RESUMO
Chronic obstructive pulmonary disease (COPD) is characterized by an irreversible loss of lung function and is one of the most prevalent and severe diseases worldwide. A major feature of COPD is emphysema, which is the progressive loss of alveolar tissue. Coactivator-associated arginine methyltransferase-1 (CARM1) regulates histone methylation and the transcription of genes involved in senescence, proliferation, and differentiation. Complete loss of CARM1 leads to disrupted differentiation and maturation of alveolar epithelial type II (ATII) cells. We thus hypothesized that CARM1 regulates the development and progression of emphysema. To address this, we investigated the contribution of CARM1 to alveolar rarefication using the mouse model of elastase-induced emphysema in vivo and small interfering (si)RNA-mediated knockdown in ATII-like LA4 cells in vitro. We demonstrate that emphysema progression in vivo is associated with a time-dependent down-regulation of CARM1. Importantly, elastase-treated CARM1 haploinsufficient mice show significantly increased airspace enlargement (52.5 ± 9.6 µm versus 38.8 ± 5.5 µm; P < 0.01) and lung compliance (2.8 ± 0.32 µl/cm H2O versus 2.4 ± 0.4 µl/cm H2O; P < 0.04) compared with controls. The knockdown of CARM1 in LA4 cells led to decreased sirtuin 1 expression (0.034 ± 0.003 versus 0.022 ± 0.001; P < 0.05) but increased expression of p16 (0.27 ± 0.013 versus 0.31 ± 0.010; P < 0.5) and p21 (0.81 ± 0.088 versus 1.28 ± 0.063; P < 0.01) and higher ß-galactosidase-positive senescent cells (50.57 ± 7.36% versus 2.21 ± 0.34%; P < 0.001) compared with scrambled siRNA. We further demonstrated that CARM1 haploinsufficiency impairs transdifferentiation and wound healing (32.18 ± 0.9512% versus 8.769 ± 1.967%; P < 0.001) of alveolar epithelial cells. Overall, these results reveal a novel function of CARM1 in regulating emphysema development and premature lung aging via alveolar senescence as well as impaired regeneration, repair, and differentiation of ATII cells.
Assuntos
Células Epiteliais Alveolares/enzimologia , Proteína-Arginina N-Metiltransferases/fisiologia , Enfisema Pulmonar/enzimologia , Animais , Diferenciação Celular , Linhagem Celular , Senescência Celular , Feminino , Predisposição Genética para Doença , Haploinsuficiência , Camundongos Endogâmicos C57BL , Elastase Pancreática , Enfisema Pulmonar/induzido quimicamenteRESUMO
Chronic obstructive lung disease determines morbidity and mortality of patients with cystic fibrosis (CF). CF airways are characterized by a nonresolving neutrophilic inflammation. After pathogen contact or prolonged activation, neutrophils release DNA fibres decorated with antimicrobial proteins, forming neutrophil extracellular traps (NETs). NETs have been described to act in a beneficial way for innate host defense by bactericidal, fungicidal, and virucidal actions. On the other hand, excessive NET formation has been linked to the pathogenesis of autoinflammatory and autoimmune disease conditions. We quantified free DNA structures characteristic of NETs in airway fluids of CF patients and a mouse model with CF-like lung disease. Free DNA levels correlated with airflow obstruction, fungal colonization, and CXC chemokine levels in CF patients and CF-like mice. When viewed in combination, our results demonstrate that neutrophilic inflammation in CF airways is associated with abundant free DNA characteristic for NETosis, and suggest that free DNA may be implicated in lung function decline in patients with CF.
Assuntos
Fibrose Cística/metabolismo , DNA/química , Inflamação/microbiologia , Neutrófilos/metabolismo , Pseudomonas aeruginosa/imunologia , Adolescente , Adulto , Obstrução das Vias Respiratórias/metabolismo , Animais , Líquido da Lavagem Broncoalveolar/química , Quimiocinas CXC/metabolismo , Criança , Modelos Animais de Doenças , Feminino , Humanos , Inflamação/metabolismo , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Neutrófilos/imunologia , Adulto JovemRESUMO
In early stages of various pulmonary diseases, such as emphysema and fibrosis, the change in X-ray attenuation is not detectable with absorption-based radiography. To monitor the morphological changes that the alveoli network undergoes in the progression of these diseases, we propose using the dark-field signal, which is related to small-angle scattering in the sample. Combined with the absorption-based image, the dark-field signal enables better discrimination between healthy and emphysematous lung tissue in a mouse model. All measurements have been performed at 36 keV using a monochromatic laser-driven miniature synchrotron X-ray source (Compact Light Source). In this paper we present grating-based dark-field images of emphysematous vs. healthy lung tissue, where the strong dependence of the dark-field signal on mean alveolar size leads to improved diagnosis of emphysema in lung radiographs.
