Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 10 de 10
Filtrar
1.
Eur J Clin Invest ; 46(10): 853-63, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27513763

RESUMO

BACKGROUND: Several activities are attributed to antimicrobial peptides (AMPs), including bacterial killing, leucocyte recruitment and angiogenesis. Despite promises of advanced cellular therapies for treatment of diabetic foot ulcer, it is currently accepted that paracrine factors rather than cellular components are causative for the observed effects. Whether AMPs are present in the mononuclear cell (MNC) secretome (MNC-sec) of white blood cells that are beneficial in experimental wound healing is not known. MATERIALS AND METHODS: Antimicrobial activity of the secretomes of nonirradiated (MNC-sec) and γ-irradiated MNCs (MNC-sec rad) was analysed by microdilution assay. AMPs were determined by quantitative real-time PCR (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). Whether human MNC-sec rad causes AMP secretion in vivo was examined in an experimental rat model. Image flow cytometry was used to determine the type of cell death induced in MNCs after exposure to γ-radiation. RESULTS: The antimicrobial activity assay revealed a bactericidal activity of MNC-sec rad and to a lesser degree also of MNC-sec. Image flow cytometry showed that γ-irradiation of MNCs induced early apoptosis followed mainly by necroptosis. RT-PCR and ELISA revealed a high abundance of different AMPs in the secretome of MNCs. In addition, human MNC-sec elicited an increase in de novo endogenous AMP production in rats in vivo. CONCLUSION: We provide evidence that the secretome of MNCs has direct and indirect positive effects on the immune defence system, including augmentation of antibacterial properties. Our data further suggest that necroptosis could play a key role for the release of paracrine factors and the therapeutic action of MNC-sec rad.


Assuntos
Anti-Infecciosos/farmacocinética , Peptídeos Catiônicos Antimicrobianos/farmacologia , Infecções por Bactérias Gram-Negativas/tratamento farmacológico , Infecções por Bactérias Gram-Positivas/tratamento farmacológico , Leucócitos Mononucleares/metabolismo , Adolescente , Adulto , Animais , Peptídeos Catiônicos Antimicrobianos/metabolismo , Apoptose/efeitos da radiação , Morte Celular , Raios gama , Humanos , Leucócitos Mononucleares/fisiologia , Masculino , Ratos Sprague-Dawley , Adulto Jovem
2.
Exp Cell Res ; 339(1): 1-9, 2015 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-26453936

RESUMO

Combination therapy of cancer is based on the cumulative effects mediated by several drugs. Although molecular mechanisms of action of each particular drug are partially elucidated, understanding of the dynamic cross-talk between different cell death pathways at the quantitative level induced by combination therapy is still missing. Here, we exemplified this question for the death receptor (DR) networks in pancreatic cancer cells. We demonstrate that the combined action of CD95L and gemcitabine in pancreatic cancer cells leads to the simultaneous induction of caspase-dependent and caspase-independent cell death. The pro-apoptotic effects are mediated through down-regulation of the anti-apoptotic proteins c-FLIP and Mcl-1, while caspase-independent cell death was blocked by inhibition of the kinase activity of RIP1. Furthermore, gemcitabine co-treatment strongly increased the amount of cells undergoing CD95-induced RIP1-regulated necrosis. Imaging flow cytometry has enabled us to get the quantitative insights into the apoptosis-necroptosis network and reveal that the majority of the cells upon the CD95L/gemcitabine co-treatment undergoes necroptosis. Our data underlie the importance of the quantitative understanding of the interplay between different cell death modalities, which is essential for the development of anti-cancer therapies. Taken together, our results are important for combination therapy of pancreatic cancer comprising chemotherapeutics and DR-agonists and offer a possibility to sensitize cells with defects in the apoptotic machinery towards necroptosis-type-mediated death.


Assuntos
Apoptose/efeitos dos fármacos , Desoxicitidina/análogos & derivados , Proteína Ligante Fas/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Neoplasias Pancreáticas/patologia , Proteínas de Ligação a RNA/metabolismo , Trifosfato de Adenosina/metabolismo , Antimetabólitos Antineoplásicos/farmacologia , Western Blotting , Caspases/metabolismo , Proliferação de Células/efeitos dos fármacos , Terapia Combinada , Desoxicitidina/farmacologia , Humanos , Necrose , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células Tumorais Cultivadas , Receptor fas/metabolismo , Gencitabina
3.
Lung ; 193(6): 947-53, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26319657

RESUMO

PURPOSE: Chronic lung inflammation commonly induces a multitude of structural and functional adaptations within the lung tissue and airspaces. Yet the impact of a persistent inflammatory environment on alveolar macrophages is still incompletely understood. Here, we examined morphology and function of alveolar macrophages in a transgenic mouse model of chronic lung disease. METHODS: Imaging flow cytometry, flow cytometry, and microscopic evaluation of alveolar macrophages isolated from healthy and inflamed lungs were performed. Gene expression of polarization markers was compared by quantitative real-time RT-PCR. The pro-inflammatory immune response of alveolar macrophages toward bacterial ligands was assessed in in vivo clodronate-liposome depletion studies. RESULTS: Chronic lung inflammation is associated with a substantially altered, activated alveolar macrophage morphology, and blunted TNF-α response by these cells following stimulation with ligands derived from the respiratory pathogen Streptococcus pneumoniae. CONCLUSIONS: These results demonstrate pleiotropic effects of pulmonary inflammation on alveolar macrophage phenotype and function and suggest a functional impairment of these cells during infection with airborne pathogens.


Assuntos
Doenças Autoimunes/imunologia , Inflamação/imunologia , Peptídeos e Proteínas de Sinalização Intercelular/imunologia , Pneumopatias/imunologia , Macrófagos Alveolares/imunologia , Receptores Depuradores Classe A/imunologia , Infecções Estreptocócicas/imunologia , Fator de Necrose Tumoral alfa/imunologia , Animais , Doença Crônica , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Macrófagos Alveolares/metabolismo , Camundongos , Camundongos Transgênicos , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Receptores Depuradores Classe A/genética , Streptococcus pneumoniae/imunologia
4.
Oncogene ; 43(25): 1955-1971, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38730267

RESUMO

Procaspase-8 is a key mediator of death receptor (DR)-mediated pathways. Recently, the role of post-translational modifications (PTMs) of procaspase-8 in controlling cell death has received increasing attention. Here, using mass spectrometry screening, pharmacological inhibition and biochemical assays, we show that procaspase-8 can be targeted by the PRMT5/RIOK1/WD45 methylosome complex. Furthermore, two potential methylation sites of PRMT5 on procaspase-8, R233 and R435, were identified in silico. R233 and R435 are highly conserved in mammals and their point mutations are among the most common mutations of caspase-8 in cancer. The introduction of mutations at these positions resulted in inhibitory effects on CD95L-induced caspase-8 activity, effector caspase activation and apoptosis. In addition, we show that procaspase-8 can undergo symmetric di-methylation. Finally, the pharmacological inhibition of PRMT5 resulted in the inhibitory effects on caspase activity and apoptotic cell death. Taken together, we have unraveled the additional control checkpoint in procaspase-8 activation and the arginine methylation network in the extrinsic apoptosis pathway.


Assuntos
Apoptose , Arginina , Caspase 8 , Proteína-Arginina N-Metiltransferases , Caspase 8/metabolismo , Caspase 8/genética , Arginina/metabolismo , Humanos , Metilação , Proteína-Arginina N-Metiltransferases/metabolismo , Proteína-Arginina N-Metiltransferases/genética , Processamento de Proteína Pós-Traducional
6.
Bio Protoc ; 7(1): e2081, 2017 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-34458412

RESUMO

Apoptosis or programmed cell death is important for multicellular organisms to keep cell homeostasis and for the clearance of mutated or infected cells. Apoptosis can be induced by intrinsic or extrinsic stimuli. The first event in extrinsic apoptosis is the formation of the Death-Inducing Signalling Complex (DISC), where the initiator caspases-8 and -10 are fully activated by several proteolytic cleavage steps and induce the caspase cascade leading to apoptotic cell death. Analysing the processing of procaspases-8 and -10 by Western blot is a commonly used method to study the induction of apoptosis by death receptor stimulation. To analyse procaspase-8 and -10 cleavage, cells are stimulated with a death ligand for different time intervals, lysed and subjected to Western blot analysis using anti-caspase-8 and anti-caspase-10 antibodies. This allows monitoring the caspase cleavage products and thereby induction of apoptosis.

7.
Acta Neuropathol Commun ; 4: 25, 2016 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-26984535

RESUMO

INTRODUCTION: Alzheimer's disease (AD) is associated with the accumulation of ß-amyloid (Aß) as senile plaques in the brain, thus leading to neurodegeneration and cognitive impairment. Plaque formation depends not merely on the amount of generated Aß peptides, but more importantly on their effective removal. Chronic infections with neurotropic pathogens, most prominently the parasite Toxoplasma (T.) gondii, are frequent in the elderly, and it has been suggested that the resulting neuroinflammation may influence the course of AD. In the present study, we investigated how chronic T. gondii infection and resulting neuroinflammation affect plaque deposition and removal in a mouse model of AD. RESULTS: Chronic infection with T. gondii was associated with reduced Aß and plaque load in 5xFAD mice. Upon infection, myeloid-derived CCR2(hi) Ly6C(hi) monocytes, CCR2(+) Ly6C(int), and CCR2(+) Ly6C(low) mononuclear cells were recruited to the brain of mice. Compared to microglia, these recruited mononuclear cells showed highly increased phagocytic capacity of Aß ex vivo. The F4/80(+) Ly6C(low) macrophages expressed high levels of Triggering Receptor Expressed on Myeloid cells 2 (TREM2), CD36, and Scavenger Receptor A1 (SCARA1), indicating phagocytic activity. Importantly, selective ablation of CCR2(+) Ly6C(hi) monocytes resulted in an increased amount of Aß in infected mice. Elevated insulin-degrading enzyme (IDE), matrix metalloproteinase 9 (MMP9), as well as immunoproteasome subunits ß1i/LMP2, ß2i/MECL-1, and ß5i/LMP7 mRNA levels in the infected brains indicated increased proteolytic Aß degradation. Particularly, LMP7 was highly expressed by the recruited mononuclear cells in the brain, suggesting a novel mechanism of Aß clearance. CONCLUSIONS: Our results indicate that chronic Toxoplasma infection ameliorates ß-amyloidosis in a murine model of AD by activation of the immune system, specifically by recruitment of Ly6C(hi) monocytes and by enhancement of phagocytosis and degradation of soluble Aß. Our findings provide evidence for a modulatory role of inflammation-induced Aß phagocytosis and degradation by newly recruited peripheral immune cells in the pathophysiology of AD.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Macrófagos/metabolismo , Monócitos/fisiologia , Fagocitose/fisiologia , Toxoplasmose/metabolismo , Toxoplasmose/patologia , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Animais , Anticorpos/farmacologia , Antígenos CD/metabolismo , Antígenos Ly/metabolismo , Encéfalo/parasitologia , Encéfalo/patologia , Proteínas de Ligação ao Cálcio/metabolismo , Doença Crônica , Modelos Animais de Doenças , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas dos Microfilamentos/metabolismo , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Monócitos/patologia , Fagocitose/genética , Placa Amiloide/metabolismo , Placa Amiloide/patologia , Receptores CCR2/genética , Receptores CCR2/imunologia
8.
J Immunol Methods ; 423: 99-103, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25975759

RESUMO

Precisely identifying the type of programmed cell death is one of the key questions in contemporary biomedical research. We developed a straightforward approach allowing quantitative discrimination between two types of cell death on the single cell level: apoptosis and necroptosis. This method uses the combination of imaging flow cytometry with classical Annexin V/propidium iodide staining, which allows for the ascertainment of typical features of dying cells: exposure of the phospholipid phosphatidylserine and the loss of membrane integrity. Image-based analysis of nuclear morphology enables us to distinguish between secondary necrotic/late apoptotic and necroptotic cells directly in one assay. This is a major advantage compared to other contemporary approaches of necroptosis detection, which require a parallel application of several methods. This approach can be used for the quantitative assessment of cell death in cell and systems biology studies of signal transduction networks.


