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1.
Photochem Photobiol Sci ; 18(12): 3008-3015, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31696896

RESUMO

A quinoline moiety was used as a building block for designing a probe for the selective detection of copper ions in a partially aqueous medium. We have developed a molecular sensing system which gives insight into the complex physiological and redox aspects of labile copper. The probe provides a colorimetric approach for distinguishing cuprous and cupric ions along with their simultaneous discrimination from other metal ions in the visible range of the spectrum. The chemosensor showed a remarkable fluorescence enhancement along with a significant bathochromic shift of about 35 nm. The detection limit of the probe was found to be 1.03 µM which is optimally favorable to be applied in real-time monitoring. Fabrication of paper strips with the probe was done to detect the presence of cuprous ions in the real sample. The value of the binding constant (1.37 × 104 M-1) suggests stable complex formation between the metal ion and the sensing probe. The photoluminescence and structural aspects of the chemosensor were characterized by using fluorescence, absorption, ESI-MS, and 1H NMR spectroscopy. Furthermore, the cytotoxic nature and bioimaging properties of the probe were interpreted in vitro on RAW 264.7 macrophage cell lines and peripheral blood mononuclear cells (PBMCs) respectively.


Assuntos
Colorimetria , Cobre/análise , Quinolinas/química , Animais , Corantes Fluorescentes/química , Humanos , Íons/química , Leucócitos Mononucleares/química , Leucócitos Mononucleares/citologia , Limite de Detecção , Macrófagos/química , Macrófagos/citologia , Camundongos , Microscopia de Fluorescência , Células RAW 264.7
2.
BMC Complement Altern Med ; 19(1): 314, 2019 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-31744490

RESUMO

BACKGROUNDS: Inflammation is recognized as the key pathological mechanism of type 2 diabetes. The hypoglyceamic effects of berberine (BBR) are related to the inhibition of the inflammatory response, but the mechanism is not completely clear. METHODS: The inflammatory polarization of Raw264.7 cells and primary peritoneal macrophages were induced by LPS, and then effects and underlying mechanisms of BBR were explored. An inflammatory model was established by LPS treatment at different concentrations for different treatment time. An ELISA assay was used to detect the secretions of TNF-α. RT-PCR was applied to detect M1 inflammatory factors. The F4/80+ ratio and CD11c+ ratio of primary peritoneal macrophages were determined by flow cytometry. The expressions of p-AMPK and TLR4 were detected by Western blot. The cytoplasmic and nuclear distributions of NFκB p65 were observed by confocal microscopy. The binding of TLR4 to MyD88 was tested by CoIP, and the affinity of BBR for TLR4 was assessed by molecular docking. RESULTS: Upon exposure to LPS, the secretion of TNF-α and transcription of inflammatory factors in macrophages increased, cell morphology changed and protrusions appeared gradually, the proportion of F4/80+CD11c+ M1 macrophages increased, and the nuclear distribution of NFκB p65 increased. BBR pretreatment partially inhibited the changes mentioned above. However, the expression of TLR4 and p-AMPK did not change significantly after LPS intervention for 3 h. Meanwhile, CoIP showed that the interaction between TLR4 and MyD88 increased, and BBR inhibited the binding. Molecular docking suggested that BBR might interact with TLR4. CONCLUSIONS: Inflammatory changes were induced in macrophages after LPS stimulation for 3 h, and BBR pretreatment inhibited inflammatory polarization. BBR might interact with TLR4 and disturb TLR4/MyD88/NFκB signalling pathway, and it might be the mechanism by which BBR attenuated inflammation in the early phase.


Assuntos
Berberina/farmacologia , Macrófagos/efeitos dos fármacos , Fator 88 de Diferenciação Mieloide/metabolismo , Receptor 4 Toll-Like/metabolismo , Animais , Berberina/química , Polaridade Celular/efeitos dos fármacos , Células Cultivadas , Feminino , Humanos , Lipopolissacarídeos/farmacologia , Macrófagos/química , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Simulação de Acoplamento Molecular , Fator 88 de Diferenciação Mieloide/química , Fator 88 de Diferenciação Mieloide/genética , Ligação Proteica/efeitos dos fármacos , Células RAW 264.7 , Receptor 4 Toll-Like/química , Receptor 4 Toll-Like/genética , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo
3.
J Agric Food Chem ; 67(49): 13568-13576, 2019 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-31709793

RESUMO

Astaxanthin (AST) is a fat-soluble and non-vitamin A source of carotenoid that can quench reactive oxygen species and it has strong antioxidant and anti-inflammatory abilities. Herein, we have used H2O2 to establish a model of oxidative damage to RAW 264.7 cells and cells treated with vitamin C as the positive control group. The changes in metabolome were examined using 1H NMR and the results demonstrated that H2O2 treatment and various metabolic pathways such as amino acid, glucose, and glycerolipid metabolism were downregulated, which in turn affected citric acid cycle and energy status. AST could reverse downregulation of some of these metabolic pathways to a certain extent, and reduce cellular oxidative stress and death. The AST group differed from the vitamin C group in regulating d-glutamine, d-glutamic acid, pyruvate, and glycerolipid metabolism. The experimental results help to further understand the antioxidant effects of AST.


Assuntos
Antioxidantes/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Animais , Peróxido de Hidrogênio/toxicidade , Macrófagos/química , Redes e Vias Metabólicas/efeitos dos fármacos , Metabolômica , Camundongos , Estresse Oxidativo/efeitos dos fármacos , Espectroscopia de Prótons por Ressonância Magnética , Células RAW 264.7 , Xantofilas/farmacologia
4.
Biochemistry (Mosc) ; 84(7): 729-745, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31509725

RESUMO

Despite the progress of modern medicine, oncological diseases are still among the most common causes of death of adult populations in developed countries. The current therapeutic approaches are imperfect, and the high mortality of oncological patients under treatment, the lack of personalized strategies, and severe side effects arising as a result of treatment force seeking new approaches to therapy of malignant tumors. During the last decade, cancer immunotherapy, an approach that relies on activation of the host antitumor immune response, has been actively developing. Cancer immunotherapy is the most promising trend in contemporary fundamental and practical oncology, and restoration of the pathologically altered tumor microenvironment is one of its key tasks, in particular, the reprogramming of tumor macrophages from the immunosuppressive M2-phenotype into the proinflammatory M1-phenotype is pivotal for eliciting antitumor response. This review describes the current knowledge about macrophage classification, mechanisms of their polarization, their role in formation of the tumor microenvironment, and strategies for changing the functional activity of M2-macrophages, as well as problems of targeted delivery of immunostimulatory signals to tumor macrophages using nanoparticles.


Assuntos
Imunoterapia , Macrófagos/metabolismo , Nanopartículas/metabolismo , Neoplasias/terapia , Animais , Polaridade Celular/fisiologia , Humanos , Fatores Imunológicos/farmacologia , Fatores Imunológicos/uso terapêutico , Microscopia Intravital , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/química , Macrófagos/classificação , Camundongos , Nanopartículas/química , Fenótipo , Coroa de Proteína/imunologia , Microambiente Tumoral/imunologia
5.
Anal Chim Acta ; 1080: 104-115, 2019 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-31409459

RESUMO

We have implemented a linear ion trap (LIT)-based SIM-stitching method for ultra-high-resolution Fourier transform mass spectrometry (FTMS) that increases the S/N over a wide m/z range compared to non-segmented wide full-scan (WFS) spectra. Here we described an improved segmented spectral scan stitching method that was based on quadrupole mass filter (QMF)-SIM, which overcame previous limitations of ion signal loss in LIT. This allowed for accurate representation of isotopologue distributions, both at natural abundance and in stable isotope-resolved metabolomics (SIRM)-based experiments. We also introduced a new spectral binning method that provided more precise and resolution-independent bins for irreversibly noise-suppressed FTMS spectra. We demonstrated a substantial improvement in S/N and sensitivity (typically > 10-fold) for 13C labeled lipid extracts of human macrophages grown as three-dimensional (3D) cell culture, with detection of an increased number of 13C isotopologue ions. The method also enabled analysis of extracts from very limited biological samples.


Assuntos
Lipídeos/análise , Macrófagos/química , Isótopos de Carbono/química , Análise de Fourier , Glucose/química , Glucose/metabolismo , Humanos , Marcação por Isótopo , Macrófagos/metabolismo , Espectrometria de Massas/métodos , Metabolômica/métodos , Esferoides Celulares/química , Esferoides Celulares/metabolismo
6.
PLoS Negl Trop Dis ; 13(8): e0007691, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31469835

RESUMO

Lung disease is regularly reported in human filarial infections but the molecular pathogenesis of pulmonary filariasis is poorly understood. We used Litomosoides sigmodontis, a rodent filaria residing in the pleural cavity responsible for pleural inflammation, to model responses to human filarial infections and probe the mechanisms. Wild-type and Th2-deficient mice (ΔdblGata1 and Il-4receptor(r)a-/-/IL-5-/-) were infected with L. sigmodontis. Survival and growth of adult filariae and prevalence and density of microfilariae were evaluated. Cells and cytokines in the pleural cavity and bronchoalveolar space were characterized by imaging, flow cytometry and ELISA. Inflammatory pathways were evaluated by transcriptomic microarrays and lungs were isolated and analyzed for histopathological signatures. 40% of WT mice were amicrofilaremic whereas almost all mutant mice display blood microfilaremia. Microfilariae induced pleural, bronchoalveolar and lung-tissue inflammation associated with an increase in bronchoalveolar eosinophils and perivascular macrophages, production of mucus, visceral pleura alterations and fibrosis. Inflammation and pathology were decreased in Th2-deficient mice. An IL-4R-dependent increase of CD169 was observed on pleural and bronchoalveolar macrophages in microfilaremic mice. CD169+ tissue-resident macrophages were identified in the lungs with specific localizations. Strikingly, CD169+ macrophages increased significantly in the perivascular area in microfilaremic mice. These data describe lung inflammation and pathology in chronic filariasis and emphasize the role of Th2 responses according to the presence of microfilariae. It is also the first report implicating CD169+ lung macrophages in response to a Nematode infection.


Assuntos
Filariose/patologia , Filarioidea/imunologia , Inflamação/patologia , Pulmão/imunologia , Macrófagos/imunologia , Receptores de Interleucina-4/metabolismo , Lectina 1 Semelhante a Ig de Ligação ao Ácido Siálico/análise , Animais , Modelos Animais de Doenças , Feminino , Filariose/imunologia , Inflamação/imunologia , Pulmão/patologia , Macrófagos/química , Camundongos Endogâmicos BALB C , Células Th2/imunologia
7.
Nanoscale ; 11(30): 14123-14133, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31322633

RESUMO

Quantum dots, derived from two-dimensional (2D) materials, have shown promise in bioimaging, sensing and photothermal applications, and in white light emitting devices (WLEDs). Herein, nitrogen and phosphorus functionalized Ti3C2 MXene based quantum dots (N,P-MQDs) were successfully prepared through a top-bottom hydrothermal method. This type of photoluminescent quantum dots has realized green fluorescence for the first time at around 560 nm with a photoluminescence quantum yield (PLQY) of 20.1%, the highest ever reported; meanwhile, it also exhibits excellent photostability and pH resistance capacities. Comprehensive characterization and well-resolved density functional theory (DFT) calculation were implemented to determine the mechanism of fluorescence shift and enhancement. Furthermore, the N,P-MQDs have been proved to efficiently act as fluorescent probes for macrophage labeling. In addition, the high sensitivity of the N,P-MQDs toward Cu2+ ions made them a low cost, sensitive, environment-friendly, and label-free fluorescence platform for Cu2+ detection. The outstanding performance of Ti3C2 MXene based quantum dots has demonstrated their great potential to be used as promising fluorescent probes in the fields of biological imaging, optical sensing, photoelectric conversion, etc.


Assuntos
Cobre/análise , Macrófagos/química , Microscopia de Fluorescência , Pontos Quânticos/química , Titânio/química , Linhagem Celular , Teoria da Densidade Funcional , Corantes Fluorescentes/química , Humanos , Concentração de Íons de Hidrogênio , Íons/química , Macrófagos/metabolismo , Espectrometria de Fluorescência
8.
Biosens Bioelectron ; 141: 111430, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31299629

RESUMO

Reactive oxygen species are highly reactive molecules that as well as being ubiquitously expressed throughout the body, are also known to be involved in many diseases and disorders including bacterial infection. Current technology has limited success in the accurate detection and identification of specific reactive oxygen species. To combat this, we have developed an electrochemical biosensor that is constructed from single walled carbon nanotubes that have been immobilised on an indium tin oxide surface functionalised with osmium-based compound. This sensor was integrated within mouse macrophage cells (RAW 264.7) with multiple serotypes of bacteria used to initiate an immune response. Intracellular hydrogen peroxide was then measured in response to the interaction of the lipopolysaccharides, present on the outer wall of Gram-negative bacteria, with the Toll-like Receptor 4. Additional controls of n-acetylcysteine and sodium pyruvate were implemented to prove the specificity of the sensor towards hydrogen peroxide. The sensors were found to have a lower limit of detection of 368 nM hydrogen peroxide. An increase in intracellular hydrogen peroxide was detected within 3 seconds of interaction of the bacteria with the macrophage cells. This low limit of detection combined with the rapid response of the sensor resulted in the unprecedented detection of hydrogen peroxide on a temporal level not previously seen in response to a bacterial threat. From the three serotypes of Gram-negative bacteria that were tested, there were distinct differences in hydrogen peroxide production. This proves that the innate immune system has the ability to respond dynamically and rapidly, after infection prior to the activation of the adaptive immune system.


Assuntos
Técnicas Biossensoriais/métodos , Bactérias Gram-Negativas/imunologia , Peróxido de Hidrogênio/análise , Macrófagos/química , Macrófagos/imunologia , Animais , Técnicas Eletroquímicas/métodos , Infecções por Bactérias Gram-Negativas/imunologia , Peróxido de Hidrogênio/imunologia , Imunidade Inata , Limite de Detecção , Lipopolissacarídeos/imunologia , Macrófagos/microbiologia , Camundongos , Nanotubos de Carbono/química , Células RAW 264.7 , Espécies Reativas de Oxigênio/análise , Espécies Reativas de Oxigênio/imunologia , Receptor 4 Toll-Like/imunologia
9.
ACS Appl Mater Interfaces ; 11(26): 23018-23025, 2019 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-31252477

RESUMO

Macromolecular protein drugs are promising anti-neoplastic agents based on their precise tumor affinity and innocuousness to normal tissues. Although direct delivery of protein drugs remains impractical due to its short half-life in circulation, inefficiency in tumor accumulation, and poor penetrability in intratumoral distribution. Recently, biogenetic cell-based drug vectors have been widely reported for antitumor drug delivery. Macrophage is naturally independent with endogenous proteolysis, elimination of reticuloendothelial system, and immune surveillance. Meanwhile, its innate recruitment behaviors responsive to chronic inflammation signals make it a potential cellular vector for tumor targeting drug delivery. In this study, we develop a trained macrophage bioreactor for tumor homing and an in situ expression of fused antitumor protein. The recombinant tumor necrosis factor related apoptosis-inducing ligand is coded on a plasmid vector with penetrating domain on the C terminus, which improves the intratumoral distribution by facilitating protein dispersion in tumor tissue after in situ secretion. The combination of tumor-infiltrating macrophage bioreactor and multifunctional fused protein drug embodies a new effective tumor homing system for antitumor protein delivery.


Assuntos
Antineoplásicos/farmacologia , Peptídeos Penetradores de Células/farmacologia , Inflamação/tratamento farmacológico , Macrófagos/química , Animais , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Reatores Biológicos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Peptídeos Penetradores de Células/química , Humanos , Camundongos
10.
J Agric Food Chem ; 67(22): 6407-6413, 2019 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-31083940

RESUMO

The precise cellular function of peroxynitrite (ONOO-) in biosystems remains elusive, primarily owing to being short of ultrasensitive techniques for monitoring its intracellular distribution. In this work, a novel rhodamine B cyclic 1,2-dimethylhydrazine fluorescent chemodosimeter RDMH-PN for highly specific and ultrasensitive monitoring of basal ONOO- in biosystems was rationally designed. The fluorescence titration experiments demonstrated that RDMH-PN was capable of quantitatively detecting 0-100 nM ONOO- (limit of detection = 0.68 nM). In addition, RDMH-PN has outstanding performances of ultrafast measurement, naked-eye detection, and preeminent selectivity toward ONOO- to accurately detect intracellular basal ONOO-. Finally, it has been confirmed that RDMH-PN could not only map the intracellular basal ONOO- level by inhibition tests but also trace the fluctuations of endogenous and exogenous ONOO- levels with diverse stimulations in live cells and zebrafish.


Assuntos
Dimetilidrazinas/química , Corantes Fluorescentes/química , Ácido Peroxinitroso/análise , Espectrometria de Fluorescência/métodos , Peixe-Zebra/metabolismo , Animais , Macrófagos/química , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Ácido Peroxinitroso/biossíntese , Células RAW 264.7 , Rodaminas/química , Espectrometria de Fluorescência/instrumentação
11.
J Am Soc Mass Spectrom ; 30(7): 1276-1283, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30972724

RESUMO

Selenium (Se) functions as a cellular redox gatekeeper through its incorporation into proteins as the 21st amino acid, selenocysteine (Sec). Supplementation of macrophages with exogenous Se (as sodium selenite) downregulates inflammation and intracellular oxidative stress by effectively restoring redox homeostasis upon challenge with bacterial endotoxin lipopolysaccharide (LPS). Here, we examined the use of a standard Tandem Mass Tag (TMT)-labeling mass spectrometry-based proteomic workflow to quantitate and examine temporal regulation of selenoproteins in such inflamed cells. Se-deficient murine primary bone marrow-derived macrophages (BMDMs) exposed to LPS in the presence or absence of selenite treatment for various time periods (0-20 h) were used to analyze the selenoproteome expression using isobaric labeling and shotgun proteomic workflow. To overcome the challenge of identification of Sec peptides, we used the identification of non-Sec containing peptides downstream of Sec as a reliable evidence of ribosome readthrough indicating efficient decoding of Sec codon. Results indicated a temporal regulation of the selenoproteome with a general increase in their expression in inflamed cells in a Se-dependent manner. Selenow, Gpx1, Msrb1, and Selenom were highly upregulated upon stimulation with LPS when compared to other selenoproteins. Interestingly, Selenow appeared to be one amongst the highly regulated selenoproteins in macrophages that was previously thought to be mainly restricted to myocytes. Collectively, TMT-labeling method of non-Sec peptides offers a reliable method to quantitate and study temporal regulation of selenoproteins; however, further optimization to include Sec-peptides could make this strategy more robust and sensitive compared to other semi-quantitative or qualitative methods. Graphical Abstract.


Assuntos
Macrófagos/química , Selenoproteínas/análise , Sequência de Aminoácidos , Animais , Inflamação/imunologia , Lipopolissacarídeos/imunologia , Macrófagos/imunologia , Masculino , Camundongos Endogâmicos C57BL , Proteômica/métodos , Selenoproteínas/imunologia , Espectrometria de Massas em Tandem/métodos
12.
Nano Lett ; 19(5): 3040-3048, 2019 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-30968694

RESUMO

Exosomes, also known as extracellular vesicles, are naturally occurring, biocompatible, and bioacive nanoparticles ranging from 40 to 150 nm in diameter. Bone-secreted exosomes play important roles in bone homeostasis, the interruption of which can lead to diseases such as osteoporosis, rheumatoid arthritis, and osteopetrosis. Though the relationship between vascular and bone homeostasis has been recognized recently, the role of vascular endothelial cell (EC)-secreted exosomes (EC-Exos) in bone homeostasis is not well understood. Herein, we found that EC-Exos show more efficient bone targeting than osteoblast-derived exosomes or bone marrow mesenchymal stem cell-derived exosomes. We also found that EC-Exos can be internalized by bone marrow-derived macrophages (BMMs) to alter their morphology. EC-Exos can inhibit osteoclast activity in vitro and inhibit osteoporosis in an ovariectomized mouse model. Sequencing of exosome miRNA revealed that miR-155 was highly expressed in EC-Exos-treated BMMs. The miR-155 level in EC-Exos was much higher than that in BMMs and ECs, indicating that miR-155 was endogenous cargo of EC-derived vesicles. Blockage of BMMs miR-155 levels reversed the suppression by EC-Exos of osteoclast induction, confirming that exosomal miR-155 may have therapeutic potential against osteoporosis. Taken together, our findings suggest that EC-Exos may be utilized as a bone targeting and nontoxic nanomedicine for the treatment of bone resorption disorders.


Assuntos
Exossomos/química , Homeostase/efeitos dos fármacos , MicroRNAs/genética , Osteoporose/tratamento farmacológico , Animais , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Endoteliais/química , Células Endoteliais/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Homeostase/genética , Humanos , Macrófagos/química , Macrófagos/efeitos dos fármacos , Células-Tronco Mesenquimais/química , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos , MicroRNAs/química , Osteoblastos/efeitos dos fármacos , Osteoclastos/efeitos dos fármacos , Osteócitos/química , Osteócitos/efeitos dos fármacos , Osteoporose/patologia
13.
Artigo em Inglês | MEDLINE | ID: mdl-30895174

RESUMO

We have previously reported that Rickettsia conorii and Rickettsia montanensis have distinct intracellular fates within THP-1 macrophages, suggesting that the ability to proliferate within macrophages may be a distinguishable factor between pathogenic and non-pathogenic Spotted fever group (SFG) members. To start unraveling the molecular mechanisms underlying the capacity (or not) of SFG Rickettsia to establish their replicative niche in macrophages, we have herein used quantitative proteomics by SWATH-MS to profile the alterations resulted by the challenge of THP-1 macrophages with R. conorii and R. montanensis. We show that the pathogenic, R. conorii, and the non-pathogenic, R. montanensis, member of SFG Rickettsia trigger differential proteomic signatures in macrophage-like cells upon infection. R. conorii specifically induced the accumulation of several enzymes of the tricarboxylic acid cycle, oxidative phosphorylation, fatty acid ß-oxidation, and glutaminolysis, as well as of several inner and outer membrane mitochondrial transporters. These results suggest a profound metabolic rewriting of macrophages by R. conorii toward a metabolic signature of an M2-like, anti-inflammatory activation program. Moreover, several subunits forming the proteasome and immunoproteasome are found in lower abundance upon infection with both rickettsial species, which may help bacteria to escape immune surveillance. R. conorii-infection specifically induced the accumulation of several host proteins implicated in protein processing and quality control in ER, suggesting that this pathogenic Rickettsia may be able to increase the ER protein folding capacity. This work reveals novel aspects of macrophage-Rickettsia interactions, expanding our knowledge of how pathogenic rickettsiae explore host cells to their advantage.


Assuntos
Interações Hospedeiro-Patógeno , Macrófagos/química , Macrófagos/microbiologia , Proteoma/análise , Rickettsia/crescimento & desenvolvimento , Humanos , Metabolismo , Proteômica , Células THP-1
14.
Microb Pathog ; 130: 44-53, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30831227

RESUMO

Johne's disease is a chronic wasting disease of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP). Closely related pathogenic mycobacteria such as M. tuberculosis are capable of altering host lipid metabolism, highlighting the need to explore the role of lipid metabolism contributing to intracellular survival. This study aimed to identify whether MAP is able to manipulate host lipid metabolic pathways and accumulate host cholesterol during early infection. Macrophages were exposed to four different MAP strains and non-pathogenic M. phlei for up to 72 h, with changes to lipid metabolism examined using fluorescent microscopy and gene expression. MAP-infected macrophages displayed strain-dependent differences to intracellular cholesterol levels during early infection, however showed similarly increased intracellular cholesterol at later timepoints. Gene expression revealed that MAP strains similarly activate the host immune response in a conserved manner compared to M. phlei. MAP significantly upregulated host genes associated with lipid efflux and endocytosis. Moreover, lipid biosynthesis genes were differentially regulated in a strain-dependent manner following MAP infection. Collectively, these results demonstrate that MAP manipulates host lipid metabolism during early infection, however the extent of these modulations are strain-dependent. These findings reflect a conserved pathway contributing to intracellular MAP survival.


Assuntos
Colesterol/análise , Interações Hospedeiro-Patógeno , Metabolismo dos Lipídeos , Macrófagos/química , Macrófagos/microbiologia , Mycobacterium avium subsp. paratuberculosis/crescimento & desenvolvimento , Mycobacterium avium subsp. paratuberculosis/metabolismo , Animais , Endocitose , Perfilação da Expressão Gênica , Camundongos , Microscopia de Fluorescência , Células RAW 264.7
15.
Methods Mol Biol ; 1936: 23-36, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30820891

RESUMO

Remyelination is the regenerative process whereby myelin sheaths are restored around axons following nervous system injury, allowing reinstatement of electrical impulse conduction, trophic/metabolic support, and axon health. Failure of remyelination in progressive multiple sclerosis is considered to contribute to axon loss, a correlate of clinical decline. Lack of approved pro-regenerative therapies for MS highlights the need to understand the cellular and molecular mechanisms underpinning successful remyelination. One approach is to conduct nonbiased gene expression analyses of cell types which regulate remyelination, such as microglia and monocyte-derived macrophages. Recent technological advances address the challenges of RNA sequencing of small tissue samples, thus allowing relatively small numbers of cells to be isolated from discrete lesions for analysis. Here, we present methods for FACS-based isolation of cells from focal remyelinating lesions of the adult mouse brain and subsequent RNA extraction for sequencing, using isolation of microglia/macrophages as an example.


Assuntos
Encéfalo/citologia , Remielinização , Análise de Sequência de RNA/métodos , Animais , Separação Celular , Sistema Nervoso Central/química , Citometria de Fluxo , Regulação da Expressão Gênica , Macrófagos/química , Macrófagos/citologia , Camundongos , Microglia/química , Microglia/citologia
16.
Nano Lett ; 19(3): 1963-1975, 2019 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-30740982

RESUMO

Material implants trigger host reactions generated by cells, such as macrophages, which display dynamic adhesion and polarization including M1 inflammatory state and M2 anti-inflammatory state. Creating materials that enable diverse nanoscale display of integrin-binding groups, such as RGD ligand, can unravel nanoscale recruitment and ligation of integrin, which modulate cellular adhesion and activation. Here, we synthesized gold nanorods (GNRs) with various nanoscale anisotropies (i.e., aspect ratios, ARs), but in similar surface areas, and controlled their substrate conjugation to display an anisotropic ligand nanogeometry without modulating ligand density. Using nanoscale immunolabeling, we demonstrated that highly anisotropic ligand-coated GNRs ("AR4" and "AR7") facilitated the recruitment of integrin ß1 on macrophages to their nanoscale surfaces. Consequently, highly anisotropic GNRs (e.g., "AR4" and "AR7") elevated the adhesion and M2 state of macrophages, with the inhibition of their M1 state in the culture and mice, entailing rho-associated protein kinase. This nanoscale anisotropic nanogeometry provides a novel and critical parameter to be considered in the generation of biomaterials to potentially modulate host reactions to the implants for immunomodulatory tissue regeneration.


Assuntos
Integrina beta1/metabolismo , Macrófagos/efeitos dos fármacos , Nanopartículas/química , Próteses e Implantes , Animais , Anisotropia , Materiais Biocompatíveis/administração & dosagem , Materiais Biocompatíveis/química , Adesão Celular/efeitos dos fármacos , Polaridade Celular/efeitos dos fármacos , Células Cultivadas , Humanos , Integrina beta1/química , Ligantes , Macrófagos/química , Camundongos , Nanopartículas/administração & dosagem , Nanotubos/química , Oligopeptídeos/química , Quinases Associadas a rho/genética
17.
Future Microbiol ; 14: 293-313, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30757918

RESUMO

AIM: To investigate the formation of Mycobacterium avium membrane vesicles (MVs) within macrophage phagosomes. MATERIALS & METHODS: A phagosome model was utilized to characterize proteomics and lipidomics of MVs. A click chemistry-based enrichment assay was employed to examine the presence of MV proteins in the cytosol of host cells. RESULTS: Exposure to metals at concentrations present in phagosomes triggers formation of bacterial MVs. Proteomics identified several virulence factors, including enzymes involved in the cell wall synthesis, lipid and fatty acid metabolism. Some of MV proteins were also identified in the cytosol of infected macrophages. MVs harbor dsDNA. CONCLUSION: M. avium produces MVs within phagosomes. MVs carry products with potential roles in modulation of host immune defenses and intracellular survival.


Assuntos
Macrófagos/metabolismo , Infecções por Mycobacterium/microbiologia , Mycobacterium avium/metabolismo , Fagossomos/metabolismo , Vesículas Transportadoras/metabolismo , Proteínas de Bactérias/análise , Proteínas de Bactérias/metabolismo , Ácidos Graxos/análise , Ácidos Graxos/metabolismo , Humanos , Macrófagos/química , Mycobacterium avium/química , Fagossomos/química , Proteômica , Vesículas Transportadoras/química
18.
Environ Int ; 125: 75-81, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30710802

RESUMO

BACKGROUND: Urinary lead (Pb) is generally considered to have limited use in biomonitoring environmental exposure to lead. Carbon load in airway macrophages (AM BC) is an internal marker to assess long-term exposure to combustion-derived aerosol particles. In urban environments, atmospheric Pb and black carbon may have common sources. We aimed to study the temporal change of urinary Pb (U-Pb) when exposure to outdoor air pollution changes, and the relationship between U-Pb and AM BC. METHODS: A panel of 50 young healthy adults [mean (SD) 26.7 (5.2) years], including 17 long-term (>1 year) residents in Leuven, Belgium (BE), 15 and 18 newcomers (arrived <3 weeks) from low- and middle-income countries (LMIC) and high-income countries (HIC), respectively, underwent 8 repeated measurements at 6 weeks intervals. In urine spot samples obtained at 5 time points (T1, T2, T4, T6, T8), 24 trace elements were quantified by inductively coupled plasma-mass spectrometry. At each time point, AM BC was quantified as the median surface of black inclusions (in µm2) by means of image analysis of 25 macrophages obtained by induced sputum. Changes in urinary metal concentrations (with and without creatinine correction) and the relationship between U-Pb and AM BC were estimated using linear mixed models adjusted for covariates and potential confounders. RESULTS: Only U-Pb differed between groups and exhibited significant time trends. Participants from the LMIC group had significantly higher initial U-Pb (1.18 µg/g creat) than the HIC group (0.44 µg/g creat) and BE group (0.45 µg/g creat). In the LMIC group, U-Pb decreased significantly with time by 0.061 µg/g creatinine per 30 days [95% confidence interval (CI): 0.034, 0.088]. U-Pb remained unchanged in the other two groups. An increase in AM BC of 1 µm2 was associated with an increase in U-Pb of 0.369 µg/g creat (95% CI: 0.145, 0.593). CONCLUSION: This panel study demonstrates that U-Pb may be a valid alternative to blood Pb for biomonitoring changes in exposure to lead, at least at group level. In addition, we identified a positive association between U-Pb and AM BC, a biomarker of exposure to traffic-related air pollution, suggesting the existence of common sources of Pb and black carbon in urban environments.


Assuntos
Poluentes Atmosféricos/urina , Chumbo/urina , Macrófagos/química , Fuligem/análise , Adulto , Poluição do Ar , Bélgica , Cidades , Monitoramento Ambiental , Feminino , Humanos , Estudos Longitudinais , Masculino , Emissões de Veículos , Adulto Jovem
19.
J Chromatogr A ; 1590: 104-112, 2019 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-30630618

RESUMO

Split-flow fractionation (SPLITT) is a family of techniques that separates in the absence of labeling using very low flow rates and force fields, and is therefore expected to minimize cell damage. Although it has been documented that separation methods cause physiological changes in immune cells that are attributable to mechanical stress and antibody labeling, SPLITT has not yet been examined for possible damaging effects of hydrodynamic stress, partly because it is assumed that the low flow rates and weak forces used in this technique do not generate significant mechanical stress. The aim of this study was to investigate the effects of SPLITT on cell function of a murine macrophage cell, and to compare these effects with those induced by centrifugation. Macrophages J774.2 were cultured in RPMI-enriched media, then detached from the culture flask and resuspended for 12 h. Cell suspensions were diluted in a buffered saline solution and exposed to SPLITT (flow rates 1-10 ml/min) or centrifugation (100-1500g) for 10 min. Cell viability, diameter, membrane potential, and nitric oxide production were measured. Under the operating conditions employed, cell viability was above 98% after SPLITT and centrifugation but cells suffered immediate hydrodynamic cell damage, including decreased cell diameter and membrane hyperpolarization which was inhibitable by 4-aminopyridine; nitric oxide production was not affected. Pressure values during SPLITT and centrifugation correlated with diameter and membrane potential. Our data do not support the assumption that SPLITT is innocuous to cell function. Some changes in SPLITT channel design are suggested to minimize cell damage. Membrane potential and cell diameter are sensitive indicators for the evaluation of sublethal damage in different cell models, and allow identification of optimal operating conditions on different scales.


Assuntos
Fracionamento Químico/métodos , Macrófagos , Animais , Linhagem Celular , Centrifugação , Macrófagos/química , Macrófagos/citologia , Camundongos
20.
Dalton Trans ; 48(4): 1489-1503, 2019 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-30632585

RESUMO

Overexpression of cysteine cathepsins proteases has been documented in a wide variety of cancers, and enhances the l-cysteine concentration in tumor cells. We report the synthesis and characterization of copper(ii) complexes [Cu(L1)2(H2O)](SO3CF3)2, 1, L1 = 3-phenyl-1-(pyridin-2-yl)imidazo[1,5-a]pyridine, [Cu(L2)2(SO3CF3)]SO3CF3, 2, L2 = 3-(4-methoxyphenyl)-1-pyridin-2-yl-imidazo[1,5-a]pyridine, [Cu(L3)2(H2O)](SO3CF3)2, 3, L3 = 3-(3,4-dimethoxy-phenyl)-1-pyridin-2-yl-imidazo[1,5-a]pyridine and [Cu(L4)2(H2O)](SO3CF3)2, 4, L4 = dimethyl-[4-(1-pyridin-2-yl-imidazo[1,5-a]pyridin-3-yl)phenyl]amine as 'turn-on' optical imaging probes for l-cysteine in cancer cells. The molecular structure of complexes adopted distorted trigonal pyramidal geometry (τ, 0.68-0.87). Cu-Npy bonds (1.964-1.989 Å) were shorter than Cu-Nimi bonds (2.024-2.074 Å) for all complexes. Geometrical distortion was strongly revealed in EPR spectra, showing g‖ (2.26-2.28) and A‖ values (139-163 × 10-4 cm-1) at 70 K. The d-d transitions appeared around 680-741 and 882-932 nm in HEPES, which supported the existence of five-coordinate geometry in solution. The Cu(ii)/Cu(i) redox potential of 1 (0.221 V vs. NHE) was almost identical to that of 2 and 3 but lower than that of 4 (0.525 V vs. NHE) in HEPES buffer. The complexes were almost non-emissive in nature, but became emissive by the interaction of l-cysteine in 100% HEPES at pH 7.34 via reduction of Cu(ii) to Cu(i). Among the probes, probe 2 showed selective and efficient turn-on fluorescence behavior towards l-cysteine over natural amino acids with a limit of detection of 9.9 × 10-8 M and binding constant of 2.3 × 105 M-1. The selectivity of 2 may have originated from a nearly perfect trigonal plane adopted around a copper(ii) center (∼120.70°), which required minimum structural change during the reduction of Cu(ii) to Cu(i) while imaging Cys. The other complexes, with their distorted trigonal planes, required more reorganizational energy, which resulted in poor selectivity. Probe 2 was employed for optical imaging of l-cysteine in HeLa cells and macrophages. It exhibited brighter fluorescent images by visualizing Cys at pH 7.34 and 37 °C. It showed relatively less toxicity for these cell lines as ascertained by the MTT assay.


Assuntos
Complexos de Coordenação/farmacologia , Cobre/farmacologia , Cisteína/análise , Corantes Fluorescentes/farmacologia , Imidazóis/farmacologia , Imagem Óptica , Piridinas/farmacologia , Neoplasias do Colo do Útero/diagnóstico por imagem , Sobrevivência Celular/efeitos dos fármacos , Complexos de Coordenação/síntese química , Complexos de Coordenação/química , Cobre/química , Relação Dose-Resposta a Droga , Espectroscopia de Ressonância de Spin Eletrônica , Feminino , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/química , Células HeLa , Humanos , Imidazóis/química , Macrófagos/química , Macrófagos/efeitos dos fármacos , Estrutura Molecular , Piridinas/química , Relação Estrutura-Atividade , Neoplasias do Colo do Útero/química
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