Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 68
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Eur J Pharmacol ; 854: 398-405, 2019 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-31039344

RESUMO

Hemoglobinopathies, such as ß-thalassemia, and sickle cell disease (SCD) are caused by abnormal structure or reduced production of ß-chains and affect millions of people worldwide. Hereditary persistence of fetal hemoglobin (HPFH) is a condition which is naturally occurring and characterized by a considerable elevation of fetal hemoglobin (HbF) in adult red blood cells. Individuals with compound heterozygous ß-thalassemia or SCD and HPFH have milder clinical symptoms. So, HbF reactivation has long been sought as an approach to mitigate the clinical symptoms of ß-thalassemia and SCD. Using CRISPR-Cas9 genome-editing strategy, we deleted a 200bp genomic region within the human erythroid-specific BCL11A (B-cell lymphoma/leukemia 11A) enhancer in KU-812, KG-1, and K562 cell lines. In our study, deletion of 200bp of BCL11A erythroid enhancer including GATAA motif leads to strong induction of γ-hemoglobin expression in K562 cells, but not in KU-812 and KG-1 cells. Altogether, our findings highlight the therapeutic potential of CRISPR-Cas9 as a precision genome editing tool for treating ß-thalassemia. In addition, our data indicate that KU-812 and KG-1 cell lines are not good models for studying HbF reactivation through inactivation of BCL11A silencing pathway.


Assuntos
Sistemas CRISPR-Cas/genética , Proteínas de Transporte/genética , Hemoglobina Fetal/metabolismo , Deleção de Genes , Terapia Genética/métodos , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Talassemia beta/terapia , Sequência de Bases , Edição de Genes , Humanos , Células K562 , Proteínas Repressoras , Talassemia beta/genética , Talassemia beta/metabolismo , gama-Globinas/genética
2.
J Mol Med (Berl) ; 97(7): 889-896, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31028417

RESUMO

Bone marrow chimeras are used routinely in immunology research as well as in other fields of biology. Here, we provide a concise state-of-the-art review about the types of chimerisms that can be achieved and the type of information that each model generates. We include separate sections for caveats and future developments. We provide examples from the literature in which different types of chimerism were employed to answer specific questions. While simple bone marrow chimeras allow to dissect the role of genes in distinct cell populations such as the hematopoietic cells versus non-hematopoietic cells, mixed bone marrow chimeras can provide detailed information about hematopoietic cell types and the intrinsic and extrinsic roles of individual genes. The advantages and caveats of bone marrow chimerism for the study of microglia are addressed, as well as alternatives to irradiation that minimize blood-brain-barrier disruption. Elementary principles are introduced and their potential is exemplified through summarizing recent studies.

3.
J Mol Med (Berl) ; 97(6): 871-877, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30980104

RESUMO

Disease occurrence, clinical manifestations, and outcomes differ between men and women. Yet, women and men are most of the time treated similarly, which is often based on experimental data over-representing one sex. Accounting for persisting sex bias in biomedical research is the misconception that the analysis of sex-specific effects would double sample size and costs. We designed an analysis to test the potential benefits of a factorial study design in the context of a study including male and female animals. We chose a 2 × 2 factorial design approach to study the effect of treatment, sex, and an interaction term of treatment and sex in a hypothetical situation. We calculated the sample sizes required to detect an effect of a given magnitude with sufficient power and under different experimental setups. We demonstrated that the inclusion of both sexes in experimental setups, without testing for sex effects, requires no or few additional animals in our scenarios. These experimental designs still allow for the exploration of sex effects at low cost. In a confirmatory instead of an exploratory design, we observed an increase in total sample sizes by 33%, at most. Since the complexities associated with this mathematical model require statistical expertise, we generated and provide a sample size calculator for planning factorial design experiments. For the inclusion of sex, a factorial design is advisable, and a sex-specific analysis can be performed without excessive additional effort. Our easy-to-use calculation tool provides help in designing studies with both sexes and addresses the current sex bias in preclinical studies. KEY MESSAGES: • Both sexes should be included into animal studies. • Exploratory study of sex effects can be conducted with no or small increase in animal number. • Confirmatory analysis of sex effects requires maximum 33% more animals per study. • Our calculation tool supports the design of studies with both sexes.

4.
Cell Host Microbe ; 25(3): 389-403.e6, 2019 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-30870621

RESUMO

Commensal fungi of the mammalian skin, such as those of the genus Malassezia, are associated with atopic dermatitis and other common inflammatory skin disorders. Understanding of the causative relationship between fungal commensalism and disease manifestation remains incomplete. By developing a murine epicutaneous infection model, we found Malassezia spp. selectively induce IL-17 and related cytokines. This response is key in preventing fungal overgrowth on the skin, as disruption of the IL-23-IL-17 axis compromises Malassezia-specific cutaneous immunity. Under conditions of impaired skin integrity, mimicking a hallmark of atopic dermatitis, the presence of Malassezia dramatically aggravates cutaneous inflammation, which again was IL-23 and IL-17 dependent. Consistently, we found a CCR6+ Th17 subset of memory T cells to be Malassezia specific in both healthy individuals and atopic dermatitis patients, whereby the latter showed enhanced frequency of these cells. Thus, the Malassezia-induced type 17 response is pivotal in orchestrating antifungal immunity and in actively promoting skin inflammation.


Assuntos
Citocinas/metabolismo , Dermatite Atópica/patologia , Dermatomicoses/microbiologia , Dermatomicoses/patologia , Malassezia/imunologia , Células Th17/imunologia , Adulto , Animais , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Adulto Jovem
5.
Digestion ; 100(2): 127-138, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30423561

RESUMO

BACKGROUND/AIMS: The gut microbiota is altered in irritable bowel syndrome (IBS), and microbiota manipulations by diet or antibiotics can reduce its symptoms. As fecal microbiota transfer (FMT) in IBS is still controversial, we investigated the clinical and side effects of FMT in a cohort of IBS patients with recurrent, treatment refractory symptoms, and studied gut microbiota signatures. METHODS: Using an observational, prospective study design, we applied FMTs from one unrelated, healthy donor to 13 IBS patients. Fecal samples of patients and the donor were analyzed by 16S ribosomal RNA amplicon sequencing. RESULTS: On a symptom level, primarily abdominal pain symptoms were reduced after FMT, and no adverse effects were observed. Studying the microbiome, we found an increase in alpha diversity and changes in the composition of the gut microbiota after FMT. Beta diversity changes after FMT were prominent in a subset of 7 patients with microbiota profiles coming very close to the donor. These patients also showed most pronounced visceral pain reduction. The relative abundance of Akkermansia muciniphila was inversely correlated with pain reduction in our cohort. CONCLUSION: Although exploratory in nature and with a pilot character, this study highlights the potential role of microbiota manipulations in IBS and describes a novel association of intestinal Akkermansia and pain modulation.


Assuntos
Dor Abdominal/terapia , Transplante de Microbiota Fecal/métodos , Fezes/microbiologia , Síndrome do Intestino Irritável/terapia , Verrucomicrobia/isolamento & purificação , Dor Abdominal/diagnóstico , Dor Abdominal/etiologia , Adulto , DNA Bacteriano/isolamento & purificação , Feminino , Microbioma Gastrointestinal/genética , Humanos , Mucosa Intestinal/microbiologia , Síndrome do Intestino Irritável/complicações , Síndrome do Intestino Irritável/microbiologia , Masculino , Medição da Dor , Projetos Piloto , Estudos Prospectivos , RNA Ribossômico 16S/genética , Resultado do Tratamento , Verrucomicrobia/genética , Adulto Jovem
6.
Nature ; 564(7735): E9, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30410124

RESUMO

In this Article, the pCaMIN construct consisted of 'mouse MYC and mouse NrasG12V' instead of 'mouse Myc and human NRASG12V; and the pCAMIA construct consisted of 'mouse Myc and human AKT1' instead of 'mouse Myc and Akt1' this has been corrected online.

7.
Nature ; 562(7725): 69-75, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30209397

RESUMO

Primary liver cancer represents a major health problem. It comprises hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC), which differ markedly with regards to their morphology, metastatic potential and responses to therapy. However, the regulatory molecules and tissue context that commit transformed hepatic cells towards HCC or ICC are largely unknown. Here we show that the hepatic microenvironment epigenetically shapes lineage commitment in mosaic mouse models of liver tumorigenesis. Whereas a necroptosis-associated hepatic cytokine microenvironment determines ICC outgrowth from oncogenically transformed hepatocytes, hepatocytes containing identical oncogenic drivers give rise to HCC if they are surrounded by apoptotic hepatocytes. Epigenome and transcriptome profiling of mouse HCC and ICC singled out Tbx3 and Prdm5 as major microenvironment-dependent and epigenetically regulated lineage-commitment factors, a function that is conserved in humans. Together, our results provide insight into lineage commitment in liver tumorigenesis, and explain molecularly why common liver-damaging risk factors can lead to either HCC or ICC.