Assuntos
Anexina A5/metabolismo , Apoptose/fisiologia , Necrose/patologia , Propídio/metabolismo , Análise de Célula Única/métodos , Morte Celular/fisiologia , Linhagem Celular Tumoral , Citometria de Fluxo/métodos , Humanos , Citometria por Imagem/métodos , Células Jurkat , Necrose/metabolismo , Fosfatidilserinas/metabolismo , Coloração e Rotulagem/métodos
9.
J Immunol Methods ; 423: 12-7, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25967949

RESUMO

CD95/Fas/APO-1 is a member of the death receptor (DR) family. Stimulation of CD95 leads to the induction of apoptosis as well as to NF-κB signaling. Crosstalk between these two pathways plays a central role in cell fate. Defects in the regulation of apoptosis and of NF-κB are connected to a number of chronic inflammatory diseases and cancer. For a better understanding of the life/death decisions in the cell and their contribution to disease progression, the development of new technologies is required. Using imaging flow cytometry we developed a method that enables a quantitative detection of different CD95 signaling pathways in the single cell. The important advantage of this method compared to other approaches is that it allows quantifying a large number of single cells undergoing apoptosis and NF-κB activation. This technology could provide new insights into the quantitative characterization of apoptosis and NF-κB at the single cell level and could be used for the quantitative network analysis in systems biology studies.


Assuntos
Apoptose/fisiologia , NF-kappa B/metabolismo , Receptor fas/metabolismo , Linhagem Celular Tumoral , Citometria de Fluxo/métodos , Células HeLa , Humanos , Análise de Célula Única/métodos , Biologia de Sistemas/métodos
10.
PLoS One ; 8(4): e61438, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23593480

RESUMO

Proteasome inhibitors (PIs) potently induce apoptosis in a variety of tumor cells, but the underlying mechanisms are not fully elucidated. Comparing PI-induced apoptosis susceptibilities of various mouse embryonic fibroblast (MEF) lines differing in their c-jun N-terminal kinase (JNK) 1 and 2 status, we show that several hallmarks of apoptosis were most rapidly detectable in JNK2-/- cells, whereas they appeared only delayed and severely reduced in their intensities in cells expressing JNK2. Consistent with our finding that PI-induced apoptosis requires de novo protein synthesis, the proteasomal inhibitor MG-132 induced expression of the BH3-only protein Noxa at the transcriptional level in a JNK1-dependent, but JNK2-opposing manner. As the knockdown of Noxa blocked only the rapid PI-induced apoptosis of JNK2-/- cells, but not the delayed death occurring in JNK1-/- and JNK1+/+ cells, our data uncover a novel PI-induced apoptosis pathway that is regulated by the JNK1/2-dependent expression of Noxa. Furthermore, several transcription factors known to modulate Noxa expression including ATF3, ATF4, c-Jun, c-Myc, HIF1α, and p53 were found upregulated following MG-132 exposure. From those, only knockdown of c-Myc rescued JNK2-/- cells from PI-induced apoptosis, however, without affecting expression of Noxa. Together, our data not only show that a rapid execution of PI-induced apoptosis requires JNK1 for upregulation of Noxa via an as yet unknown transcription factor, but also that JNK2 controls this event in an oppositional manner.


Assuntos
Apoptose/fisiologia , Regulação da Expressão Gênica/fisiologia , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Proteína Quinase 9 Ativada por Mitógeno/metabolismo , Inibidores de Proteassoma/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Análise de Variância , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Linhagem Celular , Fluorometria , Técnicas de Inativação de Genes , Leupeptinas , Camundongos , Proteína Quinase 8 Ativada por Mitógeno/genética , Proteína Quinase 9 Ativada por Mitógeno/genética , Reação em Cadeia da Polimerase
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA