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1.
J Phys Chem A ; 127(10): 2351-2366, 2023 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-36877868

RESUMO

Ammonia (NH3) is a promising fuel, because it is carbon-free and easier to store and transport than hydrogen (H2). However, an ignition enhancer such as H2 might be needed for technical applications, because of the rather poor ignition properties of NH3. The combustion of pure NH3 and H2 has been explored widely. However, for mixtures of both gases, mostly only global parameters such as ignition delay times or flame speeds were reported. Studies with extensive experimental species profiles are scarce. Therefore, we experimentally investigated the interactions in the oxidation of different NH3/H2 mixtures in the temperature range of 750-1173 K at 0.97 bar in a plug-flow reactor (PFR), as well as in the temperature range of 1615-2358 K with an average pressure of 3.16 bar in a shock tube. In the PFR, temperature-dependent mole fraction profiles of the main species were obtained via electron ionization molecular-beam mass spectrometry (EI-MBMS). Additionally, for the first time, tunable diode laser absorption spectroscopy (TDLAS) with a scanned-wavelength method was adapted to the PFR for the quantification of nitric oxide (NO). In the shock tube, time-resolved NO profiles were also measured by TDLAS using a fixed-wavelength approach. The experimental results both in PFR and shock tube reveal the reactivity enhancement by H2 on ammonia oxidation. The extensive sets of results were compared with predictions by four NH3-related reaction mechanisms. None of the mechanisms can well predict all experimental results, but the Stagni et al. [React. Chem. Eng. 2020, 5, 696-711] and Zhu et al. [Combust. Flame 2022, 246, 115389] mechanisms perform best for the PFR and shock tube conditions, respectively. Exploratory kinetic analysis was conducted to identify the effect of H2 addition on ammonia oxidation and NO formation, as well as sensitive reactions in different temperature regimes. The results presented in this study can provide valuable information for further model development and highlight relevant properties of H2-assisted NH3 combustion.

2.
Nat Commun ; 12(1): 1119, 2021 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-33602930

RESUMO

Regulatory CD4+ T cells (Treg) prevent tumor clearance by conventional T cells (Tconv) comprising a major obstacle of cancer immune-surveillance. Hitherto, the mechanisms of Treg repertoire formation in human cancers remain largely unclear. Here, we analyze Treg clonal origin in breast cancer patients using T-Cell Receptor and single-cell transcriptome sequencing. While Treg in peripheral blood and breast tumors are clonally distinct, Tconv clones, including tumor-antigen reactive effectors (Teff), are detected in both compartments. Tumor-infiltrating CD4+ cells accumulate into distinct transcriptome clusters, including early activated Tconv, uncommitted Teff, Th1 Teff, suppressive Treg and pro-tumorigenic Treg. Trajectory analysis suggests early activated Tconv differentiation either into Th1 Teff or into suppressive and pro-tumorigenic Treg. Importantly, Tconv, activated Tconv and Treg share highly-expanded clones contributing up to 65% of intratumoral Treg. Here we show that Treg in human breast cancer may considerably stem from antigen-experienced Tconv converting into secondary induced Treg through intratumoral activation.


Assuntos
Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Linfócitos T Reguladores/imunologia , Antígenos de Neoplasias/metabolismo , Neoplasias da Mama/sangue , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Proliferação de Células , Células Clonais , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Ativação Linfocitária/imunologia , Estadiamento de Neoplasias , Receptores de Antígenos de Linfócitos T/imunologia , Análise de Célula Única , Células Th1/imunologia , Transcriptoma/genética
3.
Cytokine ; 133: 155153, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32554157

RESUMO

INTRODUCTION: Congenital heart surgery with cardiopulmonary bypass (CPB) initiates an immune response which frequently leads to organ dysfunction and a systemic inflammatory response. Complications associated with exacerbated immune responses may severely impact the postoperative recovery. The objective was to describe the characteristics of monocyte subpopulations and neutrophils at the level of pattern recognition receptors (PRR) and the cytokine response after CPB in infants. METHODS: An observational cohort study was conducted between June 2016 and June 2017 of infants < 2 years of age, electively admitted for surgical correction of acyanotic congenital heart defects using CPB. Fourteen blood samples were collected sequentially and processed immediately during and up to 48 h following cardiac surgery for each patient. Flow cytometry analysis comprised monocytic and granulocytic surface expression of CD14, CD16, CD64, TLR2, TLR4 and Dectin-1 (CLEC7A). Monocyte subpopulations were further defined as classical (CD14++/CD16-), intermediate (CD14++/CD16+) and nonclassical (CD14+/CD16++) monocytes. Plasma concentrations of 14 cytokines, including G-CSF, GM-CSF, IL-1ß, IL-1RA, IL-4, IL-6, IL-8, IL-10, IL-12p40, IL-12p70, TNF-α, IFN-γ, MIP-1ß (CCL4) and TGF-ß1, were measured using multiplex immunoassay for seven points in time. RESULTS: Samples from 21 infants (median age 7.4 months) were analyzed by flow cytometry and from 11 infants, cytokine concentrations were measured. Classical and intermediate monocytes showed first receptor upregulation with an increase in CD64 expression four hours post CPB. CD64-expression on intermediate monocytes almost tripled 48 h post CPB (p < 0.0001). TLR4 was only increased on intermediate monocytes, occurring 12 h post CPB (p = 0.0406) along with elevated TLR2 levels (p = 0.0002). TLR4 expression on intermediate monocytes correlated with vasoactive-inotropic score (rs = 0.642, p = 0.0017), duration of ventilation (rs = 0.485, p = 0.0259), highest serum creatinine (rs = 0.547, p = 0.0102), postsurgical transfusion (total volume per kg bodyweight) (rs = 0.469, p = 0.0321) and lowest mean arterial pressure (rs = -0.530, p = 0.0135). Concentrations of IL-10, MIP-1ß, IL-8, G-CSF and IL-6 increased one hour post CPB. Methylprednisolone administration in six patients had no significant influence on the studied surface receptors but led to lower IL-8 and higher IL-10 plasma concentrations. CONCLUSIONS: Congenital heart surgery with CPB induces a systemic inflammatory process including cytokine response and changes in PRR expression. Intermediate monocytes feature specific inflammatory characteristics in the 48 h after pediatric CPB and TLR4 correlates with poorer clinical course, which might provide a potential diagnostic or even therapeutic target.


Assuntos
Cardiopatias Congênitas/metabolismo , Monócitos/metabolismo , Receptores de IgG/metabolismo , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Pressão Arterial/fisiologia , Citocinas/metabolismo , Feminino , Cardiopatias Congênitas/cirurgia , Humanos , Lactente , Recém-Nascido , Inflamação/metabolismo , Masculino , Neutrófilos/metabolismo , Estudos Prospectivos
5.
BMC Cancer ; 16(1): 896, 2016 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-27855654

RESUMO

BACKGROUND: The polyether antibiotic Salinomycin (Sal) is regarded as an inhibitor of cancer stem cells. Its effectiveness on human colorectal cancer (CRC) cells in vitro has been demonstrated before. The aim of this study was to establish a murine model to investigate the effectiveness of Sal in vivo. Furthermore, we investigated the impact of Sal on Wnt/ß-catenin signaling in human CD133+ CRC cells. METHODS: The two murine CRC cell lines MC38 and CT26 were used to analyze the impact of Sal on tumor cell proliferation, viability, migration, cell cycle progression and cell death in vitro. For in vivo studies, CT26 cells were injected into syngeneic BALB/c mice to initiate (i) subcutaneous, (ii) orthotopic, or (iii) metastatic CRC growth. Sal was administered daily, 5-Fluoruracil served as a control. For mechanistic studies, the CD133+and CD133- subpopulations of human CRC cells were separated by flow cytometry and separately exposed to increasing concentrations of Sal. The impact on Wnt/ß-catenin signaling was determined by Western blotting and quantitative PCR. RESULTS: Sal markedly impaired tumor cell viability, proliferation and migration, and induced necrotic cell death in vitro. CRC growth in vivo was likewise inhibited upon Sal treatment. Interference with Wnt signaling and reduced expression of the Wnt target genes Fibronectin and Lgr5 indicates a novel molecular mechanism, mediating anti-tumoral effects of Sal in CRC. CONCLUSION: Sal effectively impairs CRC growth in vivo. Furthermore, Sal acts as an inhibitor of Wnt/ß-catenin signaling. Thus, Salinomycin represents a promising candidate for clinical CRC treatment.


Assuntos
Antígeno AC133/metabolismo , Neoplasias Colorretais/tratamento farmacológico , Piranos/administração & dosagem , Via de Sinalização Wnt/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Neoplasias Colorretais/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Metástase Neoplásica , Piranos/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
6.
Blood ; 128(9): 1246-59, 2016 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-27268087

RESUMO

Clonal evolution is believed to be a main driver for progression of various types of cancer and implicated in facilitating resistance to drugs. However, the hierarchical organization of malignant clones in the hematopoiesis of myelodysplastic syndromes (MDS) and its impact on response to drug therapy remain poorly understood. Using high-throughput sequencing of patient and xenografted cells, we evaluated the intratumoral heterogeneity (n= 54) and reconstructed mutational trajectories (n = 39) in patients suffering from MDS (n = 52) and chronic myelomonocytic leukemia-1 (n = 2). We identified linear and also branching evolution paths and confirmed on a patient-specific level that somatic mutations in epigenetic regulators and RNA splicing genes frequently constitute isolated disease-initiating events. Using high-throughput exome- and/or deep-sequencing, we analyzed 103 chronologically acquired samples from 22 patients covering a cumulative observation time of 75 years MDS disease progression. Our data revealed highly dynamic shaping of complex oligoclonal architectures, specifically upon treatment with lenalidomide and other drugs. Despite initial clinical response to treatment, patients' marrow persistently remained clonal with rapid outgrowth of founder-, sub-, or even fully independent clones, indicating an increased dynamic rate of clonal turnover. The emergence and disappearance of specific clones frequently correlated with changes of clinical parameters, highlighting their distinct and far-reaching functional properties. Intriguingly, increasingly complex mutational trajectories are frequently accompanied by clinical progression during the course of disease. These data substantiate a need for regular broad molecular monitoring to guide clinical treatment decisions in MDS.


Assuntos
Hematopoese/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/terapia , Mutação , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/terapia , Animais , Feminino , Xenoenxertos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Transplante de Neoplasias
7.
BMC Cancer ; 13: 569, 2013 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-24304513

RESUMO

BACKGROUND: Biomarkers allowing the characterization of malignancy and therapy response of oral squamous cell carcinomas (OSCC) or other types of carcinomas are still outstanding. The biochemical suicide molecule endonuclease DNaseX (DNaseI-like 1) has been used to identify the Apo10 protein epitope that marks tumor cells with abnormal apoptosis and proliferation. The transketolase-like protein 1 (TKTL1) represents the enzymatic basis for an anaerobic glucose metabolism even in the presence of oxygen (aerobic glycolysis/Warburg effect), which is concomitant with a more malignant phenotype due to invasive growth/metastasis and resistance to radical and apoptosis inducing therapies. METHODS: Expression of Apo10 and TKTL1 was analysed retrospectively in OSCC specimen (n = 161) by immunohistochemistry. Both markers represent independent markers for poor survival. Furthermore Apo10 and TKTL1 have been used prospectively for epitope detection in monocytes (EDIM)-blood test in patients with OSCC (n = 50), breast cancer (n = 48), prostate cancer (n = 115), and blood donors/controls (n = 74). RESULTS: Positive Apo10 and TKTL1 expression were associated with recurrence of the tumor. Multivariate analysis demonstrated Apo10 and TKTL1 expression as an independent prognostic factor for reduced tumor-specific survival. Apo10+/TKTL1+ subgroup showed the worst disease-free survival rate in OSCC.EDIM-Apo10 and EDIM-TKTL1 blood tests allowed a sensitive and specific detection of patients with OSCC, breast cancer and prostate cancer before surgery and in after care. A combined score of Apo10+/TKTL1+ led to a sensitivity of 95.8% and a specificity of 97.3% for the detection of carcinomas independent of the tumor entity. CONCLUSIONS: The combined detection of two independent fundamental biophysical processes by the two biomarkers Apo10 and TKTL1 allows a sensitive and specific detection of neoplasia in a noninvasive and cost-effective way. Further prospective trials are warranted to validate this new concept for the diagnosis of neoplasia and tumor recurrence.


Assuntos
Biomarcadores Tumorais/sangue , Carcinoma de Células Escamosas/sangue , Desoxirribonuclease I/sangue , Neoplasias Bucais/sangue , Proteínas Musculares/sangue , Transcetolase/sangue , Anticorpos Monoclonais Murinos/química , Carcinoma de Células Escamosas/mortalidade , Carcinoma de Células Escamosas/secundário , Carcinoma de Células Escamosas/cirurgia , Estudos de Casos e Controles , Linhagem Celular Tumoral , Desoxirribonuclease I/imunologia , Intervalo Livre de Doença , Feminino , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Linfonodos/patologia , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Monócitos/metabolismo , Neoplasias Bucais/mortalidade , Neoplasias Bucais/patologia , Neoplasias Bucais/cirurgia , Análise Multivariada , Proteínas Musculares/imunologia , Pescoço , Estadiamento de Neoplasias , Prognóstico , Curva ROC , Estudos Retrospectivos , Transcetolase/imunologia , Carga Tumoral
8.
EMBO Mol Med ; 5(12): 1804-20, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24124051

RESUMO

The t(8;21) chromosomal translocation activates aberrant expression of the AML1-ETO (AE) fusion protein and is commonly associated with core binding factor acute myeloid leukaemia (CBF AML). Combining a conditional mouse model that closely resembles the slow evolution and the mosaic AE expression pattern of human t(8;21) CBF AML with global transcriptome sequencing, we find that disease progression was characterized by two principal pathogenic mechanisms. Initially, AE expression modified the lineage potential of haematopoietic stem cells (HSCs), resulting in the selective expansion of the myeloid compartment at the expense of normal erythro- and lymphopoiesis. This lineage skewing was followed by a second substantial rewiring of transcriptional networks occurring in the trajectory to manifest leukaemia. We also find that both HSC and lineage-restricted granulocyte macrophage progenitors (GMPs) acquired leukaemic stem cell (LSC) potential being capable of initiating and maintaining the disease. Finally, our data demonstrate that long-term expression of AE induces an indolent myeloproliferative disease (MPD)-like myeloid leukaemia phenotype with complete penetrance and that acute inactivation of AE function is a potential novel therapeutic option.


Assuntos
Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Neoplásicas/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Animais , Antibióticos Antineoplásicos/farmacologia , Antibióticos Antineoplásicos/uso terapêutico , Linhagem da Célula , Modelos Animais de Doenças , Doxorrubicina/farmacologia , Doxorrubicina/uso terapêutico , Regulação da Expressão Gênica/efeitos dos fármacos , Células Progenitoras de Granulócitos e Macrófagos/citologia , Células Progenitoras de Granulócitos e Macrófagos/metabolismo , Células-Tronco Hematopoéticas/citologia , Imunofenotipagem , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Neoplásicas/citologia , Fenótipo , Análise de Sequência de RNA , Translocação Genética/efeitos dos fármacos
9.
Pathology ; 43(3): 220-7, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21436631

RESUMO

AIMS: In colorectal cancer (CRC), CD133 expression is an independent prognostic marker associated with adverse clinical outcome. The CD133 epitope AC133 allowed isolating stem cells from normal and cancerous tissues, although its use in colon was questioned. We aimed to identify differences between AC133 and AC133 cells. METHODS: We analysed the gene expression profiles of EpCAM/CEA/AC133 and EpCAM/CEA/AC133 cells from primary CRC and liver metastasis tissues (n = 5). Immunohistochemistry confirmed these results in a validation set. RESULTS: We identified 68 genes differentially expressed between both populations, including genes of notorious importance in CRC pathogenesis, and several candidates not previously shown to play a major role in CRC. Notably, EGR1 belonged to the most highly expressed genes in AC133 cells. In the validation set, the presence of EGR1 and CD133 correlated (r = 0.625). Since EGR1 regulates Wnt through up-regulation of TCF4, which induces stem cell marker LGR5, the potential association between LGR5, EGR1 and CD133 was investigated. The presence of LGR5 correlated with the presence of EGR1 and CD133. Strong signals for LGR5 were detected throughout tumour invasion fronts. CONCLUSIONS: The study suggests a connection between CD133 and EGR1 and emphasises the importance of the EGR1/TCF4/CD133/LGR5 network in CRC.


Assuntos
Antígenos CD/genética , Neoplasias Colorretais/genética , Proteína 1 de Resposta de Crescimento Precoce/genética , Glicoproteínas/genética , Peptídeos/genética , Antígeno AC133 , Antígenos CD/metabolismo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Epitopos/genética , Epitopos/metabolismo , Citometria de Fluxo/métodos , Perfilação da Expressão Gênica , Glicoproteínas/metabolismo , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/secundário , Instabilidade de Microssatélites , Peptídeos/metabolismo , RNA Mensageiro/metabolismo , RNA Neoplásico/análise , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais
10.
Cancer Biol Ther ; 10(12): 1280-9, 2010 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-21124075

RESUMO

Treatment of cancers by means of viruses, that specifically replicate in (oncotropism) and kill (oncolysis) neoplastic cells, is increasingly gaining acceptance in the clinic. Among these agents, parvoviruses have been shown to possess not only direct oncolytic but also immunomodulating properties, serving as an adjuvant to prime the immune system to react against infected tumors. Here, we aimed to establish whether immunomodulating mechanisms participate in the recently reported therapeutic potential of parvoviruses against pancreatic carcinoma. Using adoptive transfer experiments we discovered that the transfer of splenocytes of donor rats harboring H-1PV-treated orthotopic PDAC tumors could significantly prolong the survival of naïve tumor-bearing recipients, compared to those receiving cells from mock-treated donors. Closer investigation of immunological parameters in infected donor rats revealed that virus-induced interferon gamma production and cellular immune response played an important role in this effect. These data have also preclinical relevance since abortive H-1PV infection of human peripheral blood mononuclear cells or cocultivation of these cells with H-1PV-preinfected pancreatic cancer cells, resulted in enhancement of innate and adaptive immune reactivity. Taken together our data reveal that oncolytic H-1PV modulates the immune system into an anticancer state, and further support the concept of using parvoviruses in the fight against pancreatic cancer.


Assuntos
Carcinoma Ductal Pancreático/imunologia , Carcinoma Ductal Pancreático/terapia , Parvovirus H-1/imunologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/virologia , Terapia Viral Oncolítica/métodos , Transferência Adotiva , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Citocinas/metabolismo , Citometria de Fluxo , Parvovirus H-1/fisiologia , Humanos , Imunidade Celular/imunologia , Imunomodulação , Interferon gama/imunologia , Vírus Oncolíticos , Reação em Cadeia da Polimerase , Ratos , Baço/imunologia , Baço/virologia , Equilíbrio Th1-Th2
11.
Mech Dev ; 126(10): 863-72, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19631738

RESUMO

One of the principal issues facing biomedical research is to elucidate developmental pathways and to establish the fate of stem and progenitor cells in vivo. Hematopoiesis, the process of blood cell formation, provides a powerful experimental system for investigating this process. Here, we employ transcriptional regulatory elements from the stem cell leukemia (SCL) gene to selectively label primitive and definitive hematopoiesis. We report that SCL-labelled cells arising in the mid to late streak embryo give rise to primitive red blood cells but fail to contribute to the vascular system of the developing embryo. Restricting SCL-marking to different stages of foetal development, we identify a second population of multilineage progenitors, proficient in contributing to adult erythroid, myeloid and lymphoid cells. The distinct lineage-restricted potential of SCL-labelled early progenitors demonstrates that primitive erythroid cell fate specification is initiated during mid gastrulation. Our data also suggest that the transition from a hemangioblastic precursors with endothelial and blood forming potential to a committed hematopoietic progenitor must have occurred prior to SCL-marking of definitive multilineage blood precursors.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Linhagem da Célula , Hematopoese , Proteínas Proto-Oncogênicas/fisiologia , Células-Tronco/citologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Desenvolvimento Embrionário , Citometria de Fluxo , Técnicas de Introdução de Genes , Camundongos , Microscopia Confocal , Proteínas Proto-Oncogênicas/genética , Proteína 1 de Leucemia Linfocítica Aguda de Células T
12.
J Immunol ; 181(11): 7751-8, 2008 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-19017964

RESUMO

In allergic airway disease, Treg may play an important role in the modulation of airway hyperreactivity (AHR) and inflammation. We therefore investigated the therapeutic potential of Treg in an Ag-dependent murine asthma model. We here describe that AHR can be completely suppressed by adoptive transfer of Treg overexpressing active TGF-beta1. Using mice with impaired TGF-beta signaling in T cells, we could demonstrate that TGF-beta signaling in recipient effector T cells or transferred Treg themselves is not required for the protective effects on AHR. However, the expression of IL-10 by Treg was found to be essential for the suppression of AHR, since Treg overexpressing active TGF-beta1 but deficient in IL-10 lacked protective effects. Airway inflammation could not be significantly suppressed by wild-type or transgenic Treg. In conclusion, modulation of cytokine expression by Treg may have therapeutic potential for the treatment of AHR in asthma. The mechanisms of the effects of Treg on airway inflammation require further clarification.


Assuntos
Transferência Adotiva , Interleucina-10/imunologia , Hipersensibilidade Respiratória/terapia , Linfócitos T Reguladores/transplante , Fator de Crescimento Transformador beta1/imunologia , Animais , Modelos Animais de Doenças , Expressão Gênica , Inflamação/genética , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/terapia , Interleucina-10/biossíntese , Interleucina-10/genética , Camundongos , Camundongos Transgênicos , Hipersensibilidade Respiratória/genética , Hipersensibilidade Respiratória/imunologia , Hipersensibilidade Respiratória/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Linfócitos T Reguladores/imunologia , Fator de Crescimento Transformador beta1/biossíntese , Fator de Crescimento Transformador beta1/genética
13.
PLoS One ; 3(10): e3302, 2008 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-18827879

RESUMO

CD4+CD25+ regulatory T cells (Treg) are important mediators of immune tolerance. A subset of Treg can be generated in the periphery by TGF-beta dependent conversion of conventional CD4+CD25- T cells into induced Treg (iTreg). In chronic viral infection or malignancy, such induced iTreg, which limit the depletion of aberrant or infected cells, may be of pathogenic relevance. To identify potential targets for therapeutic intervention, we investigated the TGF-beta signaling in Treg. In contrast to conventional CD4+ T cells, Treg exhibited marked activation of the p38 MAP kinase pathway. Inhibition of p38 MAP kinase activity prevented the TGF-beta-dependent conversion of CD4+CD25- T cells into Foxp3+ iTreg in vitro. Of note, the suppressive capacity of nTreg was not affected by inhibiting p38 MAP kinase. Our findings indicate that signaling via p38 MAP kinase seems to be important for the peripheral generation of iTreg; p38 MAP kinase could thus be a therapeutic target to enhance immunity to chronic viral infection or cancer.


Assuntos
Antígenos CD4/imunologia , Subunidade alfa de Receptor de Interleucina-2/imunologia , Transdução de Sinais , Linfócitos T Reguladores/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Western Blotting , Citometria de Fluxo , Camundongos
14.
J Allergy Clin Immunol ; 121(4): 992-9.e6, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18329088

RESUMO

BACKGROUND: The transcriptional regulation of cytokines released and controlled by memory T cells is not well understood. Defective IFN-gamma production in allergic asthma correlates in human beings with the risk of wheezing in childhood. OBJECTIVE: To understand the role of the transcription factor nuclear factor of activated T cells 2 (NFATc2) in memory and effector T cells in the airways in experimental allergic asthma. METHODS: We used murine models of allergic asthma and adoptive cell transfer of fluorescence-activated sorted cells in a disease model. RESULTS: Mice lacking NFATc2 developed an increase in airwayhyperresponsiveness (AHR), remodeling, and serum IgE levelson ovalbumin sensitization. This phenotype was associated withCD81CD1222 T cells deficient in IFN-g production in theairways. The origin of this phenotype in NFATc2(2/2) mice wasrelated to an expanded population of lung CD81CD1221(IL-2Rb chain) CD127hi (IL-7 receptor [R] a chain1) long-livedmemory cells. Adoptive transfer of ovalbumin-specific CD81NFATc2(2/2) T cells enhanced the AHR generated byNFATc2(2/2) CD41 T cells in immunodeficient mice, increasedIL-17, and reduced IFN-g production in the reconstituted mice. Depletion of the memory CD81CD1221IL-7Rhigh T-cellpopulation corrected the defect in IFN-g production by lungNFATc2(2/2) CD81CD1222 cells and abrogated the increasedAHR observed in NFATc2(2/2) CD81 T-cell-reconstituted micewith a severe combined immunodeficiency disorder. CONCLUSION: Taken together, our results suggest that NFATc2 expression in long-lived memory CD8+ T cells controls IL-2 and IFN-gamma production in lung CD8+ T cells, which then limits TH17 and TH2 development in the airways during allergen challenge.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Hipersensibilidade/imunologia , Hipersensibilidade/prevenção & controle , Memória Imunológica , Fatores de Transcrição NFATC/fisiologia , Subpopulações de Linfócitos T/imunologia , Transferência Adotiva , Animais , Hiper-Reatividade Brônquica/imunologia , Hiper-Reatividade Brônquica/metabolismo , Hiper-Reatividade Brônquica/prevenção & controle , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/transplante , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Feminino , Inibidores do Crescimento/deficiência , Inibidores do Crescimento/genética , Inibidores do Crescimento/fisiologia , Hipersensibilidade/metabolismo , Interferon gama/antagonistas & inibidores , Interferon gama/deficiência , Interferon gama/genética , Interleucina-17/biossíntese , Subunidade beta de Receptor de Interleucina-2/biossíntese , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Camundongos SCID , Fatores de Transcrição NFATC/deficiência , Fatores de Transcrição NFATC/genética , Receptores de Interleucina-7/biossíntese , Subpopulações de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/transplante , Regulação para Cima/imunologia
15.
J Exp Med ; 204(6): 1303-10, 2007 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-17502663

RESUMO

Naturally occurring regulatory T cells (T reg cells) are a thymus-derived subset of T cells, which are crucial for the maintenance of peripheral tolerance by controlling potentially autoreactive T cells. However, the underlying molecular mechanisms of this strictly cell contact-dependent process are still elusive. Here we show that naturally occurring T reg cells harbor high levels of cyclic adenosine monophosphate (cAMP). This second messenger is known to be a potent inhibitor of proliferation and interleukin 2 synthesis in T cells. Upon coactivation with naturally occurring T reg cells the cAMP content of responder T cells is also strongly increased. Furthermore, we demonstrate that naturally occurring T reg cells and conventional T cells communicate via cell contact-dependent gap junction formation. The suppressive activity of naturally occurring T reg cells is abolished by a cAMP antagonist as well as by a gap junction inhibitor, which blocks the cell contact-dependent transfer of cAMP to responder T cells. Accordingly, our results suggest that cAMP is crucial for naturally occurring T reg cell-mediated suppression and traverses membranes via gap junctions. Hence, naturally occurring T reg cells unexpectedly may control the immune regulatory network by a well-known mechanism based on the intercellular transport of cAMP via gap junctions.


Assuntos
Linfócitos T CD4-Positivos/imunologia , AMP Cíclico/imunologia , Sistemas do Segundo Mensageiro/imunologia , Fatores Supressores Imunológicos/imunologia , Linfócitos T Reguladores/imunologia , Animais , Conexinas , Citocinas/metabolismo , Primers do DNA , Ensaio de Imunoadsorção Enzimática , Feminino , Junções Comunicantes/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Oligopeptídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa
16.
Physiol Genomics ; 31(1): 32-41, 2007 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-17456738

RESUMO

Ligand-activated Cre recombinases are widely used for studying gene function in vitro and in conditional mouse models. To compare ligand-dependent Cre recombinases, different Cre estrogen receptor fusions were introduced into the ROSA26 locus of embryonic stem (ES) cells and assayed for genotoxicity and recombination efficiency. Of the tested recombinases, the CreERT2 variant showed no toxicity and was highly responsive to ligand induction. To constitutively express CreERT2 in mice and also to clarify whether the CreERT2 system displays background activity, we generated a knock-in mouse line harboring the CreERT2 coding region under the control of the ROSA26 locus. Analysis of this ROSA26-CreERT2 deleter mouse with different reporter strains revealed ubiquitous recombination in the embryo and partial recombination in peripheral and hematopoietic tissues but no effective CreERT2 expression in the brain. Furthermore, using flow cytometry, we found low-level background recombination in noninduced bitransgenic ROSA26-CreERT2/EGFP reporter mice. To determine whether background activity poses a general problem for conducting conditional in vivo experiments with the ROSA26-CreERT2 deleter, we used a sensitive conditional skin cancer model. In this assay, cancer induction was completely restricted to induced bitransgenic CreERT2/K-Ras(V12) mice, whereas noninduced control animals did not show any sign of cancer, indicating the usefulness of the ROSA-CreERT2 system for regulating conditional gene expression in vivo. The ROSA26-CreERT2 deleter strain will be a convenient experimental tool for studying gene function under circumstances requiring partial induction of recombination in peripheral tissues and will be useful for uncovering previously unknown or unsuspected phenotypes.


Assuntos
Integrases/metabolismo , Mosaicismo , Animais , Encéfalo/metabolismo , DNA/metabolismo , Modelos Animais de Doenças , Citometria de Fluxo , Deleção de Genes , Genes Reporter , Genômica/métodos , Ligantes , Camundongos , Camundongos Transgênicos , Modelos Genéticos , Recombinação Genética
17.
J Gene Med ; 9(4): 308-18, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17330923

RESUMO

BACKGROUND: Conditional gene regulatory systems ensuring tight and adjustable expression of therapeutic genes are central for developing future gene therapy strategies. Among various regulatory systems, tetracycline-controlled gene expression has emerged as a safe and reliable option. Moreover, the tightness of tetracycline-regulated gene switches can be substantially improved by complementing transcriptional activators with antagonizing repressors. METHODS: To develop novel tetracycline-responsive transcriptional repressors, we fused various transcriptional silencing domains to the TetR (B/E) DNA-binding and dimerization domain of the Tn10-encoded tetracycline resistance operon (TetR (B/E)). The resulting fusion proteins were individually tested for their ability to repress transcription of the constitutively active hypoxanthine phosphoribosyltransferase (HPRT) promoter. In addition, compatibility with the commonly used reverse tetracycline-controlled transactivator system (rtTA-system) and responsiveness to the pharmacological effector doxycycline (DOX) were evaluated. Finally, inducibility, effector-dependent promoter activity and the modification of histone H3 and H4 of the active versus the repressed target promoter were determined. RESULTS: Fusion of the human deacetylase 4 (HDAC4) carboxy-terminal silencing domain to TetR (B/E) resulted in a functional transcriptional repressor. This novel repressor, termed tTS-H4, efficiently reduced the activity of the murine HPRT promoter and a constitutively active human cytomegalovirus (hCMV) minimal promoter. Furthermore, combining tTS-H4 with the rtTA transcriptional activator allowed for grading, turning off and resuming target gene expression over several orders of magnitude without background. CONCLUSIONS: The tTS-H4 repressor is compatible with the commonly used rtTA transcriptional activation system and is a versatile new tool for tightly and adjustably regulating conditional gene expression.


Assuntos
Regulação da Expressão Gênica , Terapia Genética/métodos , Proteínas Repressoras/metabolismo , Tetraciclina/metabolismo , Animais , Genes Reporter , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Histonas/metabolismo , Humanos , Camundongos , Células NIH 3T3 , Regiões Promotoras Genéticas , Proteínas Repressoras/química , Proteínas Repressoras/genética , Tetraciclina/química , Transcrição Gênica
18.
Blood ; 108(5): 1533-41, 2006 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-16675709

RESUMO

The stem cell leukemia gene SCL, also known as TAL-1, encodes a basic helix-loop-helix transcription factor expressed in erythroid, myeloid, megakaryocytic, and hematopoietic stem cells. To be able to make use of the unique tissue-restricted and spatio-temporal expression pattern of the SCL gene, we have generated a knock-in mouse line containing the tTA-2S tetracycline transactivator under the control of SCL regulatory elements. Analysis of this mouse using different tetracycline-dependent reporter strains demonstrated that switchable transgene expression was restricted to erythrocytes, megakaryocytes, granulocytes, and, importantly, to the c-kit-expressing and lineage-negative cell fraction of the bone marrow. In addition, conditional transgene activation also was detected in a very minor population of endothelial cells and in the kidney. However, no activation of the reporter transgene was found in the brain of adult mice. These findings suggested that the expression of tetracycline-responsive reporter genes recapitulated the known endogenous expression pattern of SCL. Our data therefore demonstrate that exogenously inducible and reversible expression of selected transgenes in myeloid, megakaryocytic, erythroid, and c-kit-expressing lineage-negative bone marrow cells can be directed through SCL regulatory elements. The SCL knock-in mouse presented here represents a powerful tool for studying normal and malignant hematopoiesis in vivo.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Eritrócitos/fisiologia , Granulócitos/fisiologia , Hematopoese/fisiologia , Megacariócitos/fisiologia , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas/genética , Tetraciclina/farmacologia , Animais , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos/efeitos dos fármacos , Primers do DNA , Eritrócitos/citologia , Citometria de Fluxo , Regulação da Expressão Gênica , Genes Reporter , Genótipo , Granulócitos/citologia , Megacariócitos/citologia , Camundongos , Camundongos Transgênicos , Proteínas Proto-Oncogênicas/efeitos dos fármacos , Proteínas Recombinantes/metabolismo , Proteína 1 de Leucemia Linfocítica Aguda de Células T
19.
Cancer Res ; 66(7): 3715-25, 2006 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-16585198

RESUMO

Oncogenic activation of the receptor tyrosine kinase ERBB2 is a key event in the development of a number of epithelial malignancies. In these tumors, high levels of ERBB2 are strongly associated with metastatic disease and poor prognosis. Paradoxically, an inherent cellular response to hypermitogenic signaling by ERBB2 and other oncogenes seems to be growth arrest, rather than proliferation. Molecular characterization of this yet undefined antiproliferative state in independent cell lines overexpressing either wild-type ERBB2 or the mutationally activated receptor unveiled a dramatic induction of the alpha5beta1 integrin fibronectin receptor. alpha5 Integrin up-regulation is mainly a transcriptional response mediated by the hypoxia-inducible transcription factors (HIF), leading to a massive increase in membrane-resident receptor molecules and enhanced fibronectin adhesiveness of the respective cells. Functionally, ERBB2-dependent ligation of fibronectin results in improved survival of mammary adenocarcinoma cells under adverse conditions, like serum withdrawal, hypoxia, and chemotherapy. HIF-1alpha is an independent predictor of poor overall survival in patients with breast cancer. In particular, HIF-1alpha overexpression correlates significantly with early local relapse and distant metastasis, a phenotype also highly characteristic of ERBB2-positive tumors. As HIF-1alpha is known to be stabilized by ERBB2 signaling under normoxic conditions, we propose that alpha5 integrin is a major effector in this regulatory circuit and may represent the molecular basis for the HIF-1alpha-dependent aggressiveness observed in ERBB2-overexpressing breast carcinomas. Hypermitogenic ERBB2 signaling and tumor hypoxia may act synergistically to favor the establishment of chemoresistant dormant micrometastatic cells frequently observed in patients with breast cancer. This new insight could be the basis for additional approaches complementing current cancer therapy.


Assuntos
Neoplasias da Mama/patologia , Integrina alfa5beta1/biossíntese , Receptor ErbB-2/fisiologia , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , Regulação Neoplásica da Expressão Gênica , Humanos , Integrina alfa5/biossíntese , Integrina alfa5/genética , Integrina alfa5beta1/genética , Integrina beta1/biossíntese , Integrina beta1/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptor ErbB-2/biossíntese , Receptor ErbB-2/genética , Transdução de Sinais , Transfecção , Regulação para Cima
20.
Free Radic Biol Med ; 40(8): 1340-8, 2006 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-16631524

RESUMO

Cells expressing the cytokine-inducible NO synthase are known to trigger apoptosis in neighboring cells. Paramagnetic dinitrosyl nonheme iron complexes (DNIC) were found in tumor tissue about 40 years ago; however, the role of these NO(+)-bearing species is not completely understood. In the human Jurkat leukemia cell line, the application of the model complex DNIC-thiosulfate (50-200 microM) induced apoptosis (defined by phosphatidylserine externalization) in a concentration- and time-dependent manner. In Jurkat cells, the pan-caspase inhibitor, zVADfmk (50 microM), and/or stable transfection of antiapoptotic protein, Bcl-2, was unable to afford protection against DNIC-induced apoptosis. The membrane-impermeable metal chelator, N-methyl-D-glucamine dithiocarbamate (MGD; 200 microM), in the presence of DNIC significantly increased apoptosis, but had no effect on its own. Electron paramagnetic resonance studies showed that MGD led to rapid transformation of the extracellular DNIC into the stable impermeable NO-Fe-MGD complex and to a burst-type release of nitrosonium (NO(+)) equivalents in the extracellular space. These results suggest that in Jurkat cells, DNIC-thiosulfate induces Bcl-2- and caspase-independent apoptosis, which is probably secondary to local nitrosative stress at the cell surface. We hypothesize that the local release of nonheme Fe-NO species by activated macrophages may play a role in the killing of malignant cells that have high Bcl-2 levels.


Assuntos
Apoptose/efeitos dos fármacos , Ferro/farmacologia , Óxidos de Nitrogênio/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Inibidores de Caspase , Caspases/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Regulação da Expressão Gênica , Humanos , Células Jurkat , Nitrosação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/genética
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