Assuntos
Apoptose , Carcinoma Hepatocelular/patologia , Linhagem da Célula , Colangiocarcinoma/patologia , Neoplasias Hepáticas/patologia , Necrose , Microambiente Tumoral , Animais , Apoptose/genética , Carcinogênese/genética , Carcinoma Hepatocelular/genética , Diferenciação Celular , Linhagem da Célula/genética , Colangiocarcinoma/genética , Inibidor p16 de Quinase Dependente de Ciclina/deficiência , Citocinas/metabolismo , Elementos de DNA Transponíveis/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Epigênese Genética/genética , Feminino , Perfilação da Expressão Gênica , Genes myc , Genes ras , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Neoplasias Hepáticas/genética , Masculino , Camundongos , Mosaicismo , Necrose/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
8.
Pediatr Allergy Immunol ; 29(8): 823-833, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30102794

RESUMO

BACKGROUND: Asthma is the most common chronic disease in children. Underlying immunologic mechanisms-in particular of different phenotypes-are still just partly understood. The objective of the study was the identification of distinct cellular pathways in allergic asthmatics (AA) and nonallergic asthmatics (NA) vs healthy controls (HC). METHODS: Peripheral blood mononuclear cells (PBMCs) of steroid-naïve children (n(AA/NA/HC) = 35/13/34)) from the CLARA study (n = 275) were stimulated (anti-CD3/CD28, LpA) or kept unstimulated. Gene expression was investigated by transcriptomics and quantitative RT-PCR. Differentially regulated pathways between phenotypes were assessed after adjustment for sex and age (KEGG pathways). Networks based on correlations of gene expression were built using force-directed graph drawing. RESULTS: Allergic asthmatics vs NA and asthmatics overall vs HC showed significantly different expression of Ca2+ and innate immunity-associated pathways. PCR analysis confirmed significantly increased Ca2+ -associated gene regulation (ORMDL3 and ATP2A3) in asthmatics vs HC, most prominent in AA. Innate immunity receptors (LY75, TLR7), relevant for virus infection, were also upregulated in AA and NA compared to HC. AA and NA could be differentiated by increased ATP2A3 and FPR2 in AA, decreased CLEC4E in AA, and increased IFIH1 expression in NA following anti-CD3/28 stimulation vs unstimulated (fold change). CONCLUSIONS: Ca2+ regulation and innate immunity response pattern to viruses were activated in PBMCs of asthmatics. Asthma phenotypes were differentially characterized by distinct regulation of ATP2A3 and expression of innate immune receptors (FPR2, CLEC4E, IFIH1). These genes may present promising targets for future in-depth investigation with the long-term goal of more phenotype-specific therapeutic interventions in asthmatics.


Assuntos
Asma/metabolismo , Cálcio/metabolismo , Imunidade Inata/genética , Leucócitos Mononucleares/metabolismo , Adolescente , Asma/imunologia , Técnicas de Cultura de Células , Criança , Pré-Escolar , Citocinas/metabolismo , Feminino , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Humanos , Masculino , Análise em Microsséries/métodos , Fenótipo , Reação em Cadeia da Polimerase em Tempo Real/métodos , Transdução de Sinais
10.
Am J Gastroenterol ; 113(10): 1475-1483, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29535416

RESUMO

OBJECTIVES: Variants in patatin-like phospholipase domain-containing 3 (PNPLA3; rs738409), transmembrane 6 superfamily member 2 (TM6SF2; rs58542926), and membrane bound O-acyltransferase domain containing 7 (MBOAT7; rs641738) are risk factors for the development of alcohol-related cirrhosis. Within this population, PNPLA3 rs738409 is also an established risk factor for the development of hepatocellular carcinoma (HCC). The aim of this study was to explore possible risk associations of TM6SF2 rs58542926 and MBOAT7 rs641738 with HCC. METHODS: Risk variants in PNPLA3, TM6SF2, and MBOAT7 were genotyped in 751 cases with alcohol-related cirrhosis and HCC and in 1165 controls with alcohol-related cirrhosis without HCC. Association with the risk of developing HCC was analyzed using multivariate logistic regression. RESULTS: The development of HCC was independently associated with PNPLA3 rs738409 (ORadjusted 1.84 [95% CI 1.55-2.18], p = 1.85 × 10-12) and TM6SF2 rs58542926 (ORadjusted 1.66 [1.30-2.13], p = 5.13 × 10-05), using an additive model, and controlling the sex, age, body mass index, and type 2 diabetes mellitus; the risk associated with carriage of MBOAT7 rs641738 (ORadjusted 1.04 [0.88-1.24], p = 0.61) was not significant. The population-attributable fractions were 43.5% for PNPLA3 rs738409, 11.5% for TM6SF2 rs58542926, and 49.9% for the carriage of both the variants combined. CONCLUSIONS: Carriage of TM6SF2 rs58542926 is an additional risk factor for the development of HCC in people with alcohol-related cirrhosis. Carriage of both PNPLA3 rs738409 and TM6SF2 rs58542926 accounts for half of the attributable risk for HCC in this population. Genotyping will allow for more precise HCC risk-stratification of patients with alcohol-related cirrhosis, and genotype-guided screening algorithms would optimize patient care.


Assuntos
Aciltransferases/genética , Carcinoma Hepatocelular/genética , Lipase/genética , Cirrose Hepática Alcoólica/genética , Neoplasias Hepáticas/genética , Proteínas de Membrana/genética , Idoso , Carcinoma Hepatocelular/patologia , Estudos de Casos e Controles , Progressão da Doença , Europa (Continente) , Feminino , Predisposição Genética para Doença , Genótipo , Humanos , Fígado/patologia , Cirrose Hepática Alcoólica/patologia , Neoplasias Hepáticas/patologia , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único
12.
Nature ; 552(7683): 121-125, 2017 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-29143824

RESUMO

T cell non-Hodgkin lymphomas are a heterogeneous group of highly aggressive malignancies with poor clinical outcomes. T cell lymphomas originate from peripheral T cells and are frequently characterized by genetic gain-of-function variants in T cell receptor (TCR) signalling molecules. Although these oncogenic alterations are thought to drive TCR pathways to induce chronic proliferation and cell survival programmes, it remains unclear whether T cells contain tumour suppressors that can counteract these events. Here we show that the acute enforcement of oncogenic TCR signalling in lymphocytes in a mouse model of human T cell lymphoma drives the strong expansion of these cells in vivo. However, this response is short-lived and robustly counteracted by cell-intrinsic mechanisms. A subsequent genome-wide in vivo screen using T cell-specific transposon mutagenesis identified PDCD1, which encodes the inhibitory receptor programmed death-1 (PD-1), as a master gene that suppresses oncogenic T cell signalling. Mono- and bi-allelic deletions of PDCD1 are also recurrently observed in human T cell lymphomas with frequencies that can exceed 30%, indicating high clinical relevance. Mechanistically, the activity of PD-1 enhances levels of the tumour suppressor PTEN and attenuates signalling by the kinases AKT and PKC in pre-malignant cells. By contrast, a homo- or heterozygous deletion of PD-1 allows unrestricted T cell growth after an oncogenic insult and leads to the rapid development of highly aggressive lymphomas in vivo that are readily transplantable to recipients. Thus, the inhibitory PD-1 receptor is a potent haploinsufficient tumour suppressor in T cell lymphomas that is frequently altered in human disease. These findings extend the known physiological functions of PD-1 beyond the prevention of immunopathology after antigen-induced T cell activation, and have implications for T cell lymphoma therapies and for current strategies that target PD-1 in the broader context of immuno-oncology.


Assuntos
Carcinogênese/genética , Genes Supressores de Tumor , Haploinsuficiência/genética , Linfoma de Células T/genética , Linfoma de Células T/patologia , Receptor de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/metabolismo , Animais , Células Cultivadas , Feminino , Humanos , Linfoma de Células T/metabolismo , Masculino , Camundongos , Mutação , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Transdução de Sinais/genética , Linfócitos T/metabolismo , Linfócitos T/patologia
13.
Lab Anim ; 50(6): 427-432, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27909192

