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1.
Nat Med ; 2021 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-33619368

RESUMO

B cell maturation antigen (BCMA) is a target for various immunotherapies and a biomarker for tumor load in multiple myeloma (MM). We report a case of irreversible BCMA loss in a patient with MM who was enrolled in the KarMMa trial ( NCT03361748 ) and progressed after anti-BCMA CAR T cell therapy. We identified selection of a clone with homozygous deletion of TNFRSF17 (BCMA) as the underlying mechanism of immune escape. Furthermore, we found heterozygous TNFRSF17 loss or monosomy 16 in 37 out of 168 patients with MM, including 28 out of 33 patients with hyperhaploid MM who had not been previously treated with BCMA-targeting therapies, suggesting that heterozygous TNFRSF17 deletion at baseline could theoretically be a risk factor for BCMA loss after immunotherapy.

3.
Artigo em Inglês | MEDLINE | ID: mdl-33126068

RESUMO

A simple, rapid, cost-effective and sensitive high-performance liquid chromatography method with diode array detection was developed and validated for the quantification of letermovir, a compound approved for prophylaxis of cytomegalovirus infection and disease in adult recipients of an allogeneic hematopoietic stem cell transplant. Sorafenib was used as internal standard. Samples were pre-treated by liquid-liquid extraction with tert-butyl methylether. Separation was achieved on a XTerra® RP18 column (150 × 2.1 mm, 5 µm) at 30 °C using gradient elution with a mobile phase of 20 mM ammonium bicarbonate pH 7.9 (mobile phase A) and acetonitrile:20 mM ammonium bicarbonate (9:1 v/v) (mobile phase B). Samples were eluted at a flow rate of 0.3 mL/min throughout the 20-min run. UV wavelength mode was used, letermovir and sorafenib were monitored at 260 nm. The calibration curve was linear in a concentration range of 25-5000 ng/mL with correlation coefficients ≥ 0.99. Intra-day and inter-day accuracy expressed as relative error were -11.4-20% and -7.96-10.62%, respectively. Precision expressed as coefficient of variation was 1.44-3.15% (intra-day) and 1.17-1.93% (inter-day). The method was successfully applied for analysis of 128 letermovir levels demonstrating its usefulness for letermovir monitoring in routine clinical practice.

4.
PLoS One ; 15(6): e0235160, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32579600

RESUMO

Vancomycin-resistant E. faecium (VRE) are an important cause of nosocomial infections, which are rapidly transmitted in hospitals. To identify possible transmission routes, we applied combined genomics and contact-network modeling to retrospectively evaluate routine VRE screening data generated by the infection control program of a hemato-oncology unit. Over 1 year, a total of 111 VRE isolates from 111 patients were collected by anal swabs in a tertiary care hospital in Southern Germany. All isolated VRE were whole-genome sequenced, followed by different in-depth bioinformatics analyses including genotyping and determination of phylogenetic relations, aiming to evaluate a standardized workflow. Patient movement data were used to overlay sequencing data to infer transmission events and strain dynamics over time. A predominant clone harboring vanB and exhibiting genotype ST117/CT469 (n = 67) was identified. Our comprehensive combined analyses suggested intra-hospital spread, especially of clone ST117/CT469, despite of extensive screening, single room placement, and contact isolation. A new interactive tool to visualize these complex data was designed. Furthermore, a patient-contact network-modeling approach was developed, which indicates both the periodic import of the clone into the hospital and its spread within the hospital due to patient movements. The analyzed spread of VRE was most likely due to placement of patients in the same room prior to positivity of screening. We successfully demonstrated the added value for this combined strategy to extract well-founded knowledge from interdisciplinary data sources. The combination of patient-contact modeling and high-resolution typing unraveled the transmission dynamics within the hospital department and, additionally, a constant VRE influx over time.


Assuntos
Busca de Comunicante/métodos , Infecção Hospitalar/transmissão , Infecções por Bactérias Gram-Positivas/transmissão , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Vigilância da População/métodos , Centros de Atenção Terciária/estatística & dados numéricos , Algoritmos , Antibacterianos/farmacologia , Infecção Hospitalar/microbiologia , Infecção Hospitalar/prevenção & controle , Enterococcus faecium/classificação , Enterococcus faecium/efeitos dos fármacos , Enterococcus faecium/genética , Alemanha/epidemiologia , Infecções por Bactérias Gram-Positivas/epidemiologia , Infecções por Bactérias Gram-Positivas/microbiologia , Humanos , Controle de Infecções/métodos , Modelos Teóricos , Filogenia , Dinâmica Populacional , Estudos Retrospectivos , Vancomicina/farmacologia , Enterococos Resistentes à Vancomicina/efeitos dos fármacos , Enterococos Resistentes à Vancomicina/genética , Enterococos Resistentes à Vancomicina/fisiologia
6.
Br J Haematol ; 187(3): 386-395, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31273765

RESUMO

Hereditary spherocytosis (HS) is characterised by increased osmotic fragility and enhanced membrane loss of red blood cells (RBC) due to defective membrane protein complexes. In our diagnostic laboratory, we observed that pyruvate kinase (PK) activity in HS was merely slightly elevated with respect to the amount of reticulocytosis. In order to evaluate whether impaired PK activity is a feature of HS, we retrospectively analysed laboratory data sets from 172 unrelated patients with HS, hereditary elliptocytosis (HE), glucose-6-phosphate dehydrogenase (G6PD) or PK deficiency, sickle cell or haemoglobin C disease, or ß-thalassaemia minor. Results from linear regression analysis provided proof that PK activity decreases with rising reticulocyte counts in HS (R2  = 0·15; slope = 9·09) and, less significantly, in HE (R2  = 0·021; slope = 8·92) when compared with other haemolytic disorders (R2  ≥ 0·65; slopes ≥ 78·6). Reticulocyte-adjusted erythrocyte PK activity levels were significantly lower in HS and even declined with increasing reticulocytes (R2  = 0·48; slope = -9·74). In this report, we describe a novel association between HS and decreased PK activity that is apparently caused by loss of membrane-bound PK due to impaired structural integrity of the RBC membrane and may aggravate severity of haemolysis in HS.


Assuntos
Membrana Eritrocítica/enzimologia , Eritrócitos Anormais/enzimologia , Piruvato Quinase/metabolismo , Esferocitose Hereditária/enzimologia , Adolescente , Adulto , Idoso , Anemia Hemolítica Congênita não Esferocítica/enzimologia , Anemia Hemolítica Congênita não Esferocítica/patologia , Anemia Falciforme/enzimologia , Anemia Falciforme/patologia , Criança , Pré-Escolar , Membrana Eritrocítica/patologia , Eritrócitos Anormais/patologia , Feminino , Doença da Hemoglobina C/enzimologia , Doença da Hemoglobina C/patologia , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Piruvato Quinase/deficiência , Erros Inatos do Metabolismo dos Piruvatos/enzimologia , Erros Inatos do Metabolismo dos Piruvatos/patologia , Reticulócitos/enzimologia , Reticulócitos/patologia , Esferocitose Hereditária/patologia , Talassemia beta/enzimologia , Talassemia beta/patologia
7.
Plant Physiol ; 172(4): 2471-2490, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27789739

RESUMO

A variety of eukaryotes, in particular plants, do not contain the required number of tRNAs to support the translation of mitochondria-encoded genes and thus need to import tRNAs from the cytosol. This study identified two Arabidopsis (Arabidopsis thaliana) proteins, Tric1 and Tric2 (for tRNA import component), which on simultaneous inactivation by T-DNA insertion lines displayed a severely delayed and chlorotic growth phenotype and significantly reduced tRNA import capacity into isolated mitochondria. The predicted tRNA-binding domain of Tric1 and Tric2, a sterile-α-motif at the C-terminal end of the protein, was required to restore tRNA uptake ability in mitochondria of complemented plants. The purified predicted tRNA-binding domain binds the T-arm of the tRNA for alanine with conserved lysine residues required for binding. T-DNA inactivation of both Tric proteins further resulted in an increase in the in vitro rate of in organello protein synthesis, which was mediated by a reorganization of the nuclear transcriptome, in particular of genes encoding a variety of proteins required for mitochondrial gene expression at both the transcriptional and translational levels. The characterization of Tric1/2 provides mechanistic insight into the process of tRNA import into mitochondria and supports the theory that the tRNA import pathway resulted from the repurposing of a preexisting protein import apparatus.


Assuntos
Sistemas de Transporte de Aminoácidos/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Mitocôndrias/metabolismo , Transporte de RNA , RNA de Transferência/metabolismo , Sequência de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/química , Deleção de Genes , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Mitocôndrias/ultraestrutura , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Folhas de Planta/metabolismo , Folhas de Planta/ultraestrutura , Ligação Proteica , Biossíntese de Proteínas , Domínios Proteicos , RNA de Transferência/química , Proteínas de Ligação a RNA/metabolismo , Especificidade da Espécie , Transcriptoma/genética
8.
J Exp Med ; 213(9): 1881-900, 2016 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-27526711

RESUMO

Donor CD4(+)Foxp3(+) regulatory T cells (T reg cells) suppress graft-versus-host disease (GvHD) after allogeneic hematopoietic stem cell transplantation (HCT [allo-HCT]). Current clinical study protocols rely on the ex vivo expansion of donor T reg cells and their infusion in high numbers. In this study, we present a novel strategy for inhibiting GvHD that is based on the in vivo expansion of recipient T reg cells before allo-HCT, exploiting the crucial role of tumor necrosis factor receptor 2 (TNFR2) in T reg cell biology. Expanding radiation-resistant host T reg cells in recipient mice using a mouse TNFR2-selective agonist before allo-HCT significantly prolonged survival and reduced GvHD severity in a TNFR2- and T reg cell-dependent manner. The beneficial effects of transplanted T cells against leukemia cells and infectious pathogens remained unaffected. A corresponding human TNFR2-specific agonist expanded human T reg cells in vitro. These observations indicate the potential of our strategy to protect allo-HCT patients from acute GvHD by expanding T reg cells via selective TNFR2 activation in vivo.


Assuntos
Doença Enxerto-Hospedeiro/prevenção & controle , Receptores Tipo II do Fator de Necrose Tumoral/fisiologia , Linfócitos T Reguladores/imunologia , Doença Aguda , Animais , Feminino , Doença Enxerto-Hospedeiro/imunologia , Transplante de Células-Tronco Hematopoéticas , Interleucina-2/farmacologia , Camundongos , Camundongos Endogâmicos , Células Supressoras Mieloides/fisiologia
9.
Blood ; 126(4): 437-44, 2015 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-26012567

RESUMO

Inhibition of the tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK)/fibroblast growth factor-inducible 14 (Fn14) system reduces intestinal cell death and disease development in several models of colitis. In view of the crucial role of TNF and intestinal cell death in graft-versus-host disease (GVHD) and the ability of TWEAK to enhance TNF-induced cell death, we tested here the therapeutic potential of Fn14 blockade on allogeneic hematopoietic cell transplantation (allo-HCT)-induced intestinal GVHD. An Fn14-specific blocking human immunoglobulin G1 antibody variant with compromised antibody-dependent cellular cytotoxicity (ADCC) activity strongly inhibited the severity of murine allo-HCT-induced GVHD. Treatment of the allo-HCT recipients with this monoclonal antibody reduced cell death of gastrointestinal cells but neither affected organ infiltration by donor T cells nor cytokine production. Fn14 blockade also inhibited intestinal cell death in mice challenged with TNF. This suggests that the protective effect of Fn14 blockade in allo-HCT is based on the protection of intestinal cells from TNF-induced apoptosis and not due to immune suppression. Importantly, Fn14 blockade showed no negative effect on graft-versus-leukemia/lymphoma (GVL) activity. Thus, ADCC-defective Fn14-blocking antibodies are not only possible novel GVL effect-sparing therapeutics for the treatment of GVHD but might also be useful for the treatment of other inflammatory bowel diseases where TNF-induced cell death is of relevance.


Assuntos
Anticorpos Monoclonais Murinos/uso terapêutico , Apoptose , Doença Enxerto-Hospedeiro/prevenção & controle , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Intestinos/patologia , Receptores do Fator de Necrose Tumoral/antagonistas & inibidores , Inibidores do Fator de Necrose Tumoral , Animais , Citotoxicidade Celular Dependente de Anticorpos , Western Blotting , Células Cultivadas , Citocina TWEAK , Modelos Animais de Doenças , Feminino , Imunofluorescência , Doença Enxerto-Hospedeiro/etiologia , Doença Enxerto-Hospedeiro/metabolismo , Doença Enxerto-Hospedeiro/patologia , Humanos , Imunoglobulina G/administração & dosagem , Imunoglobulina G/imunologia , Mucosa Intestinal/metabolismo , Intestinos/imunologia , Medições Luminescentes , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores de IgG/imunologia , Receptores de IgG/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 , Rituximab , Receptor de TWEAK , Fator de Necrose Tumoral alfa/farmacologia , Fatores de Necrose Tumoral/imunologia , Fatores de Necrose Tumoral/metabolismo
10.
Cell Commun Signal ; 12: 36, 2014 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-24965524

RESUMO

CCN family member 1 (CCN1), also known as cysteine-rich angiogenic inducer 61 (CYR61), belongs to the extracellular matrix-associated CCN protein family. The diverse functions of these proteins include regulation of cell migration, adhesion, proliferation, differentiation and survival/apoptosis, induction of angiogenesis and cellular senescence. Their functions are partly overlapping, largely non-redundant, cell-type specific, and depend on the local microenvironment. To elucidate the role of CCN1 in the crosstalk between stromal cells and myeloma cells, we performed co-culture experiments with primary mesenchymal stem cells (MSC) and the interleukin-6 (IL-6)-dependent myeloma cell line INA-6. Here we show that INA-6 cells display increased transcription and induction of splicing of intron-retaining CCN1 pre-mRNA when cultured in contact with MSC. Protein analyses confirmed that INA-6 cells co-cultured with MSC show increased levels of CCN1 protein consistent with the existence of a pre-mature stop codon in intron 1 that abolishes translation of unspliced mRNA. Addition of recombinant CCN1-Fc protein to INA-6 cells was also found to induce splicing of CCN1 pre-mRNA in a concentration-dependent manner. Only full length CCN1-Fc was able to induce mRNA splicing of all introns, whereas truncated recombinant isoforms lacking domain 4 failed to induce intron splicing. Blocking RGD-dependent integrins on INA-6 cells resulted in an inhibition of these splicing events. These findings expand knowledge on splicing of the proangiogenic, matricellular factor CCN1 in the tumor microenvironment. We propose that contact with MSC-derived CCN1 leads to splicing and enhanced transcription of CCN1 which further contributes to the translation of angiogenic factor CCN1 in myeloma cells, supporting tumor viability and myeloma bone disease.


Assuntos
Proteína Rica em Cisteína 61/metabolismo , Células-Tronco Mesenquimais/metabolismo , Mieloma Múltiplo/metabolismo , Processamento de RNA , RNA Mensageiro/metabolismo , Transcrição Genética , Linhagem Celular Tumoral , Células Cultivadas , Técnicas de Cocultura , Meios de Cultivo Condicionados/farmacologia , Proteína Rica em Cisteína 61/genética , Proteína Rica em Cisteína 61/farmacologia , Humanos , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , RNA Mensageiro/genética , Proteínas Recombinantes/farmacologia
11.
J Cell Mol Med ; 18(7): 1444-59, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24779367

RESUMO

Members of the transforming growth factor (TGF)-ß family govern a wide range of mechanisms in brain development and in the adult, in particular neuronal/glial differentiation and survival, but also cell cycle regulation and neural stem cell maintenance. This clearly created some discrepancies in the field with some studies favouring neuronal differentiation/survival of progenitors and others favouring cell cycle exit and neural stem cell quiescence/maintenance. Here, we provide a unifying hypothesis claiming that through its regulation of neural progenitor cell (NPC) proliferation, TGF-ß signalling might be responsible for (i) maintaining stem cells in a quiescent stage, and (ii) promoting survival of newly generated neurons and their functional differentiation. Therefore, we performed a detailed histological analysis of TGF-ß1 signalling in the hippocampal neural stem cell niche of a transgenic mouse that was previously generated to express TGF-ß1 under a tetracycline regulatable Ca-Calmodulin kinase promoter. We also analysed NPC proliferation, quiescence, neuronal survival and differentiation in relation to elevated levels of TGF-ß1 in vitro and in vivo conditions. Finally, we performed a gene expression profiling to identify the targets of TGF-ß1 signalling in adult NPCs. The results demonstrate that TGF-ß1 promotes stem cell quiescence on one side, but also neuronal survival on the other side. Thus, considering the elevated levels of TGF-ß1 in ageing and neurodegenerative diseases, TGF-ß1 signalling presents a molecular target for future interventions in such conditions.


Assuntos
Diferenciação Celular , Hipocampo/citologia , Neurogênese/fisiologia , Neurônios/citologia , Nicho de Células-Tronco , Células-Tronco/citologia , Fator de Crescimento Transformador beta/metabolismo , Animais , Biomarcadores/metabolismo , Western Blotting , Proliferação de Células , Células Cultivadas , Senescência Celular , Eletrofisiologia , Feminino , Perfilação da Expressão Gênica , Hipocampo/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Neurônios/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , Ratos , Ratos Endogâmicos F344 , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células-Tronco/metabolismo , Fator de Crescimento Transformador beta/genética
12.
PLoS One ; 8(12): e81320, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24349055

RESUMO

To promote cancer research and to develop innovative therapies, refined pre-clinical mouse tumor models that mimic the actual disease in humans are of dire need. A number of neoplasms along the B cell lineage are commonly initiated by a translocation recombining c-myc with the immunoglobulin heavy-chain gene locus. The translocation is modeled in the C.129S1-Igha(tm1(Myc)Janz)/J mouse which has been previously engineered to express c-myc under the control of the endogenous IgH promoter. This transgenic mouse exhibits B cell hyperplasia and develops diverse B cell tumors. We have isolated tumor cells from the spleen of a C.129S1-Igha(tm1(Myc)Janz)/J mouse that spontaneously developed a plasmablastic lymphoma-like disease. These cells were cultured, transduced to express eGFP and firefly luciferase, and gave rise to a highly aggressive, transplantable B cell lymphoma cell line, termed IM380. This model bears several advantages over other models as it is genetically induced and mimics the translocation that is detectable in a number of human B cell lymphomas. The growth of the tumor cells, their dissemination, and response to treatment within immunocompetent hosts can be imaged non-invasively in vivo due to their expression of firefly luciferase. IM380 cells are radioresistant in vivo and mice with established tumors can be allogeneically transplanted to analyze graft-versus-tumor effects of transplanted T cells. Allogeneic hematopoietic stem cell transplantation of tumor-bearing mice results in prolonged survival. These traits make the IM380 model very valuable for the study of B cell lymphoma pathophysiology and for the development of innovative cancer therapies.


Assuntos
Diagnóstico por Imagem/métodos , Medições Luminescentes/métodos , Linfoma de Células B/patologia , Animais , Linfócitos B , Linhagem Celular Tumoral , Feminino , Citometria de Fluxo , Transplante de Células-Tronco Hematopoéticas , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Transplante Homólogo
13.
PLoS One ; 8(9): e75737, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24098720

RESUMO

Multiple activities are ascribed to the cytokine tumor necrosis factor (TNF) in health and disease. In particular, TNF was shown to affect carcinogenesis in multiple ways. This cytokine acts via the activation of two cell surface receptors, TNFR1, which is associated with inflammation, and TNFR2, which was shown to cause anti-inflammatory signaling. We assessed the effects of TNF and its two receptors on the progression of pancreatic cancer by in vivo bioluminescence imaging in a syngeneic orthotopic tumor mouse model with Panc02 cells. Mice deficient for TNFR1 were unable to spontaneously reject Panc02 tumors and furthermore displayed enhanced tumor progression. In contrast, a fraction of wild type (37.5%), TNF deficient (12.5%), and TNFR2 deficient mice (22.2%) were able to fully reject the tumor within two weeks. Pancreatic tumors in TNFR1 deficient mice displayed increased vascular density, enhanced infiltration of CD4(+) T cells and CD4(+) forkhead box P3 (FoxP3)(+) regulatory T cells (Treg) but reduced numbers of CD8(+) T cells. These alterations were further accompanied by transcriptional upregulation of IL4. Thus, TNF and TNFR1 are required in pancreatic ductal carcinoma to ensure optimal CD8(+) T cell-mediated immunosurveillance and tumor rejection. Exogenous systemic administration of human TNF, however, which only interacts with murine TNFR1, accelerated tumor progression. This suggests that TNFR1 has basically the capability in the Panc02 model to trigger pro-and anti-tumoral effects but the spatiotemporal availability of TNF seems to determine finally the overall outcome.


Assuntos
Carcinoma Ductal/fisiopatologia , Regulação da Expressão Gênica/imunologia , Neoplasias Pancreáticas/fisiopatologia , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Linfócitos T CD4-Positivos/imunologia , Carcinoma Ductal/imunologia , Carcinoma Ductal/metabolismo , Linhagem Celular Tumoral , Primers do DNA/genética , Citometria de Fluxo , Interleucina-4/metabolismo , Medições Luminescentes , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Receptores Tipo I de Fatores de Necrose Tumoral/deficiência , Receptores Tipo II do Fator de Necrose Tumoral/deficiência , Reação em Cadeia da Polimerase Via Transcriptase Reversa
14.
Glia ; 61(11): 1767-83, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24038377

RESUMO

The differentiation of adult neural progenitors (NPCs) into functional neurons is still a limiting factor in the neural stem cell field but mandatory for the potential use of NPCs in therapeutic approaches. Neuronal function requires the appropriate electrophysiological properties. Here, we demonstrate that priming of NPCs using transforming growth factor (TGF)-ß1 under conditions that usually favor NPCs' proliferation induces electrophysiological neuronal properties in adult NPCs. Gene chip array analyses revealed upregulation of voltage-dependent ion channel subunits (Kcnd3, Scn1b, Cacng4, and Accn1), neurotransmitters, and synaptic proteins (Cadps, Snap25, Grik4, Gria3, Syngr3, and Gria4) as well as other neuronal proteins (doublecortin [DCX], Nrxn1, Sept8, and Als2cr3). Patch-clamp analysis demonstrated that control-treated cells expressed only voltage-dependent K(+) -channels of the delayed-rectifier type and the A-type channels. TGF-ß1-treated cells possessed more negative resting potentials than nontreated cells owing to the presence of delayed-rectifier and inward-rectifier channels. Furthermore, TGF-ß1-treated cells expressed voltage-dependent, TTX-sensitive Na(+) channels, which showed increasing current density with TGF-ß1 treatment duration and voltage-dependent (+)BayK8644-sensitive L-Type Ca(2+) channels. In contrast to nontreated cells, TGF-ß1-treated cells responded to current injections with action-potentials in the current-clamp mode. Furthermore, TGF-ß1-treated cells responded to application of GABA with an increase in membrane conductance and showed spontaneous synaptic currents that were blocked by the GABA-receptor antagonist picrotoxine. Only NPCs, which were treated with TGF-ß1, showed Na(+) channel currents, action potentials, and GABAergic currents. In summary, stimulation of NPCs by TGF-ß1 fosters a functional neuronal phenotype, which will be of relevance for future cell replacement strategies in neurodegenerative diseases or acute CNS lesions.


Assuntos
Potenciais de Ação/fisiologia , Diferenciação Celular/fisiologia , Proliferação de Células , Canais de Potássio/metabolismo , Células-Tronco/citologia , Fator de Crescimento Transformador beta1/metabolismo , Potenciais de Ação/efeitos dos fármacos , Envelhecimento , Animais , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Feminino , Potenciais da Membrana/fisiologia , Canais de Potássio/efeitos dos fármacos , Ratos , Ratos Endogâmicos F344 , Células-Tronco/metabolismo , Tetrodotoxina/farmacologia
15.
Carcinogenesis ; 34(6): 1296-303, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23385062

RESUMO

The cytokine tumor necrosis factor (TNF) has pleiotropic functions both in normal physiology and disease. TNF signals by the virtue of two cell surface receptors, TNF receptor 1 (TNFR1) and TNF receptor 2 (TNFR2). Exogenous TNF promotes experimental metastasis in some models, yet the underlying mechanisms are poorly understood. To study the contribution of host TNFR1 and TNFR2 on tumor cell progression and metastasis, we employed a syngeneic B16F10 melanoma mouse model of lung metastasis combined with in vivo bioluminescence imaging. Treatment of tumor-bearing mice with recombinant human TNF resulted in a significant increase in tumor burden and metastatic foci. This correlated with an increase in pulmonary regulatory CD4(+)/Foxp3(+) T cells. TNF caused an expansion of regulatory T (Treg) cells in vitro in a TNFR2-dependent manner. To assess the contribution of immune cell expression of endogenous TNF and its two receptors on B16F10 metastasis, we generated bone marrow chimeras by reconstituting wild-type mice with bone marrow from different knockout mice. Loss of either TNF or TNFR2 on immune cells resulted in decreased B16F10 metastasis and lower numbers of Treg cells within the lungs of these animals. Selective depletion of Treg cells attenuated metastasis even in conjunction with TNF treatment. We propose a novel mechanism in which TNF activates TNFR2 on Treg cells and thereby expands this immunosuppressive immune cell population. Loss of either TNF or TNFR2 prevents the accumulation of Treg cells and results in a less tolerogenic environment, enabling the immune system to control B16F10 tumor metastasis and growth.


Assuntos
Neoplasias Pulmonares/metabolismo , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Linfócitos T Reguladores/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Animais , Antígenos CD4/biossíntese , Linhagem Celular Tumoral , Proliferação de Células , Fatores de Transcrição Forkhead/biossíntese , Neoplasias Pulmonares/secundário , Melanoma , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Metástase Neoplásica , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Linfócitos T Reguladores/imunologia , Fator de Necrose Tumoral alfa/metabolismo
16.
Stem Cell Rev Rep ; 7(4): 815-35, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21431886

RESUMO

It is commonly accepted that adult neurogenesis and gliogenesis follow the same principles through the mammalian class. However, it has been reported that neurogenesis might differ between species, even from the same order, like in rodents. Currently, it is not known if neural stem/progenitor cells (NSPCs) from various species differ in their cell identity and potential. NSPCs can be expanded ex vivo as neurospheres (NSph), a model widely used to study neurogenesis in vitro. Here we demonstrate that rat (r) and mouse (m) NSph display different cell identities, differentiation fate, electrophysiological function and tumorigenic potential. Adult rNSph consist mainly of oligodendroglial progenitors (OPCs), which after repeated passaging proliferate independent of mitogens, whereas adult mNSph show astroglial precursor-like characteristics and retain their mitogen dependency. Most of the cells in rNSph express OPC markers and spontaneously differentiate into oligodendrocytes after growth factor withdrawal. Electrophysiological analysis confirmed OPC characteristics. mNSph have different electrophysiological properties, they express astrocyte precursor markers and spontaneously differentiate primarily into astrocytes. Furthermore, rNSph have the potential to differentiate into oligodendrocytes and astrocytes, whereas mNSph are restricted to the astrocytic lineage. The phenotypic differences between rNSph and mNSph were not due to a distinct response to species specific derived growth factors and are probably not caused by autocrine mechanisms. Our findings suggest that NSph derived from adult rat and mouse brains display different cell identities. Thus, results urge for caution when data derived from NSph are extrapolated to other species or to the in vivo situation, especially when aimed towards the clinical use of human NSph.


Assuntos
Diferenciação Celular , Células-Tronco Neurais/citologia , Oligodendroglia/citologia , Animais , Astrócitos/citologia , Astrócitos/metabolismo , Biomarcadores/metabolismo , Adesão Celular , Agregação Celular , Contagem de Células , Técnicas de Cultura de Células , Linhagem da Célula , Proliferação de Células , Células Cultivadas , Cromossomos de Mamíferos/genética , Meios de Cultivo Condicionados , Eletrofisiologia , Feminino , Potenciais da Membrana , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Neurais/metabolismo , Oligodendroglia/metabolismo , Técnicas de Patch-Clamp , Ratos , Ratos Endogâmicos F344 , Especificidade da Espécie
17.
Mol Plant Pathol ; 11(2): 227-43, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20447272

RESUMO

Oomycete plant pathogens cause a wide variety of economically and environmentally important plant diseases. Mandipropamid (MPD) is a carboxylic acid amide (CAA) effective against downy mildews, such as Plasmopara viticola on grapes and potato late blight caused by Phytophthora infestans. Historically, the identification of the mode of action of oomycete-specific control agents has been problematic. Here, we describe how a combination of biochemical and genetic techniques has been utilized to identify the molecular target of MPD in P. infestans. Phytophthora infestans germinating cysts treated with MPD produced swelling symptoms typical of cell wall synthesis inhibitors, and these effects were reversible after washing with H(2)O. Uptake studies with (14)C-labelled MPD showed that this oomycete control agent acts on the cell wall and does not enter the cell. Furthermore, (14)C glucose incorporation into cellulose was perturbed in the presence of MPD which, taken together, suggests that the inhibition of cellulose synthesis is the primary effect of MPD. Laboratory mutants, insensitive to MPD, were raised by ethyl methane sulphonate (EMS) mutagenesis, and gene sequence analysis of cellulose synthase genes in these mutants revealed two point mutations in the PiCesA3 gene, known to be involved in cellulose synthesis. Both mutations in the PiCesA3 gene result in a change to the same amino acid (glycine-1105) in the protein. The transformation and expression of a mutated PiCesA3 allele was carried out in a sensitive wild-type isolate to demonstrate that the mutations in PiCesA3 were responsible for the MPD insensitivity phenotype.


Assuntos
Proteínas de Algas/metabolismo , Amidas/farmacologia , Ácidos Carboxílicos/farmacologia , Parede Celular/metabolismo , Glucosiltransferases/metabolismo , Phytophthora infestans/enzimologia , Plantas/microbiologia , Proteínas de Algas/química , Proteínas de Algas/genética , Sequência de Aminoácidos , Parede Celular/efeitos dos fármacos , Celulose/biossíntese , Cruzamentos Genéticos , Metanossulfonato de Etila , Dosagem de Genes/genética , Glucose/metabolismo , Glucosiltransferases/química , Glucosiltransferases/genética , Dados de Sequência Molecular , Mutagênese/efeitos dos fármacos , Mutação/genética , Phytophthora infestans/citologia , Phytophthora infestans/efeitos dos fármacos , Phytophthora infestans/genética , Plantas/efeitos dos fármacos , Transformação Genética/efeitos dos fármacos
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