Assuntos
Enfisema/diagnóstico , Lasers , Síncrotrons , Animais , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Raios XRESUMO
The nuclei of most vertebrate cells contain members of the high mobility group N (HMGN) protein family, which bind specifically to nucleosome core particles and affect chromatin structure and function, including transcription. Here, we study the biological role of this protein family by systematic analysis of phenotypes and tissue transcription profiles in mice lacking functional HMGN variants. Phenotypic analysis of Hmgn1(tm1/tm1), Hmgn3(tm1/tm1), and Hmgn5(tm1/tm1) mice and their wild type littermates with a battery of standardized tests uncovered variant-specific abnormalities. Gene expression analysis of four different tissues in each of the Hmgn(tm1/tm1) lines reveals very little overlap between genes affected by specific variants in different tissues. Pathway analysis reveals that loss of an HMGN variant subtly affects expression of numerous genes in specific biological processes. We conclude that within the biological framework of an entire organism, HMGNs modulate the fidelity of the cellular transcriptional profile in a tissue- and HMGN variant-specific manner.
Assuntos
Regulação da Expressão Gênica/fisiologia , Proteínas HMGN/metabolismo , Transcrição Gênica/fisiologia , Animais , Proteínas HMGN/genética , Camundongos , Camundongos Mutantes , Especificidade de Órgãos/fisiologiaRESUMO
PURPOSE: To test the hypothesis that the joint distribution of x-ray transmission and dark-field signals obtained with a compact cone-beam preclinical scanner with a polychromatic source can be used to diagnose pulmonary emphysema in ex vivo murine lungs. MATERIALS AND METHODS: The animal care committee approved this study. Three excised murine lungs with pulmonary emphysema and three excised murine control lungs were imaged ex vivo by using a grating-based micro-computed tomographic (CT) scanner. To evaluate the diagnostic value, the natural logarithm of relative transmission and the natural logarithm of dark-field scatter signal were plotted on a per-pixel basis on a scatterplot. Probability density function was fit to the joint distribution by using principle component analysis. An emphysema map was calculated based on the fitted probability density function. RESULTS: The two-dimensional scatterplot showed a characteristic difference between control and emphysematous lungs. Control lungs had lower average median logarithmic transmission (-0.29 vs -0.18, P = .1) and lower average dark-field signal (-0.54 vs -0.37, P = .1) than emphysematous lungs. The angle to the vertical axis of the fitted regions also varied significantly (7.8° for control lungs vs 15.9° for emphysematous lungs). The calculated emphysema distribution map showed good agreement with histologic findings. CONCLUSION: X-ray dark-field scatter images of murine lungs obtained with a preclinical scanner can be used in the diagnosis of pulmonary emphysema. SUPPLEMENTAL MATERIAL: http://radiology.rsna.org/lookup/suppl/doi:10.1148/radiol.13122413/-/DC1.
Assuntos
Enfisema Pulmonar/diagnóstico por imagem , Microtomografia por Raio-X/métodos , Animais , Desenho de Equipamento , Feminino , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos C57BL , Interpretação de Imagem Radiográfica Assistida por Computador , Microtomografia por Raio-X/instrumentaçãoRESUMO
Under the label of the German Mouse Clinic (GMC), a concept has been developed and implemented that allows the better understanding of human diseases on the pathophysiological and molecular level. This includes better understanding of the crosstalk between different organs, pleiotropy of genes, and the systemic impact of envirotypes and drugs. In the GMC, experts from various fields of mouse genetics and physiology, in close collaboration with clinicians, work side by side under one roof. The GMC is an open-access platform for the scientific community by providing phenotypic analysis in bilateral collaborations ("bottom-up projects") and as a partner and driver in international large-scale biology projects ("top-down projects"). Furthermore, technology development is a major topic in the GMC. Innovative techniques for primary and secondary screens are developed and implemented into the phenotyping pipelines (e.g., detection of volatile organic compounds, VOCs).
Assuntos
Modelos Animais , Animais , Alemanha , Camundongos , FenótipoRESUMO
RATIONALE: Chronic obstructive pulmonary disease (COPD) is a devastating disease, for which no causal therapy is available. OBJECTIVES: To characterize WNT/ß-catenin signaling in COPD in humans and elucidate its potential role as a preventive and therapeutic target in experimental emphysema in mice. METHODS: The expression, localization, and activity of WNT/ß-catenin signaling was assessed in 12 COPD and 12 transplant donor samples using quantitative reverse transcriptase polymerase chain reaction, immunohistochemistry, and Western blotting. The role of WNT/ß-catenin signaling was assessed in elastase- and cigarette smoke-induced emphysema and therapeutic modulation thereof in elastase-induced emphysema in TOPGAL reporter and wild-type mice in vivo. MEASUREMENTS AND MAIN RESULTS: No differences in the mRNA expression profile of the main WNT/ß-catenin signaling components were observed comparing COPD and donor lung homogenates. Immunohistochemical analysis revealed reduced numbers of nuclear ß-catenin-positive alveolar epithelial cells in COPD. Similarly, WNT/ß-catenin signaling was down-regulated in both experimental emphysema models. Preventive and therapeutic, WNT/ß-catenin activation by lithium chloride attenuated experimental emphysema, as assessed by decreased airspace enlargement, improved lung function, reduced collagen content, and elevated expression of alveolar epithelial cell markers. CONCLUSIONS: Decreased WNT/ß-catenin signaling is involved in parenchymal tissue destruction and impaired repair capacity in emphysema. These data indicate a crucial role of WNT/ß-catenin signaling in lung repair mechanisms in vivo, and highlight WNT/ß-catenin activation as a future therapeutic approach for emphysema.
Assuntos
Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Enfisema Pulmonar/fisiopatologia , Transdução de Sinais/fisiologia , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Adulto , Animais , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Doadores de Tecidos , Proteína Wnt1/metabolismoRESUMO
Genomic integration by the Streptomyces bacteriophage C31 integrase is a promising tool for non-viral gene therapy of various genetic disorders. We investigated the C31 integrase recombination activity in T cell derived cell lines, primary T lymphocytes and CD34(+) haematopoietic stem cells in comparison to mesenchymal stem cells and cell lines derived from lung-, liver- and cervix-tissue. In T cell lines, enhanced long-term expression above control was observed only with high amounts of integrase mRNA. Transfections of C31 integrase plasmids were not capable of mediating enhanced long-term transgene expression in T cell lines. In contrast, moderate to high efficiency could be detected in human mesenchymal stem cells, human lung, liver and cervix carcinoma cell lines. Up to 100-fold higher levels of recombination product was found in C31 integrase transfected A549 lung than Jurkat T cells. When the C31 integrase activity was normalized to the intracellular integrase mRNA levels, a 16-fold difference was found. As one possible inhibitor of the C31 integrase, we found 3- to 5-fold higher DAXX levels in Jurkat than in A549 cells, which could in addition to other yet unknown factors explain the observed discrepancy of C31 integrase activity.
Assuntos
Bacteriófagos/enzimologia , Integrases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/análise , Linhagem Celular , Linhagem Celular Tumoral , Células Cultivadas , Proteínas Correpressoras , Expressão Gênica , Genes Reporter , Humanos , Células Jurkat , Chaperonas Moleculares , Proteínas Nucleares/análise , Plasmídeos/genética , Reação em Cadeia da Polimerase , Recombinação Genética , Streptomyces/virologia , Linfócitos T/metabolismo , TransfecçãoRESUMO
BACKGROUND: Supercoiled topology of transfected plasmid DNA (pDNA) is critical for transgene expression in mammalian cells. In the present study, we analysed transgene expression of transfected supercoiled pDNA concatemers. METHODS: Jurkat T cells were transfected with a supercoiled 4.7-kb monomeric and, in parallel, a 9.4-kb dimeric pEGFP plasmid concatemer using electroporation. The absolute amounts of pDNA delivered into the cytoplasm and the nucleus were quantified by quantitative real-time polymerase chain reaction. Further, the number and mean fluorescent intensity (MFI) of enhanced green fluorescent protein (EGFP) expressing cells and the relative amounts of TOTO-1 fluorescently-labeled pDNA associated with the cell, located in the cytoplasm, and in the nucleus, were analysed by flow cytometry. RESULTS: For both constructs, significantly higher amounts of pDNA were detected in the cytoplasm compared to the nucleus. Furthermore, from FACS analysis, we could infer the relative gene copy (E(gene)) and plasmid expression efficiency (E(plasmid)) by determining the ratio of the EGFP MFI of the transfected cells to TOTO-1 MFI per nucleus on the single cell level. E(gene) and E(plasmid) were significantly 1.6-and 3.5-fold higher for EGFP-dimer than for EGFP-monomer, although the transfection rates considering the number of transfected cells were significantly lower for EGFP-dimer than for EGFP-monomer. Together with hydrodynamic plasmid diameter measurements, these observations suggest that concatemer arrangement increases relative gene expression efficiency, whereas plasmid size is important for cell and nucleus entry after electroporation. CONCLUSIONS: We propose using preferably small supercoiled plasmid concatemers as the ideal plasmid vectors to maximize both transgene expression and the number of transfected target cells.
Assuntos
DNA Concatenado/genética , DNA Super-Helicoidal/genética , Regulação da Expressão Gênica , Plasmídeos/genética , Transfecção , Transgenes/genética , Southern Blotting , Compartimento Celular , Dimerização , Eletroporação , Citometria de Fluxo , Fluorescência , Dosagem de Genes , Proteínas de Fluorescência Verde/metabolismo , Humanos , Células Jurkat , Luz , Espalhamento de RadiaçãoRESUMO
In neuropsychiatric diseases, such as major depression and anxiety, pathogenic vulnerability is partially dictated by a genetic predisposition. The search continues to define this genetic susceptibility and establish new genetic elements as potential therapeutic targets. The fibroblast growth factors (FGFs) could be interesting in this regard. This family of signaling molecules plays important roles in development while also functioning within the adult. This includes effects on aspects of brain function such as neurogenesis and synapse formation. Of this family, Fgf9 is expressed in the adult brain, but its functional role is less well defined. In this study, we examined the role of Fgf9 in different brain functions by analyzing the behavior of Fgf9 Y162C mutant mice, an Fgf9 allele without the confounding systemic effects of other Fgf9 genetic models. Here, we show that this mutation caused altered locomotor and exploratory reactivity to novel, mildly stressful environments. In addition, mutants showed heightened acoustic startle reactivity as well as impaired social discrimination memory. Notably, there was a substantial decrease in the level of adult olfactory bulb neurogenesis with no difference in hippocampal neurogenesis. Collectively, our findings indicate a role for the Fgf9 Y162C mutation in information processing and perception of aversive situations as well as in social memory. Thus, genetic alterations in Fgf9 could increase vulnerability to developing neuropsychiatric disease, and we propose the Fgf9 Y162C mutant mice as a valuable tool to study the predictive etiological aspects.
Assuntos
Fator 9 de Crescimento de Fibroblastos/genética , Memória , Mutação/genética , Comportamento Social , Análise de Variância , Anestesia , Animais , Ansiedade/genética , Ansiedade/fisiopatologia , Corticosterona/metabolismo , Discriminação Psicológica , Feminino , Hipocampo/patologia , Hipocampo/fisiopatologia , Masculino , Camundongos , Camundongos Mutantes , Atividade Motora , Neurogênese , Bulbo Olfatório/patologia , Bulbo Olfatório/fisiopatologia , Reflexo de Sobressalto , Caracteres SexuaisRESUMO
The transcription factor Zscan10 had been attributed a role as a pluripotency factor in embryonic stem cells based on its interaction with Oct4 and Sox2 in in vitro assays. Here we suggest a potential role of Zscan10 in controlling progenitor cell populations in vivo. Mice homozygous for a Zscan10 mutation exhibit reduced weight, mild hypoplasia in the spleen, heart and long bones and phenocopy an eye malformation previously described for Sox2 hypomorphs. Phenotypic abnormalities are supported by the nature of Zscan10 expression in midgestation embryos and adults suggesting a role for Zscan10 in either maintaining progenitor cell subpopulation or impacting on fate choice decisions thereof.
Assuntos
Pleiotropia Genética , Fatores de Transcrição/genética , Animais , Comportamento Animal , Peso Corporal/genética , Densidade Óssea/genética , Osso e Ossos/fisiologia , Códon de Iniciação/genética , Olho/crescimento & desenvolvimento , Feminino , Regulação da Expressão Gênica/genética , Homozigoto , Masculino , Camundongos , Mutação , Tamanho do Órgão/genética , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Gravidez , DesmameRESUMO
PURPOSE: An experimental comparison of the contrast-to-noise ratio (CNR) between transmission and dark-field signals in grating-based X-ray imaging for ex-vivo murine lung tissue. MATERIALS AND METHODS: Lungs from three healthy mice were imaged ex vivo using a laser-driven compact synchrotron X-ray source. Background noise of transmission and dark-field signal was quantified by measuring the standard deviation in a region of interest (ROI) placed in a homogeneous area outside the specimen. Image contrast was quantified by measuring the signal range in rectangular ROIs placed in central and peripheral lung parenchyma. The relative contrast gain (RCG) of dark-field over transmission images was calculated as CNRDF / CNRT. RESULTS: In all images, there was a trend for contrast-to-noise ratios of dark-field images (CNRDF) to be higher than for transmission images (CNRT) for all ROIs (median 61 vs. 38, p=0.10), but the difference was statistically significant only for peripheral ROIs (61 vs. 32, p=0.03). Median RCG was >1 for all ROIs (1.84). RCG values were significantly smaller for central ROIs than for peripheral ROIs (1.34 vs. 2.43, p=0.03). CONCLUSION: The contrast-to-noise ratio of dark-field images compares more favorably to the contrast-to-noise ratio of transmission images for peripheral lung regions as compared to central regions. For any specific specimen, a calculation of the RCG allows comparing which X-ray modality (dark-field or transmission imaging) produces better contrast-to-noise characteristics in a well-defined ROI.
Assuntos
Iluminação/métodos , Pulmão/diagnóstico por imagem , Refratometria/instrumentação , Síncrotrons , Tomografia Computadorizada por Raios X/instrumentação , Difração de Raios X/instrumentação , Animais , Cadáver , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Valores de Referência , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
PURPOSE: To assess whether grating-based X-ray dark-field imaging can increase the sensitivity of X-ray projection images in the diagnosis of pulmonary emphysema and allow for a more accurate assessment of emphysema distribution. MATERIALS AND METHODS: Lungs from three mice with pulmonary emphysema and three healthy mice were imaged ex vivo using a laser-driven compact synchrotron X-ray source. Median signal intensities of transmission (T), dark-field (V) and a combined parameter (normalized scatter) were compared between emphysema and control group. To determine the diagnostic value of each parameter in differentiating between healthy and emphysematous lung tissue, a receiver-operating-characteristic (ROC) curve analysis was performed both on a per-pixel and a per-individual basis. Parametric maps of emphysema distribution were generated using transmission, dark-field and normalized scatter signal and correlated with histopathology. RESULTS: Transmission values relative to water were higher for emphysematous lungs than for control lungs (1.11 vs. 1.06, p<0.001). There was no difference in median dark-field signal intensities between both groups (0.66 vs. 0.66). Median normalized scatter was significantly lower in the emphysematous lungs compared to controls (4.9 vs. 10.8, p<0.001), and was the best parameter for differentiation of healthy vs. emphysematous lung tissue. In a per-pixel analysis, the area under the ROC curve (AUC) for the normalized scatter value was significantly higher than for transmission (0.86 vs. 0.78, p<0.001) and dark-field value (0.86 vs. 0.52, p<0.001) alone. Normalized scatter showed very high sensitivity for a wide range of specificity values (94% sensitivity at 75% specificity). Using the normalized scatter signal to display the regional distribution of emphysema provides color-coded parametric maps, which show the best correlation with histopathology. CONCLUSION: In a murine model, the complementary information provided by X-ray transmission and dark-field images adds incremental diagnostic value in detecting pulmonary emphysema and visualizing its regional distribution as compared to conventional X-ray projections.
Assuntos
Escuridão , Enfisema Pulmonar/diagnóstico por imagem , Radiografia/métodos , Animais , Estudos de Viabilidade , Feminino , Processamento de Imagem Assistida por Computador , Pulmão/diagnóstico por imagem , Pulmão/fisiologia , Pulmão/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Enfisema Pulmonar/patologia , Enfisema Pulmonar/fisiopatologia , Testes de Função Respiratória , Espalhamento de Radiação , Sensibilidade e Especificidade , Raios XRESUMO
BACKGROUND: Nuclear myosin I (NM1) is a nuclear isoform of the well-known "cytoplasmic" Myosin 1c protein (Myo1c). Located on the 11(th) chromosome in mice, NM1 results from an alternative start of transcription of the Myo1c gene adding an extra 16 amino acids at the N-terminus. Previous studies revealed its roles in RNA Polymerase I and RNA Polymerase II transcription, chromatin remodeling, and chromosomal movements. Its nuclear localization signal is localized in the middle of the molecule and therefore directs both Myosin 1c isoforms to the nucleus. METHODOLOGY/PRINCIPAL FINDINGS: In order to trace specific functions of the NM1 isoform, we generated mice lacking the NM1 start codon without affecting the cytoplasmic Myo1c protein. Mutant mice were analyzed in a comprehensive phenotypic screen in cooperation with the German Mouse Clinic. Strikingly, no obvious phenotype related to previously described functions has been observed. However, we found minor changes in bone mineral density and the number and size of red blood cells in knock-out mice, which are most probably not related to previously described functions of NM1 in the nucleus. In Myo1c/NM1 depleted U2OS cells, the level of Pol I transcription was restored by overexpression of shRNA-resistant mouse Myo1c. Moreover, we found Myo1c interacting with Pol II. The ratio between Myo1c and NM1 proteins were similar in the nucleus and deletion of NM1 did not cause any compensatory overexpression of Myo1c protein. CONCLUSION/SIGNIFICANCE: We observed that Myo1c can replace NM1 in its nuclear functions. Amount of both proteins is nearly equal and NM1 knock-out does not cause any compensatory overexpression of Myo1c. We therefore suggest that both isoforms can substitute each other in nuclear processes.
Assuntos
Núcleo Celular/metabolismo , Miosina Tipo I/genética , Miosina Tipo I/metabolismo , Fenótipo , Animais , Western Blotting , Primers do DNA/genética , Genótipo , Imunoprecipitação , Camundongos , Camundongos Knockout , Plasmídeos/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Several barriers have to be overcome in order to achieve gene expression in target cells, e.g. cellular uptake, endosomal release and translocation to the nucleus. Nuclear localization sequences (NLS) enhance gene delivery by increasing the uptake of plasmid DNA (pDNA) to the nucleus. So far, only monopartite NLS were analysed for non-viral gene delivery. In this study, we examined the characteristics of a novel bipartite NLS like construct, namely NLS Ku70. We synthesized a dimeric structure of a modified NLS from the Ku70 protein (Ku70(2)-NLS), a nuclear transport active mutant of Ku70(2)-NLS (s1Ku70(2)-NLS) and a nuclear transport deficient mutant of Ku70(2)-NLS (s2Ku70(2)). We examined the transfection efficiency of binary Ku70(2)-NLS/DNA and ternary Ku70(2)-NLS/PEI/DNA gene vector complexes in vitro by using standard transfection protocols as well as the magnetofection method. The application of Ku70(2)-NLS and s1Ku70(2)-NLS increased gene transfer efficiency in vitro and in vivo. This study shows for the first time that the use of bipartite NLS compounds alone or in combination with cationic polymers is a promising strategy to enhance the efficiency of non-viral gene transfer.
Assuntos
Antígenos Nucleares/genética , Proteínas de Ligação a DNA/genética , Técnicas de Transferência de Genes , Sinais de Localização Nuclear/genética , Transporte Ativo do Núcleo Celular , Antígenos Nucleares/uso terapêutico , Proteínas de Ligação a DNA/uso terapêutico , Autoantígeno Ku , Sinais de Localização Nuclear/uso terapêutico , TransfecçãoRESUMO
The chitinase-like protein YKL-40 was found to be increased in patients with severe asthma and chronic obstructive pulmonary disease (COPD), two disease conditions featuring neutrophilic infiltrates. Based on these studies and a previous report indicating that neutrophils secrete YKL-40, we hypothesized that YKL-40 plays a key role in cystic fibrosis (CF) lung disease, a prototypic neutrophilic disease. The aim of this study was (i) to analyze YKL-40 levels in human and murine CF lung disease and (ii) to investigate whether YKL-40 single-nucleotide polymorphisms (SNPs) modulate CF lung disease severity. YKL-40 protein levels were quantified in serum and sputum supernatants from CF patients and control individuals. Levels of the murine homologue BRP-39 were analyzed in airway fluids from CF-like ßENaC-Tg mice. YKL-40SNPs were analyzed in CF patients. YKL-40 levels were increased in sputum supernatants and in serum from CF patients compared to healthy control individuals. Within CF patients, YKL-40 levels were higher in sputum than in serum. BRP-39 levels were increased in airways fluids from ßENaC-Tg mice compared to wild-type littermates. In both CF patients and ßENaC-Tg mice, YKL-40/BRP-39 airway levels correlated with the severity of pulmonary obstruction. Two YKL-40 SNPs (rs871799 and rs880633) were found to modulate age-adjusted lung function in CF patients. YKL-40/BRP-39 levelsare increased in human and murine CF airway fluids, correlate with pulmonary function and modulate CF lung disease severity genetically. These findings suggest YKL-40 as a potential biomarker in CF lung disease.
Assuntos
Adipocinas/metabolismo , Fibrose Cística/metabolismo , Lectinas/metabolismo , Adipocinas/sangue , Adipocinas/genética , Animais , Proteína 1 Semelhante à Quitinase-3 , Fibrose Cística/genética , Fibrose Cística/patologia , Feminino , Humanos , Lectinas/sangue , Lectinas/genética , Masculino , Camundongos , Polimorfismo de Nucleotídeo Único , Escarro/metabolismo , Adulto JovemRESUMO
Upon activation, neutrophils release DNA fibers decorated with antimicrobial proteins, forming neutrophil extracellular traps (NETs). Although NETs are bactericidal and contribute to innate host defense, excessive NET formation has been linked to the pathogenesis of autoinflammatory diseases. However, the mechanisms regulating NET formation, particularly during chronic inflammation, are poorly understood. Here we show that the G protein-coupled receptor (GPCR) CXCR2 mediates NET formation. Downstream analyses showed that CXCR2-mediated NET formation was independent of NADPH oxidase and involved Src family kinases. We show the pathophysiological relevance of this mechanism in cystic fibrosis lung disease, characterized by chronic neutrophilic inflammation. We found abundant NETs in airway fluids of individuals with cystic fibrosis and mouse cystic fibrosis lung disease, and NET amounts correlated with impaired obstructive lung function. Pulmonary blockade of CXCR2 by intra-airway delivery of small-molecule antagonists inhibited NET formation and improved lung function in vivo without affecting neutrophil recruitment, proteolytic activity or antibacterial host defense. These studies establish CXCR2 as a receptor mediating NADPH oxidase-independent NET formation and provide evidence that this GPCR pathway is operative and druggable in cystic fibrosis lung disease.
Assuntos
Fibrose Cística/fisiopatologia , NADPH Oxidases/metabolismo , Neutrófilos/fisiologia , Receptores de Interleucina-8B/fisiologia , Animais , Morte Celular , Quimiocina CXCL2/farmacologia , Fibrose Cística/metabolismo , Fibrose Cística/patologia , Inibidores Enzimáticos/farmacologia , Espaço Extracelular/fisiologia , Humanos , Inflamação/fisiopatologia , Interleucina-8/farmacologia , Interleucina-8/fisiologia , Pneumopatias/patologia , Pneumopatias/fisiopatologia , Camundongos , NADPH Oxidases/antagonistas & inibidores , Ativação de Neutrófilo/efeitos dos fármacos , Ativação de Neutrófilo/fisiologia , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Oniocompostos/farmacologiaRESUMO
Tumor necrosis factor-alpha (TNFalpha) is known to inhibit renin gene expression in juxtaglomerular cells, which are the main source of renin in vivo. In the present study we aimed to characterize the intracellular mechanisms of TNFalpha signaling to renin gene in the mouse juxtaglomerular cell line As4.1. TNFalpha was found to activate NFkappaB, which is one of the principal intracellular mediators of TNFalpha signal transduction. Constitutive activation of NFkappaB suppressed renin gene transcription, but NFkappaB appeared not to target the NFkappaB binding sites in the renin promoter. Thus, NFkappaB, but not the canonical NFkappaB binding sequences in the renin promoter, seemed to be involved in the suppression of renin transcription by TNFalpha. Deletion/mutation analysis revealed that the effect of TNFalpha on renin gene is transmitted by a cAMP-responsive element (CRE) located at -2697 to -2690. Mobility shift/supershift assays evidenced for the presence of NFkappaB proteins in the complex that binds to mouse renin CRE. Our results strongly suggest that NFkappaB mediates the effect of TNFalpha on renin transcription targeting a CRE in the mouse renin promoter.