loci Gênicos/fisiologia , Genótipo , Hidroxicolesteróis/sangue , Esteroide Hidroxilases , Vitamina D/sangue , Feminino , Humanos , Masculino , Esteroide Hidroxilases/genética , Esteroide Hidroxilases/metabolismo , Vitamina D/genética
14.
Nucleic Acids Res ; 42(5): 3119-24, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24371280

RESUMO

DNA mismatches that occur between vector homology arms and chromosomal target sequences reduce gene targeting frequencies in several species; however, this has not been reported in human cells. Here we demonstrate that even a single mismatched base pair can significantly decrease human gene targeting frequencies. In addition, we show that homology arm polymorphisms can be used to direct allele-specific targeting or to improve unfavorable vector designs that introduce deletions.


Assuntos
Marcação de Genes , Polimorfismo Genético , Pareamento Incorreto de Bases , Linhagem Celular Tumoral , Cromossomos/química , Loci Gênicos , Vetores Genéticos/química , Humanos , Polimorfismo de Nucleotídeo Único
15.
J Lipid Res ; 56(3): 722-736, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25598080

RESUMO

The spectrum of nonalcoholic fatty liver disease (NAFLD) includes steatosis, nonalcoholic steatohepatitis (NASH), and cirrhosis. Recognition and timely diagnosis of these different stages, particularly NASH, is important for both potential reversibility and limitation of complications. Liver biopsy remains the clinical standard for definitive diagnosis. Diagnostic tools minimizing the need for invasive procedures or that add information to histologic data are important in novel management strategies for the growing epidemic of NAFLD. We describe an "omics" approach to detecting a reproducible signature of lipid metabolites, aqueous intracellular metabolites, SNPs, and mRNA transcripts in a double-blinded study of patients with different stages of NAFLD that involves profiling liver biopsies, plasma, and urine samples. Using linear discriminant analysis, a panel of 20 plasma metabolites that includes glycerophospholipids, sphingolipids, sterols, and various aqueous small molecular weight components involved in cellular metabolic pathways, can be used to differentiate between NASH and steatosis. This identification of differential biomolecular signatures has the potential to improve clinical diagnosis and facilitate therapeutic intervention of NAFLD.


Assuntos
Lipídeos/sangue , Lipídeos/urina , Hepatopatia Gordurosa não Alcoólica , Polimorfismo de Nucleotídeo Único , Adulto , Biomarcadores/metabolismo , Biomarcadores/urina , Método Duplo-Cego , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Hepatopatia Gordurosa não Alcoólica/sangue , Hepatopatia Gordurosa não Alcoólica/epidemiologia , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/urina
16.
Proc Natl Acad Sci U S A ; 109(28): 11264-9, 2012 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-22733778

RESUMO

The distinct phenotypic and prognostic subclasses of human hepatocellular carcinoma (HCC) are difficult to reproduce in animal experiments. Here we have used in vivo gene targeting to insert an enhancer-promoter element at an imprinted chromosome 12 locus in mice, thereby converting ∼1 in 20,000 normal hepatocytes into a focus of HCC with a single genetic modification. A 300-kb chromosomal domain containing multiple mRNAs, snoRNAs, and microRNAs was activated surrounding the integration site. An identical domain was activated at the syntenic locus in a specific molecular subclass of spontaneous human HCCs with a similar histological phenotype, which was associated with partial loss of DNA methylation. These findings demonstrate the accuracy of in vivo gene targeting in modeling human cancer and suggest future applications in studying various tumors in diverse animal species. In addition, similar insertion events produced by randomly integrating vectors could be a concern for liver-directed human gene therapy.


Assuntos
Carcinoma Hepatocelular/etiologia , Carcinoma Hepatocelular/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/etiologia , Neoplasias Hepáticas/genética , Animais , Metilação de DNA , Dependovirus/metabolismo , Terapia Genética/métodos , Hepatócitos/citologia , Humanos , Íntrons , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Genéticos , Mutagênicos , Fenótipo , Prognóstico , RNA Mensageiro/metabolismo , Transcrição Gênica
17.
J Biol Chem ; 288(50): 35812-23, 2013 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-24189069

RESUMO

25-Hydroxycholesterol (25OHC) is an enzymatically derived oxidation product of cholesterol that modulates lipid metabolism and immunity. 25OHC is synthesized in response to interferons and exerts broad antiviral activity by as yet poorly characterized mechanisms. To gain further insights into the basis for antiviral activity, we evaluated time-dependent responses of the macrophage lipidome and transcriptome to 25OHC treatment. In addition to altering specific aspects of cholesterol and sphingolipid metabolism, we found that 25OHC activates integrated stress response (ISR) genes and reprograms protein translation. Effects of 25OHC on ISR gene expression were independent of liver X receptors and sterol-response element-binding proteins and instead primarily resulted from activation of the GCN2/eIF2α/ATF4 branch of the ISR pathway. These studies reveal that 25OHC activates the integrated stress response, which may contribute to its antiviral activity.


Assuntos
Hidroxicolesteróis/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Animais , Células da Medula Óssea/citologia , Ésteres do Colesterol/metabolismo , Perfilação da Expressão Gênica , Hidroxicolesteróis/metabolismo , Receptores X do Fígado , Macrófagos/citologia , Macrófagos/virologia , Camundongos , Camundongos Endogâmicos C57BL , Muromegalovirus/fisiologia , Receptores Nucleares Órfãos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Esfingolipídeos/metabolismo , Proteínas de Ligação a Elemento Regulador de Esterol/antagonistas & inibidores
18.
Gastroenterology ; 144(5): 945-955.e6; quiz e14-5, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23415802

RESUMO

BACKGROUND & AIMS: The final step in bile acid synthesis involves conjugation with glycine and taurine, which promotes a high intraluminal micellar concentration to facilitate lipid absorption. We investigated the clinical, biochemical, molecular, and morphologic features of a genetic defect in bile acid conjugation in 10 pediatric patients with fat-soluble vitamin deficiency, some with growth failure or transient neonatal cholestatic hepatitis. METHODS: We identified the genetic defect that causes this disorder using mass spectrometry analysis of urine, bile, and serum samples and sequence analysis of the genes encoding bile acid-CoA:amino acid N-acyltransferase (BAAT) and bile acid-CoA ligase (SLC27A5). RESULTS: Levels of urinary bile acids were increased (432 ± 248 µmol/L) and predominantly excreted in unconjugated forms (79.4% ± 3.9%) and as sulfates and glucuronides. Glycine or taurine conjugates were absent in the urine, bile, and serum. Unconjugated bile acids accounted for 95.7% ± 5.8% of the bile acids in duodenal bile, with cholic acid accounting for 82.4% ± 5.5% of the total. Duodenal bile acid concentrations were 12.1 ± 5.9 mmol/L, which is too low for efficient lipid absorption. The biochemical profile was consistent with defective bile acid amidation. Molecular analysis of BAAT confirmed 4 different homozygous mutations in 8 patients tested. CONCLUSIONS: Based on a study of 10 pediatric patients, genetic defects that disrupt bile acid amidation cause fat-soluble vitamin deficiency and growth failure, indicating the importance of bile acid conjugation in lipid absorption. Some patients developed liver disease with features of a cholangiopathy. These findings indicate that patients with idiopathic neonatal cholestasis or later onset of unexplained fat-soluble vitamin deficiency should be screened for defects in bile acid conjugation.


Assuntos
Deficiência de Vitaminas/genética , Ácidos e Sais Biliares/metabolismo , Coenzima A Ligases/genética , DNA/genética , Predisposição Genética para Doença , Mutação de Sentido Incorreto , Aciltransferases/genética , Aciltransferases/metabolismo , Deficiência de Vitaminas/metabolismo , Deficiência de Vitaminas/patologia , Biópsia , Criança , Pré-Escolar , Coenzima A Ligases/metabolismo , Análise Mutacional de DNA , Proteínas de Transporte de Ácido Graxo/genética , Proteínas de Transporte de Ácido Graxo/metabolismo , Feminino , Homozigoto , Humanos , Lactente , Fígado/patologia , Masculino , Espectrometria de Massas
19.
Mol Ther ; 21(6): 1232-41, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23629003

RESUMO

The clinical use of human pluripotent stem cells and their derivatives is limited by the rejection of transplanted cells due to differences in their human leukocyte antigen (HLA) genes. This has led to the proposed use of histocompatible, patient-specific stem cells; however, the preparation of many different stem cell lines for clinical use is a daunting task. Here, we develop two distinct genetic engineering approaches that address this problem. First, we use a combination of gene targeting and mitotic recombination to derive HLA-homozygous embryonic stem cell (ESC) subclones from an HLA-heterozygous parental line. A small bank of HLA-homozygous stem cells with common haplotypes would match a significant proportion of the population. Second, we derive HLA class I-negative cells by targeted disruption of both alleles of the Beta-2 Microglobulin (B2M) gene in ESCs. Mixed leukocyte reactions and peptide-specific HLA-restricted CD8(+) T cell responses were reduced in class I-negative cells that had undergone differentiation in embryoid bodies. These B2M(-/-) ESCs could act as universal donor cells in applications where the transplanted cells do not express HLA class II genes. Both approaches used adeno-associated virus (AAV) vectors for efficient gene targeting in the absence of potentially genotoxic nucleases, and produced pluripotent, transgene-free cell lines.


Assuntos
Antígenos HLA/genética , Células-Tronco Pluripotentes/citologia , Alelos , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/metabolismo , Diferenciação Celular , Linhagem Celular , Células Cultivadas , Dependovirus/genética , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Marcação de Genes , Engenharia Genética , Vetores Genéticos , Antígenos HLA/metabolismo , Haplótipos , Histocompatibilidade/genética , Homozigoto , Humanos , Células-Tronco Pluripotentes/metabolismo , Recombinação Genética , Microglobulina beta-2/genética , Microglobulina beta-2/metabolismo
20.
Mol Ther ; 21(5): 964-72, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23531552

RESUMO

The development of leukemia following gammaretroviral vector-mediated gene therapy for X-linked severe combined immunodeficiency disease and chronic granulomatous disease (CGD) has emphasized the need for long-term follow-up in animals treated with hematopoietic stem cell gene therapy. In this study, we report the long-term follow-up (4-7 years) of four dogs with canine leukocyte adhesion deficiency (CLAD) treated with foamy viral (FV) vector-mediated gene therapy. All four CLAD dogs previously received nonmyeloablative conditioning with 200 cGy total body irradiation followed by infusion of autologous, CD34(+) hematopoietic stem cells transduced by a FV vector expressing canine CD18 from an internal Murine Stem Cell Virus (MSCV) promoter. CD18(+) leukocyte levels were >2% following infusion of vector-transduced cells leading to ongoing reversal of the CLAD phenotype for >4 years. There was no clinical development of lymphoid or myeloid leukemia in any of the four dogs and integration site analysis did not reveal insertional oncogenesis. These results showing disease correction/amelioration of disease in CLAD without significant adverse events provide support for the use of a FV vector to treat children with leukocyte adhesion deficiency type 1 (LAD-1) in a human gene therapy clinical trial.


Assuntos
Terapia Genética , Vetores Genéticos/genética , Síndrome da Aderência Leucocítica Deficitária/genética , Síndrome da Aderência Leucocítica Deficitária/terapia , Spumavirus/genética , Animais , Antígenos CD34/metabolismo , Medula Óssea , Antígenos CD18/metabolismo , Modelos Animais de Doenças , Cães , Feminino , Seguimentos , Técnicas de Transferência de Genes , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Humanos , Contagem de Leucócitos , Leucócitos/metabolismo , Masculino , Subpopulações de Linfócitos T/metabolismo , Transdução Genética , Integração Viral
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