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1.
Cell ; 183(5): 1264-1281.e20, 2020 11 25.
Artigo em Inglês | MEDLINE | ID: mdl-33091337

RESUMO

The HLA-DR15 haplotype is the strongest genetic risk factor for multiple sclerosis (MS), but our understanding of how it contributes to MS is limited. Because autoreactive CD4+ T cells and B cells as antigen-presenting cells are involved in MS pathogenesis, we characterized the immunopeptidomes of the two HLA-DR15 allomorphs DR2a and DR2b of human primary B cells and monocytes, thymus, and MS brain tissue. Self-peptides from HLA-DR molecules, particularly from DR2a and DR2b themselves, are abundant on B cells and thymic antigen-presenting cells. Furthermore, we identified autoreactive CD4+ T cell clones that can cross-react with HLA-DR-derived self-peptides (HLA-DR-SPs), peptides from MS-associated foreign agents (Epstein-Barr virus and Akkermansia muciniphila), and autoantigens presented by DR2a and DR2b. Thus, both HLA-DR15 allomorphs jointly shape an autoreactive T cell repertoire by serving as antigen-presenting structures and epitope sources and by presenting the same foreign peptides and autoantigens to autoreactive CD4+ T cells in MS.


Assuntos
Subtipos Sorológicos de HLA-DR/imunologia , Esclerose Múltipla/imunologia , Linfócitos T/imunologia , Adulto , Idoso , Alelos , Antígenos/imunologia , Linfócitos B/imunologia , Linfócitos T CD4-Positivos/imunologia , Células Cultivadas , Reações Cruzadas/imunologia , Feminino , Humanos , Memória Imunológica , Masculino , Pessoa de Meia-Idade , Monócitos/imunologia , Peptídeos/imunologia , Proteoma/metabolismo , Adulto Jovem
2.
Proc Natl Acad Sci U S A ; 117(11): 6189-6195, 2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-32123116

RESUMO

Neurofibromatosis 1 (NF1) is caused by mutations in the NF1 gene, which encodes the protein, neurofibromin, an inhibitor of Ras activity. Cortical GABAergic interneurons (CINs) are implicated in NF1 pathology, but the cellular and molecular changes to CINs are unknown. We deleted mouse Nf1 from the medial ganglionic eminence, which gives rise to both oligodendrocytes and CINs that express somatostatin and parvalbumin. Nf1 loss led to a persistence of immature oligodendrocytes that prevented later-generated oligodendrocytes from occupying the cortex. Moreover, molecular and cellular properties of parvalbumin (PV)-positive CINs were altered by the loss of Nf1, without changes in somatostatin (SST)-positive CINs. We discovered that loss of Nf1 results in a dose-dependent decrease in Lhx6 expression, the transcription factor necessary to establish SST+ and PV+ CINs, which was rescued by the MEK inhibitor SL327, revealing a mechanism whereby a neurofibromin/Ras/MEK pathway regulates a critical CIN developmental milestone.


Assuntos
Córtex Cerebral/patologia , Neurônios GABAérgicos/patologia , Interneurônios/patologia , Proteínas com Homeodomínio LIM/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurofibromatose 1/patologia , Neurofibromina 1/genética , Fatores de Transcrição/metabolismo , Aminoacetonitrila/administração & dosagem , Aminoacetonitrila/análogos & derivados , Animais , Células Cultivadas , Córtex Cerebral/citologia , Modelos Animais de Doenças , Embrião de Mamíferos , Feminino , Neurônios GABAérgicos/metabolismo , Humanos , Interneurônios/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Eminência Mediana/citologia , Camundongos , Camundongos Knockout , Neurofibromatose 1/genética , Neurofibromina 1/metabolismo , Neuroglia/citologia , Parvalbuminas/metabolismo , Cultura Primária de Células , Somatostatina/metabolismo , Proteínas Ativadoras de ras GTPase/metabolismo
4.
Nucleic Acids Res ; 33(20): e179, 2005 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-16314309

RESUMO

A novel microRNA (miRNA) quantification method has been developed using stem-loop RT followed by TaqMan PCR analysis. Stem-loop RT primers are better than conventional ones in terms of RT efficiency and specificity. TaqMan miRNA assays are specific for mature miRNAs and discriminate among related miRNAs that differ by as little as one nucleotide. Furthermore, they are not affected by genomic DNA contamination. Precise quantification is achieved routinely with as little as 25 pg of total RNA for most miRNAs. In fact, the high sensitivity, specificity and precision of this method allows for direct analysis of a single cell without nucleic acid purification. Like standard TaqMan gene expression assays, TaqMan miRNA assays exhibit a dynamic range of seven orders of magnitude. Quantification of five miRNAs in seven mouse tissues showed variation from less than 10 to more than 30,000 copies per cell. This method enables fast, accurate and sensitive miRNA expression profiling and can identify and monitor potential biomarkers specific to tissues or diseases. Stem-loop RT-PCR can be used for the quantification of other small RNA molecules such as short interfering RNAs (siRNAs). Furthermore, the concept of stem-loop RT primer design could be applied in small RNA cloning and multiplex assays for better specificity and efficiency.


Assuntos
MicroRNAs/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Animais , Linhagem Celular , Primers do DNA/química , Humanos , Camundongos , MicroRNAs/metabolismo , Conformação de Ácido Nucleico , Precursores de RNA/análise
5.
Arterioscler Thromb Vasc Biol ; 24(9): 1676-81, 2004 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-15242861

RESUMO

OBJECTIVE: Mitogen-activated protein kinase phosphatase-1 (MKP-1) is one of several oxidized-l-alpha-1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (Ox-PAPC)-induced genes identified in human aortic endothelial cells (HAEC). We previously reported that MKP-1 activity is required for Ox-PAPC-mediated endothelial/monocyte interactions; however, an in vivo role of MKP-1 in atherogenesis has not been investigated. METHODS AND RESULTS: We now report that MKP-1 protein is expressed in the atherosclerotic lesions of mice. MKP-1 mRNA expression is highly induced in C57BL6/J mice on an atherogenic diet, low-density lipoprotein receptor (LDLR) (-/-) mice on a Western diet, and 10-week or older ApoE (-/-) mice on a chow diet. In ApoE (-/-) mice treated with 1 mg/mL of sodium orthovanadate (NaOV), a specific inhibitor of tyrosine phosphatases including MKP-1, total phosphatase activity and MKP-1 protein were decreased in both the aortic lesions and liver lysates. In 3 animal models of atherosclerosis [C57BL6/J mice on an atherogenic diet for 15 weeks, LDLR (-/-) mice on a Western diet for 10 weeks, and ApoE (-/-) mice on a chow diet for 8 weeks], mice treated with NaOV had significantly smaller atherosclerotic lesions when compared with the control group. CONCLUSIONS: MKP-1 expression is associated with hypercholesterolemia and atherosclerosis, and inhibition of MKP-1 activity may prevent atherosclerotic lesion development in mice. MKP-1 is required for Ox-PAPC-mediated endothelial/monocyte interactions; however, an in vivo role of MKP-1 in atherogenesis has not been investigated. We now report that MKP-1 protein is expressed in the atherosclerotic lesions of mice and inhibition of tyrosine phosphatase activity and MKP-1 protein reduce atherosclerotic lesions in mouse models.


Assuntos
Arteriosclerose/enzimologia , Proteínas de Ciclo Celular/fisiologia , Proteínas Imediatamente Precoces/fisiologia , Fosfoproteínas Fosfatases/fisiologia , Proteínas Tirosina Fosfatases/fisiologia , Fatores Etários , Animais , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Doenças da Aorta/enzimologia , Doenças da Aorta/etiologia , Doenças da Aorta/patologia , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Proteínas de Ciclo Celular/biossíntese , Proteínas de Ciclo Celular/genética , Dieta Aterogênica , Dimiristoilfosfatidilcolina/farmacologia , Fosfatase 1 de Especificidade Dupla , Indução Enzimática/efeitos dos fármacos , Proteínas Imediatamente Precoces/biossíntese , Proteínas Imediatamente Precoces/genética , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Fígado/metabolismo , MAP Quinase Quinase 4 , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Modelos Animais , Fosfoproteínas Fosfatases/biossíntese , Fosfoproteínas Fosfatases/genética , Fosforilação , Proteína Fosfatase 1 , Processamento de Proteína Pós-Traducional , Proteínas Tirosina Fosfatases/biossíntese , Proteínas Tirosina Fosfatases/genética , RNA Mensageiro/genética , Receptores de LDL/deficiência , Receptores de LDL/genética , Fatores Sexuais , Vanadatos/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
6.
Mol Imaging Biol ; 5(4): 248-56, 2003.
Artigo em Inglês | MEDLINE | ID: mdl-14499140

RESUMO

The cyclooxygenase-2 (COX-2) gene plays a role in a wide variety of normal physiologic pathways and is a major target of pharmacologic intervention in a large number of pathophysiologic contexts, including pain, fever, inflammation, and cancer. Expression of the COX-2 gene is induced in a wide range of cells, in response to an ever-increasing number of stimuli. The regulation of the COX-2 gene has been the subject of extensive study, using traditional transfection techniques with reporter gene constructs. Regulation of the COX-2 gene in living animals, however, requires sacrifice of the animal and in situ hybridization and/or immunohistochemical studies. We have utilized in vivo optical imaging technology with a cooled charged coupled device camera to image the expression of the firefly luciferase gene in tumor xenografts that are stably transfected with a chimeric gene containing the first kilobase of the murine COX-2 promoter. Induction of luciferase gene expression following systemic lipopolysaccharide/endotoxin administration can be robustly demonstrated; both a dose-response relationship and a time course for luciferase expression from the COX-2 promoter can be noninvasively analyzed in the tumor xenografts. These data suggest expression from the COX-2 promoter will be easily analyzed in transgenic mice, in knock-in mice, and in somatic cell and gene transfer experiments.


Assuntos
Diagnóstico por Imagem , Perfilação da Expressão Gênica , Expressão Gênica/fisiologia , Isoenzimas/genética , Prostaglandina-Endoperóxido Sintases/genética , Animais , Ciclo-Oxigenase 2 , Genes Reporter , Glioma/metabolismo , Isoenzimas/biossíntese , Camundongos , Células NIH 3T3 , Regiões Promotoras Genéticas , Prostaglandina-Endoperóxido Sintases/biossíntese , Ratos
7.
J Immunol ; 177(8): 5328-36, 2006 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-17015718

RESUMO

Galectin-1 kills immature thymocytes and activated peripheral T cells by binding to glycans on T cell glycoproteins including CD7, CD45, and CD43. Although roles for CD7 and CD45 in regulating galectin-1-induced death have been described, the requirement for CD43 remains unknown. We describe a novel role for CD43 in galectin-1-induced death, and the effects of O-glycan modification on galectin-1 binding to CD43. Loss of CD43 expression reduced galectin-1 death of murine thymocytes and human T lymphoblastoid cells, indicating that CD43 is required for maximal T cell susceptibility to galectin-1. CD43, which is heavily O-glycosylated, contributes a significant fraction of galectin-1 binding sites on T cells, as T cells lacking CD43 bound approximately 50% less galectin-1 than T cells expressing CD43. Although core 2 modification of O-glycans on other glycoprotein receptors is critical for galectin-1-induced cross-linking and T cell death, galectin-1 bound to CD43 fusion proteins modified with either unbranched core 1 or branched core 2 O-glycans and expression of core 2 O-glycans did not enhance galectin-1 binding to CD43 on T cells. Moreover, galectin-1 binding clustered CD43 modified with either core 1 or core 2 O-glycans on the T cell surface. Thus, CD43 bearing either core 1 or core 2 O-glycans can positively regulate T cell susceptibility to galectin-1, identifying a novel function for CD43 in controlling cell death. In addition, these studies demonstrate that different T cell glycoproteins on the same cell have distinct requirements for glycan modifications that allow recognition and cross-linking by galectin-1.


Assuntos
Galectina 1/metabolismo , Leucossialina/metabolismo , Linfócitos T/citologia , Animais , Morte Celular/imunologia , Glicosilação , Humanos , Leucossialina/química , Ativação Linfocitária , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Camundongos , Polissacarídeos/química , Polissacarídeos/metabolismo , Ligação Proteica/imunologia , Relação Estrutura-Atividade , Linfócitos T/imunologia , Timo/citologia
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