RESUMO

Experimental autoimmune encephalomyelitis (EAE) is a frequently used animal model for the investigation of autoimmune processes in the central nervous system. As such, EAE is useful for modelling certain aspects of multiple sclerosis, a human autoimmune disease that leads to demyelination and axonal destruction. It is an important tool for investigating pathobiology, identifying drug targets and testing drug candidates. Even though EAE is routinely used in many laboratories and is often part of the routine assessment of knockouts and transgenes, scoring of the disease course has not become standardized in the community, with at least 83 published scoring variants. Varying scales with differing parameters are used and thus limit comparability of experiments. Incorrect use of statistical analysis tools to assess EAE data is commonplace. In experimental practice the clinical score is used not only as an experimental readout, but also as a parameter to determine animal welfare actions. Often overlooked factors such as the animal's ability to sense its compromised motoric abilities, drastic though transient weight loss, and also the possibility of neuropathic pain, make the assessment of severity a difficult task and pose a problem for experimental refinement.


Assuntos
Bem-Estar do Animal , Encefalomielite Autoimune Experimental/patologia , Projetos de Pesquisa , Animais , Animais Domésticos , Animais de Laboratório , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/fisiopatologia
14.
Science ; 352(6293): 1581-6, 2016 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-27256884

RESUMO

Foxp3(+) regulatory T cells in peripheral tissues (pT(regs)) are instrumental in limiting inflammatory responses to nonself antigens. Within the intestine, pT(regs) are located primarily in the lamina propria, whereas intraepithelial CD4(+) T cells (CD4(IELs)), which also exhibit anti-inflammatory properties and depend on similar environmental cues, reside in the epithelium. Using intravital microscopy, we show distinct cell dynamics of intestinal T(regs) and CD4(IELs) Upon migration to the epithelium, T(regs) lose Foxp3 and convert to CD4(IELs) in a microbiota-dependent manner, an effect attributed to the loss of the transcription factor ThPOK. Finally, we demonstrate that pT(regs) and CD4(IELs) perform complementary roles in the regulation of intestinal inflammation. These results reveal intratissue specialization of anti-inflammatory T cells shaped by discrete niches of the intestine.


Assuntos
Animais , Movimento Celular , Rastreamento de Células , Colite , Fator 3-gama Nuclear de Hepatócito , Mucosa Intestinal , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microbiota , Linfócitos T Reguladores , Fatores de Transcrição
15.
Nat Immunol ; 17(5): 593-603, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26950238

RESUMO

Persistent viral infections are characterized by the simultaneous presence of chronic inflammation and T cell dysfunction. In prototypic models of chronicity--infection with human immunodeficiency virus (HIV) or lymphocytic choriomeningitis virus (LCMV)--we used transcriptome-based modeling to reveal that CD4(+) T cells were co-exposed not only to multiple inhibitory signals but also to tumor-necrosis factor (TNF). Blockade of TNF during chronic infection with LCMV abrogated the inhibitory gene-expression signature in CD4(+) T cells, including reduced expression of the inhibitory receptor PD-1, and reconstituted virus-specific immunity, which led to control of infection. Preventing signaling via the TNF receptor selectively in T cells sufficed to induce these effects. Targeted immunological interventions to disrupt the TNF-mediated link between chronic inflammation and T cell dysfunction might therefore lead to therapies to overcome persistent viral infection.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Infecções por HIV/imunologia , HIV/imunologia , Coriomeningite Linfocítica/imunologia , Vírus da Coriomeningite Linfocítica/imunologia , Fator de Necrose Tumoral alfa/imunologia , Adolescente , Adulto , Idoso , Animais , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/virologia , Citometria de Fluxo , Células HEK293 , HIV/fisiologia , Infecções por HIV/genética , Infecções por HIV/virologia , Interações Hospedeiro-Patógeno/imunologia , Humanos , Immunoblotting , Coriomeningite Linfocítica/genética , Coriomeningite Linfocítica/virologia , Vírus da Coriomeningite Linfocítica/fisiologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Receptor de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/imunologia , Receptor de Morte Celular Programada 1/metabolismo , Receptores do Fator de Necrose Tumoral/genética , Receptores do Fator de Necrose Tumoral/imunologia , Receptores do Fator de Necrose Tumoral/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcriptoma/efeitos dos fármacos , Transcriptoma/genética , Transcriptoma/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Adulto Jovem
16.
Nat Commun ; 7: 11032, 2016 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-27010430

RESUMO

The RNA-binding protein Roquin is required to prevent autoimmunity. Roquin controls T-helper cell activation and differentiation by limiting the induced expression of costimulatory receptors such as tumor necrosis factor receptor superfamily 4 (Tnfrs4 or Ox40). A constitutive decay element (CDE) with a characteristic triloop hairpin was previously shown to be recognized by Roquin. Here we use SELEX assays to identify a novel U-rich hexaloop motif, representing an alternative decay element (ADE). Crystal structures and NMR data show that the Roquin-1 ROQ domain recognizes hexaloops in the SELEX-derived ADE and in an ADE-like variant present in the Ox40 3'-UTR with identical binding modes. In cells, ADE-like and CDE-like motifs cooperate in the repression of Ox40 by Roquin. Our data reveal an unexpected recognition of hexaloop cis elements for the posttranscriptional regulation of target messenger RNAs by Roquin.


Assuntos
Regiões 3' não Traduzidas/genética , Conformação de Ácido Nucleico , Receptores OX40/genética , Ubiquitina-Proteína Ligases/metabolismo , Animais , Sequência de Bases , Cristalografia por Raios X , Análise Mutacional de DNA , Ensaio de Desvio de Mobilidade Eletroforética , Ligantes , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Motivos de Nucleotídeos/genética , Ligação Proteica , Estrutura Terciária de Proteína , Espectroscopia de Prótons por Ressonância Magnética , RNA/química , RNA/metabolismo , Técnica de Seleção de Aptâmeros , Ubiquitina-Proteína Ligases/química
17.
Sci Rep ; 6: 21377, 2016 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-26903281

RESUMO

The CRISPR/Cas9 system can be used to mutate target sequences by introduction of double-strand breaks followed by imprecise repair. To test its use for conditional gene editing we generated mice transgenic for CD4 promoter-driven Cas9 combined with guide RNA targeting CD2. We found that within CD4(+) and CD8(+) lymphocytes from lymph nodes and spleen 1% and 0.6% were not expressing CD2, respectively. T cells lacking CD2 carryied mutations, which confirmed that Cas9 driven by cell-type specific promoters can edit genes in the mouse and may thus allow targeted studies of gene function in vivo.


Assuntos
Antígenos CD2/genética , Sistemas CRISPR-Cas , Edição de Genes/métodos , Inativação Gênica , RNA Guia/genética , Animais , Sequência de Bases , Antígenos CD2/imunologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Engenharia Genética , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Imunofenotipagem , Linfonodos/citologia , Linfonodos/imunologia , Camundongos , Camundongos Transgênicos , Mutação , Regiões Promotoras Genéticas , RNA Guia/metabolismo , Baço/citologia , Baço/imunologia
18.
Oncotarget ; 7(2): 1451-63, 2016 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-26623725

RESUMO

Ewing sarcomas (ES) are highly malignant bone or soft tissue tumors. Genetically, ES are defined by balanced chromosomal EWS/ETS translocations, which give rise to chimeric proteins (EWS-ETS) that generate an oncogenic transcriptional program associated with altered epigenetic marks throughout the genome. By use of an inhibitor (JQ1) blocking BET bromodomain binding proteins (BRDs) we strikingly observed a strong down-regulation of the predominant EWS-ETS protein EWS-FLI1 in a dose dependent manner. This was further enhanced by co-treatment with an inhibitor of the PI3K pathway. Microarray analysis further revealed JQ1 treatment to block a typical ES associated expression program. The effect on this expression program was mimicked by RNA interference with BRD3 or BRD4 expression, indicating that the EWS-FLI1 mediated expression profile is at least in part mediated via such epigenetic readers. Consequently, contact dependent and independent proliferation of different ES lines was strongly inhibited. Mechanistically, treatment of ES resulted in a partial arrest of the cell cycle as well as induction of apoptosis. Tumor development was suppressed dose dependently in a xeno-transplant model in immune deficient mice, overall indicating that ES may be susceptible to treatment with epigenetic inhibitors blocking BET bromodomain activity and the associated pathognomonic EWS-ETS transcriptional program.


Assuntos
Antineoplásicos/farmacologia , Azepinas/farmacologia , Neoplasias Ósseas/tratamento farmacológico , Proteínas Nucleares/antagonistas & inibidores , Proteínas de Fusão Oncogênica/metabolismo , Proteína Proto-Oncogênica c-fli-1/metabolismo , Proteína EWS de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/antagonistas & inibidores , Sarcoma de Ewing/tratamento farmacológico , Fatores de Transcrição/antagonistas & inibidores , Transcrição Genética/efeitos dos fármacos , Triazóis/farmacologia , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias Ósseas/genética , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Regulação para Baixo , Epigênese Genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Terapia de Alvo Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas de Fusão Oncogênica/genética , Fosfatidilinositol 3-Quinase/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteína Proto-Oncogênica c-fli-1/genética , Interferência de RNA , Proteína EWS de Ligação a RNA/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Sarcoma de Ewing/genética , Sarcoma de Ewing/metabolismo , Sarcoma de Ewing/patologia , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transfecção , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
19.
Dtsch Med Wochenschr ; 140(16): 1232-6, 2015 Aug.
Artigo em Alemão | MEDLINE | ID: mdl-26261935

RESUMO

Irritable bowel syndrome is a disorder of the gastrointestinal tract with unknown etiology. Recent clinical data support a link between changes in fecal microbiota with decreased biodiversity and the development of irritable bowel syndrome. Whether these changes of the microbiota are caused by the disease or whether they develop during the course of the disease remains unclear. Several studies demonstrated that fecal microbiota transfer (FMT) successfully attenuates Clostridium difficile infection by restoring the disturbed bacterial flora of the gut and case reports suggest that FMT may relief symptoms in patients with irritable bowel syndrome (IBS). Here we report a 47-year-old male patient with longstanding refractory diarrhea predominant IBS, who was successfully treated with a single FMT. The beneficial effect on the patient's symptoms was associated with changes of the stool microbiome. Post-FMT the recipient's microbiome resembled the donor's microbiome.


Assuntos
Terapia Biológica/métodos , Fezes/microbiologia , Síndrome do Intestino Irritável/terapia , Colonoscopia , Diagnóstico Diferencial , Seguimentos , Humanos , Síndrome do Intestino Irritável/diagnóstico , Masculino , Pessoa de Meia-Idade
20.
Clin Cancer Res ; 21(21): 4935-46, 2015 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-26179511

RESUMO

PURPOSE: A low mutation rate seems to be a general feature of pediatric cancers, in particular in oncofusion gene-driven tumors. Genetically, Ewing sarcoma is defined by balanced chromosomal EWS/ETS translocations, which give rise to oncogenic chimeric proteins (EWS-ETS). Other contributing somatic mutations involved in disease development have only been observed at low frequency. EXPERIMENTAL DESIGN: Tumor samples of 116 Ewing sarcoma patients were analyzed here. Whole-genome sequencing was performed on two patients with normal, primary, and relapsed tissue. Whole-exome sequencing was performed on 50 Ewing sarcoma and 22 matched normal tissues. A discovery dataset of 14 of these tumor/normal pairs identified 232 somatic mutations. Recurrent nonsynonymous mutations were validated in the 36 remaining exomes. Transcriptome analysis was performed in a subset of 14 of 50 Ewing sarcomas and DNA copy number gain and expression of FGFR1 in 63 of 116 Ewing sarcomas. RESULTS: Relapsed tumors consistently showed a 2- to 3-fold increased number of mutations. We identified several recurrently mutated genes at low frequency (ANKRD30A, CCDC19, KIAA0319, KIAA1522, LAMB4, SLFN11, STAG2, TP53, UNC80, ZNF98). An oncogenic fibroblast growth factor receptor 1 (FGFR1) mutation (N546K) was detected, and the FGFR1 locus frequently showed copy number gain (31.7%) in primary tumors. Furthermore, high-level FGFR1 expression was noted as a characteristic feature of Ewing sarcoma. RNA interference of FGFR1 expression in Ewing sarcoma lines blocked proliferation and completely suppressed xenograft tumor growth. FGFR1 tyrosine kinase inhibitor (TKI) therapy in a patient with Ewing sarcoma relapse significantly reduced 18-FDG-PET activity. CONCLUSIONS: FGFR1 may constitute a promising target for novel therapeutic approaches in Ewing sarcoma.


Assuntos
Regulação Neoplásica da Expressão Gênica , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Sarcoma de Ewing/genética , Sarcoma de Ewing/metabolismo , Transdução de Sinais , Adolescente , Adulto , Animais , Linhagem Celular Tumoral , Criança , Variações do Número de Cópias de DNA , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica , Frequência do Gene , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Camundongos Knockout , Mutação , Recidiva Local de Neoplasia , Estadiamento de Neoplasias , Sarcoma de Ewing/patologia , Adulto Jovem
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA