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1.
Front Immunol ; 12: 636289, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33763080

RESUMO

Although widely prevalent, Lyme disease is still under-diagnosed and misunderstood. Here we followed 73 acute Lyme disease patients and uninfected controls over a period of a year. At each visit, RNA-sequencing was applied to profile patients' peripheral blood mononuclear cells in addition to extensive clinical phenotyping. Based on the projection of the RNA-seq data into lower dimensions, we observe that the cases are separated from controls, and almost all cases never return to cluster with the controls over time. Enrichment analysis of the differentially expressed genes between clusters identifies up-regulation of immune response genes. This observation is also supported by deconvolution analysis to identify the changes in cell type composition due to Lyme disease infection. Importantly, we developed several machine learning classifiers that attempt to perform various Lyme disease classifications. We show that Lyme patients can be distinguished from the controls as well as from COVID-19 patients, but classification was not successful in distinguishing those patients with early Lyme disease cases that would advance to develop post-treatment persistent symptoms.


Assuntos
Leucócitos Mononucleares/imunologia , Doença de Lyme/genética , Adulto , /imunologia , Citocinas/genética , Citocinas/imunologia , Feminino , Seguimentos , Humanos , Leucócitos Mononucleares/química , Doença de Lyme/sangue , Doença de Lyme/imunologia , Aprendizado de Máquina , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , RNA-Seq
2.
Mol Med Rep ; 23(1): 1, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33169172

RESUMO

Circular RNAs (circRNAs) have gained attention due to their performance in disease diagnosis. However, the characteristics of circRNAs in peripheral blood from patients with systemic lupus erythematosus (SLE) remain unknown. Therefore, the aim of the present study was to determine the expression profile and diagnostic potential of circRNAs in peripheral blood from patients with SLE. The global circRNA expression in the peripheral blood of patients with SLE and healthy controls (HCs) was detected using a circRNA microarray. Then, the expression levels of three upregulated circRNAs were selected for further validation by reverse transcription­quantitative PCR (RT­qPCR) in a training set. Moreover, the diagnostic value of these circRNAs was assessed by constructing a receiver operating characteristic curve, and then verified in a blind testing set. In total, 1,566 circRNAs were identified to be dysregulated between patients with SLE and HCs (≥2 fold change, P<0.05). Furthermore, the RT­qPCR results were consistent with the microarray data, in that all three selected circRNAs, hsa_circ_0082688, hsa_circ_0082689 and hsa_circ_0008675, were significantly upregulated in patients with SLE (P<0.05). Results from the training set demonstrated that the combination of hsa_circ_0082688­hsa_circ_0082689 may provide the most beneficial diagnostic potential. Moreover, the blind test results indicated that the combination model of hsa_circ_0082688­hsa_circ_0082689 could effectively discriminate between patients with SLE from patients with rheumatoid arthritis and HCs, with a sensitivity of 91.30%, a specificity of 78.57% and an accuracy of 82.28%. Moreover, the combination model of hsa_circ_0082688­hsa_circ_0082689 + anti­dsDNA could more effectively discriminated the SLE group from the control groups, with a sensitivity of 95.65%, a specificity of 100.00% and an accuracy of 98.73%. In addition, correlation analysis results suggested that all three circRNAs in patients with SLE did not correlate with the SLE disease activity index. In conclusion, the expression levels of hsa_circ_0082688­hsa_circ_0082689 may serve as potential biomarkers for SLE diagnosis.


Assuntos
Biomarcadores/sangue , Leucócitos Mononucleares/química , Lúpus Eritematoso Sistêmico/sangue , Lúpus Eritematoso Sistêmico/diagnóstico , RNA Circular/sangue , Adolescente , Adulto , Idoso , Artrite Reumatoide/sangue , Artrite Reumatoide/genética , Correlação de Dados , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Lúpus Eritematoso Sistêmico/genética , Masculino , Pessoa de Meia-Idade , Modelos Biológicos , Análise de Sequência com Séries de Oligonucleotídeos , RNA Circular/genética , Curva ROC , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para Cima
3.
J Vis Exp ; (160)2020 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-32658205

RESUMO

A multiplexed droplet PCR (mdPCR) workflow and detailed protocol for determining epigenetic-based white blood cell (WBC) differential count is described, along with a thermoplastic elastomer (TPE) microfluidic droplet generation device. Epigenetic markers are used for WBC subtyping which is of important prognostic value in different diseases. This is achieved through the quantification of DNA methylation patterns of specific CG-rich regions in the genome (CpG loci). In this paper, bisulfite-treated DNA from peripheral blood mononuclear cells (PBMCs) is encapsulated in droplets with mdPCR reagents including primers and hydrolysis fluorescent probes specific for CpG loci that correlate with WBC sub-populations. The multiplex approach allows for the interrogation of many CpG loci without the need for separate mdPCR reactions, enabling more accurate parametric determination of WBC sub-populations using epigenetic analysis of methylation sites. This precise quantification can be extended to different applications and highlights the benefits for clinical diagnosis and subsequent prognosis.


Assuntos
Metilação de DNA/fisiologia , Testes Hematológicos/métodos , Reação em Cadeia da Polimerase Multiplex/métodos , Polímeros/química , Humanos , Leucócitos Mononucleares/química
4.
Environ Health ; 19(1): 78, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620109

RESUMO

BACKGROUND: Asthma patients suffer from periodic acute worsening of symptoms (i.e. loss of asthma control or exacerbations), triggered by a variety of exogenous stimuli. With the growing awareness that air pollutants impact respiratory diseases, we investigated whether particulate matter (PM) derived from various livestock farms (BioPM) differentially affected innate and oxidative stress responses in asthma and health. METHODS: Peripheral blood mononuclear cells (PBMCs), collected from patients sequentially before and during loss of asthma control and from healthy individuals, were exposed to BioPM collected from chicken, goat and pig farms (1 and 5 µg/ml), with or without pre-treatment with antioxidants. Cytokine release and oxidative stress were assessed. RESULTS: PBMCs produced IFNγ, IL-1ß, IL-10 and TNFα upon stimulation with BioPM, with that from pig farms inducing the highest cytokine levels. Overall, cytokine production was irrespective of the presence or state of disease. However, PBMCs from stable asthma patients upon exposure to the three BioPM showed more extreme TNFα responses than those from healthy subjects. Furthermore, PBMCs obtained during loss of asthma control that were exposed to BioPM from pig farms showed enhanced IFNγ release as well as decreased oxidative stress levels upon pre-treatment with N-acetylcysteine (NAC) compared to stable disease. NAC, but not superoxide dismutase and catalase, also counteracted BioPM-induced cytokine release, indicating the importance of intracellular reactive oxygen species in the production of cytokines. CONCLUSIONS: BioPM triggered enhanced pro-inflammatory responses by PBMCs from both healthy subjects and asthma patients, with those from patients during loss of asthma control showing increased susceptibility to BioPM from pig farms in particular.


Assuntos
Poluentes Atmosféricos/efeitos adversos , Citocinas/metabolismo , Fazendas , Leucócitos Mononucleares/química , Estresse Oxidativo , Material Particulado/efeitos adversos , Animais , Asma/fisiopatologia , Galinhas , Saúde Ambiental , Cabras , Gado , Sus scrofa
5.
PLoS One ; 15(7): e0235214, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32614841

RESUMO

Placenta-derived extracellular vesicles (EVs) are involved in communication between the placenta and maternal immune cells possibly leading to a modulation of maternal T-cell signaling components. The ability to identify EVs in maternal blood may lead to the development of diagnostic and treatment tools for pregnancy complications. The objective of this work was to differentiate EVs from bovine placenta (trophoblast) and peripheral blood mononuclear cells (PBMC) by a label-free, non-invasive Raman spectroscopy technique. Extracellular vesicles were isolated by ultracentrifugation. Dynamic light scattering (DLS) and scanning electron microscopy (SEM) were applied to verify the presence and the size distribution of EVs. Raman peaks at 728 cm-1 (collagen) and 1573 cm-1 (protein) were observed only in PBMC-derived EVs, while the peaks 702 cm-1 (cholesterol) and 1553 cm-1 (amide) appeared only in trophoblast-derived EVs. The discrimination of the Raman spectral fingerprints for both types of EVs from different animals was performed by principal component analysis (PCA) and linear discriminant analysis (LDA). The PCA and LDA results clearly segregated the spectral clusters between the two types of EVs. Moreover, the PBMC-derived EVs from different animals were indistinguishable, while the trophoblast-derived EVs from three placental samples of different gestational ages showed separate clusters. This study reports for the first time the Raman characteristic peaks for identification of PBMC and trophoblast-derived EVs. The development of this method also provides a potential tool for further studies investigating the causes and potential treatments for pregnancy complications.


Assuntos
Vesículas Extracelulares/química , Leucócitos Mononucleares/química , Trofoblastos/química , Animais , Bovinos , Células Cultivadas , Feminino , Placenta/química , Placenta/citologia , Gravidez , Análise Espectral Raman/métodos , Trofoblastos/citologia
6.
BMC Genomics ; 21(1): 229, 2020 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-32171242

RESUMO

BACKGROUND: Gene expression regulators identified in transcriptome profiling experiments may serve as ideal targets for genetic manipulations in farm animals. RESULTS: In this study, we developed a gene expression profile of 76,000+ unique transcripts for 224 porcine samples from 28 tissues collected from 32 animals using Super deepSAGE technology. Excellent sequencing depth was achieved for each multiplexed library, and replicated samples from the same tissues clustered together, demonstrating the high quality of Super deepSAGE data. Comparison with previous research indicated that our results not only have good reproducibility but also have greatly extended the coverage of the sample types as well as the number of genes. Clustering analysis revealed ten groups of genes showing distinct expression patterns among these samples. Our analysis of over-represented binding motifs identified 41 regulators, and we demonstrated a potential application of this dataset in infectious diseases and immune biology research by identifying an LPS-dependent transcription factor, runt-related transcription factor 1 (RUNX1), in peripheral blood mononuclear cells (PBMCs). The selected genes are specifically responsible for the transcription of toll-like receptor 2 (TLR2), lymphocyte-specific protein tyrosine kinase (LCK), and vav1 oncogene (VAV1), which belong to the T and B cell signaling pathways. CONCLUSIONS: The Super deepSAGE technology and tissue-differential expression profiles are valuable resources for investigating the porcine gene expression regulation. The identified RUNX1 target genes belong to the T and B cell signaling pathways, making them novel potential targets for the diagnosis and therapy of bacterial infections and other immune disorders.


Assuntos
Biologia Computacional/métodos , Perfilação da Expressão Gênica/veterinária , Redes Reguladoras de Genes , Sus scrofa/genética , Animais , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Regulação da Expressão Gênica , Leucócitos Mononucleares/química , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/genética , Proteínas Proto-Oncogênicas c-vav/genética , Reprodutibilidade dos Testes , Suínos , Distribuição Tecidual , Receptor 2 Toll-Like/genética
7.
Environ Pollut ; 261: 113724, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32078875

RESUMO

Present study screened the toxicological assessment of airborne particulate matter (PM), mechanistic investigation, relationship between the physicochemical characteristics and its associated toxic response. The average concentration of both PM10 and PM2.5 exceeded the Indian National Ambient Air Quality Standards. In present study, PM bound metals; Fe, Cu, Cr, Ni, Mn, Pb, Cd, Zn, Sr and Co have been taken into account with total metal concentration of 0.83 and 0.44 µg m-3 of PM10 and PM2.5 mass concentrations, respectively. The contribution of redox active metals (Fe, Cu, Cr, Ni and Mn) in PM was more as compared to non-redox metals (Pb, Cd and Co) indicating significant risk to the exposed population as these metals possess the ability to produce reactive oxygen species (ROS) which are responsible for various diseases. The cytotoxicity profiles of PM samples determined by MTT assay on two different cell lines (A549 and PBMC) exhibited dose-dependent effects after 24 h exposure, but the consequences differ with respect to particle size and sampling periods. A significant decrease in cell viability with varying PM concentrations (20, 40, 60, 80 and 100 µg ml-1) with respect to control was found in both cell lines. Incubation of RBC suspension with PM samples caused pronounced disruption of RBC and thus exhibited substantial hemolytic behavior. PM samples showed a range of potency to produce reactive oxygen species (ROS). Almost all PM samples increased the level of pro-inflammatory mediator (Nitric oxide) when compared to corresponding unexposed controls suggesting the important role of reactive nitrogen species in induction of cellular toxicity.


Assuntos
Poluentes Atmosféricos/análise , Material Particulado/análise , Atmosfera , Monitoramento Ambiental , Índia , Leucócitos Mononucleares/química , Tamanho da Partícula , Espécies Reativas de Oxigênio
8.
PLoS One ; 15(2): e0226311, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32053618

RESUMO

It is not currently possible to reliably visualise and track immune cells in the human central nervous system or eye. Previous work demonstrated that indocyanine green (ICG) dye could label immune cells and be imaged after a delay during disease in the mouse retina. We report a pilot study investigating if ICG can similarly label immune cells within the human retina. Twelve adult participants receiving ICG angiography as part of routine standard of care were recruited. Baseline retinal images were obtained prior to ICG administration then repeated over a period ranging from 2 hours to 9 days. Matched peripheral blood samples were obtained to examine systemic immune cell labelling and activation from ICG by flow cytometry with human macrophage cultures as positive controls. Differences between the delayed near infrared ICG imaging and 488 nm autofluorescence was observed across pathologies, likely arising from the retinal pigment epithelium (RPE). Only one subject demonstrated ICG signal on peripheral blood myeloid cells and only three distinct cell-sized signals appeared over time within the retina of three participants. No significant increase in immune cell activation markers were detected after ICG administration. ICG accumulated in the endosomes of macrophage cultures and was detectable above a minimum concentration, suggesting cell labelling is possible. ICG can label RPE and may be used as an additional biomarker for RPE health across a range of retinal disorders. Standard clinical doses of intravenous ICG do not lead to robust immune cell labelling in human blood or retina and further optimisation in dose and route are required.


Assuntos
Corantes/administração & dosagem , Verde de Indocianina/administração & dosagem , Leucócitos Mononucleares/química , Macrófagos/química , Epitélio Pigmentado da Retina/diagnóstico por imagem , Adulto , Idoso , Corantes/química , Endossomos/química , Estudos de Viabilidade , Feminino , Citometria de Fluxo , Angiofluoresceinografia , Humanos , Verde de Indocianina/química , Injeções Intravenosas , Macrófagos/citologia , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Estudos Prospectivos , Epitélio Pigmentado da Retina/citologia , Coloração e Rotulagem/métodos , Adulto Jovem
9.
Sci Rep ; 10(1): 2064, 2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-32034172

RESUMO

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a debilitating multisystemic disease of unknown etiology, affecting thousands of individuals worldwide. Its diagnosis still relies on ruling out medical problems leading to unexplained fatigue due to a complete lack of disease-specific biomarkers. Our group and others have explored the potential value of microRNA profiles (miRNomes) as diagnostic tools for this disease. However, heterogeneity of participants, low numbers, the variety of samples assayed, and other pre-analytical variables, have hampered the identification of  disease-associated miRNomes. In this study, our team has evaluated, for the first time, ME/CFS miRNomes in peripheral blood mononuclear cells (PBMCs) and extracellular vesicles (EVs) from severely ill patients recruited at the monographic UK ME biobank to assess, using standard operating procedures (SOPs), blood fractions with optimal diagnostic power for a rapid translation of a miR-based diagnostic method into the clinic. Our results show that routine creatine kinase (CK) blood values, plasma EVs physical characteristics (including counts, size and zeta-potential), and a limited number of differentially expressed PBMC and EV miRNAs appear significantly associated with severe ME/CFS (p < 0.05). Gene enrichment analysis points to epigenetic and neuroimmune dysregulated pathways, in agreement with previous reports. Population validation by a cost-effective approach limited to these few potentially discriminating variables is granted.


Assuntos
Vesículas Extracelulares/química , Síndrome de Fadiga Crônica/diagnóstico , Leucócitos Mononucleares/química , MicroRNAs/sangue , Adolescente , Adulto , Biomarcadores/sangue , Estudos de Casos e Controles , Feminino , Nível de Saúde , Humanos , Pessoa de Meia-Idade , Qualidade de Vida , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Adulto Jovem
10.
PLoS One ; 15(1): e0223814, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31910217

RESUMO

INTRODUCTION: Chimeric antigen receptor (CAR) T-cells have been recently developed and are producing impressive outcomes in patients with hematologic malignancies. However, there is no standardized method for cell trafficking and in vivo CAR T-cell monitoring. We assessed the feasibility of real-time in vivo 89Zr-p-Isothiocyanatobenzyl-desferrioxamine (Df-Bz-NCS, DFO) labeled CAR T-cell trafficking using positron emission tomography (PET). RESULTS: The 89Zr-DFO radiolabeling efficiency of Jurkat/CAR and human peripheral blood mononuclear cells (hPBMC)/CAR T-cells was 70%-79%, and cell radiolabeling activity was 98.1-103.6 kBq/106 cells. Cell viability after radiolabeling was >95%. Cell proliferation was not significantly different during the early period after radiolabeling, compared with unlabeled cells; however, the proliferative capacity decreased over time (day 7 after labeling). IL-2 or IFN-γ secretion was not significantly different between unlabeled and labeled CAR T-cells. PET/magnetic resonance imaging in the xenograft model showed that most of the 89Zr-DFO-labeled Jurkat/CAR T-cells were distributed in the lung (24.4% ± 3.4%ID) and liver (22.9% ± 5.6%ID) by one hour after injection. The cells gradually migrated from the lung to the liver and spleen by day 1, and remained stable in these sites until day 7 (on day 7: lung 3.9% ± 0.3%ID, liver 36.4% ± 2.7%ID, spleen 1.4% ± 0.3%ID). No significant accumulation of labeled cells was identified in tumors. A similar pattern was observed in ex vivo biodistributions on day 7 (lung 3.0% ± 1.0%ID, liver 19.8% ± 2.2%ID, spleen 2.3% ± 1.7%ID). 89Zr-DFO-labeled hPBMC/CAR T-cells showed a similar distribution, compared with Jurkat/CAR T-cells, on serial PET images. CAR T cell distribution was cross-confirmed by flow cytometry, Alu polymerase chain reaction, and immunohistochemistry. CONCLUSION: Real-time in vivo cell trafficking is feasible using PET imaging of 89Zr-DFO-labeled CAR T-cells. This can be used to investigate cellular kinetics, initial in vivo biodistribution, and safety profiles in future CAR T-cell development.


Assuntos
Desferroxamina/análogos & derivados , Isotiocianatos/farmacologia , Radioisótopos/farmacologia , Receptores de Antígenos de Linfócitos T/isolamento & purificação , Receptores de Antígenos Quiméricos/isolamento & purificação , Zircônio/farmacologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Desferroxamina/farmacologia , Neoplasias Hematológicas/tratamento farmacológico , Neoplasias Hematológicas/patologia , Humanos , Imunoconjugados/farmacologia , Marcação por Isótopo , Células Jurkat , Leucócitos Mononucleares/química , Leucócitos Mononucleares/efeitos dos fármacos , Tomografia por Emissão de Pósitrons , Radioisótopos/química , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/uso terapêutico , Receptores de Antígenos Quiméricos/química , Receptores de Antígenos Quiméricos/uso terapêutico , Linfócitos T/química , Linfócitos T/imunologia , Distribuição Tecidual
11.
J Colloid Interface Sci ; 565: 278-287, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-31978790

RESUMO

The use of polymeric nanoparticles (NPs) as therapeutics has been steadily increasing over past decades. In vivo imaging of NPs is necessary to advance the therapeutic performance. 19F Magnetic Resonance Imaging (19F MRI) offers multiple advantages for in vivo imaging. However, design of a probe for both biodistribution and degradation has not been realized yet. We developed polymeric NPs loaded with two fluorocarbons as promising imaging tools to monitor NP biodistribution and degradation by 19F MRI. These 200 nm NPs consist of poly(lactic-co-glycolic acid) (PLGA) loaded with perfluoro-15-crown-5 ether (PFCE) and PERFECTA. PERFECTA/PFCE-PLGA NPs have a fractal sphere structure, in which both fluorocarbons are distributed in the polymeric matrix of the fractal building blocks, which differs from PFCE-PLGA NPs and is unique for fluorocarbon-loaded colloids. This structure leads to changes of magnetic resonance properties of both fluorocarbons after hydrolysis of NPs. PERFECTA/PFCE-PLGA NPs are colloidally stable in serum and biocompatible. Both fluorocarbons show a single resonance in 19F MRI that can be imaged separately using different excitation pulses. In the future, these findings may be used for biodistribution and degradation studies of NPs by 19F MRI in vivo using "two color" labeling leading to improvement of drug delivery agents.


Assuntos
Cor , Imagem por Ressonância Magnética de Flúor-19 , Fluorcarbonetos/metabolismo , Leucócitos Mononucleares/metabolismo , Nanopartículas/metabolismo , Sobrevivência Celular , Células Cultivadas , Fluorcarbonetos/química , Humanos , Leucócitos Mononucleares/química , Leucócitos Mononucleares/citologia , Estrutura Molecular , Nanopartículas/química , Tamanho da Partícula , Propriedades de Superfície
12.
Clin Chim Acta ; 503: 210-217, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31794770

RESUMO

Rejection and toxicity occur despite monitoring of tacrolimus blood levels during clinical routine. The intracellular concentration in lymphocytes could be a better reflection of the tacrolimus exposure. Four extraction methods for tacrolimus in peripheral blood mononuclear cells were validated and evaluated with UHPLC-MS/MS. Methods based on protein precipitation (method 1), solid phase extraction (method 2), phospholipids and proteins removal (method 3) and liquid-liquid extraction (method 4) were evaluated on linearity, lower limit of quantification (LLOQ), imprecision and bias. Validation was completed for the methods within these requirements, adding matrix effect and recovery. Linearity was 0.126 (LLOQ)-15 µg/L, 0.504 (LLOQ)-15 µg/L and 0.298 (LLOQ)-15 µg/L with method 1, 2 and 3, respectively. With method 4 non-linearity and a LLOQ higher than 0.504 µg/L were observed. Inter-day imprecision and bias were ≤4.6%, ≤10.9%; ≤6.8%, ≤-11.2%; ≤9.4%, ≤10.3% and ≤44.6%, ≤23.1%, respectively, with methods 1, 2, 3 and 4. Validation was completed for method 1 and 3 adding matrix effect (7.6%; 15.0%) and recovery (8.9%; 10.8%), respectively. The most suitable UHPLC-MS/MS method for quantification of intracellular tacrolimus was protein precipitation due to the best performance characteristics and the least time-consuming rate and complexity.


Assuntos
Leucócitos Mononucleares/química , Manejo de Espécimes/métodos , Tacrolimo/análise , Precipitação Química , Cromatografia Líquida de Alta Pressão/métodos , Humanos , Lipídeos/isolamento & purificação , Extração Líquido-Líquido/normas , Proteínas/isolamento & purificação , Extração em Fase Sólida/normas , Manejo de Espécimes/normas , Espectrometria de Massas em Tandem/métodos
13.
PLoS One ; 14(12): e0227078, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31887133

RESUMO

PURPOSE: To investigate the effect of mitochondrial dysfunction on the autoregulation of blood flow, by measuring levels of glutathione, an indicator of mitochondrial dysfunction, in glaucoma patients. METHODS: Fifty-six OAG patients and 21 age-matched controls underwent a blood assay. Mitochondrial function was measured according to the levels of total glutathione (t-GSH), reduced GSH (GSH), and oxidized GSH (GSSG, glutathione disulfide) in peripheral blood mononuclear cells. Ocular blood flow in the optic nerve head was assessed with laser speckle flowgraphy parameters, including acceleration time index (ATI). We determined correlations between these measurements and other clinical parameters. Furthermore, we investigated the association between glutathione levels and glaucoma with a logistic regression analysis. Finally, we calculated the area under the receiver operating characteristic (ROC) curve in order to determine the power of redox index (the log GSH/GSSG ratio) to distinguish the groups. RESULTS: OAG patients demonstrated significantly higher GSSG levels and a lower redox index than the controls (p = 0.01, p = 0.01, respectively), but total GSH and reduced GSH levels were similar in the OAG subjects and controls (p = 0.80, p = 0.94, respectively). Additionally, redox index was significantly correlated with mean deviation (MD) of the visual field (r = 0.29, p = 0.03) and ATI (r = -0.30, p = 0.03). Multiple linear regression analysis showed that redox index contributed to MD (p = 0.02) and ATI (p = 0.04). The receiver operating characteristic curve (AUC) analysis suggested that redox index could differentiate between control eyes and eyes with glaucoma (AUC; 0.70: 95% interval; 0.57-0.84). The cutoff point for redox index to maximize its sensitivity and specificity was 2.0 (sensitivity: 91.1%, specificity: 42.9%). CONCLUSIONS: These results suggest that redox index is lower in OAG patients than in controls. Thus, it is possible that mitochondrial dysfunction contributes to glaucoma pathogenesis by causing vascular alterations.


Assuntos
Glaucoma/patologia , Glutationa/análise , Leucócitos Mononucleares/química , Mitocôndrias/patologia , Disco Óptico/irrigação sanguínea , Idoso , Estudos de Casos e Controles , Feminino , Glaucoma/sangue , Glaucoma/fisiopatologia , Humanos , Fluxometria por Laser-Doppler , Masculino , Pessoa de Meia-Idade , Mitocôndrias/metabolismo , Disco Óptico/citologia , Disco Óptico/diagnóstico por imagem , Disco Óptico/patologia , Oxirredução , Espécies Reativas de Oxigênio/metabolismo , Fluxo Sanguíneo Regional/fisiologia
14.
Clin Lab ; 65(11)2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31710429

RESUMO

Beckground: The current study aims to investigate whether miR-937 can be used as a diagnostic biomarker in peripheral blood mononuclear cells (PBMCs) for children with Kawasaki disease (KD) with or without coronary artery dilation (CAD). METHODS: Gene chip technology was used to screen miRNAs differentially expressed between KD children and normal healthy children. Furthermore, real time PCR was carried out to validate the expression of miR-937 in 50 children with KD (25 cases with and 25 cases without CAD) and 25 healthy children. Meanwhile, target genes of miR-937 were analyzed using TargetScan and dual luciferase reporter assay. RESULTS: First, 20 miRs with significantly differentially expressed mononuclear cell (PBMCs) in peripheral blood between children with KD and normal healthy children were identified by gene chip technology. Real time PCR analysis validated that the expression of miR-937 decreased most significantly among all differentially expressed miRNAs. Secondly, miR-937 was down-regulated significantly before treatment with gamma globulin (IVIG), while its expression was significantly up-regulated after IVIG treatment. In addition, the expression of miR-937 in KD children with CAD was significantly lower than that of KD children without CAD. Person's correlation assay showed that miR-937 negatively correlated with CAD. Dual luciferase reporter assay indicated that IL-1ß was a target gene of miR-937. CONCLUSIONS: In summary, miR-937 in PBMCs was involved in the occurrence and development of KD, which provides new ideas for the prevention and treatment of KD coronary artery dilation.


Assuntos
Aneurisma Coronário/diagnóstico , Perfilação da Expressão Gênica/métodos , Leucócitos Mononucleares/química , MicroRNAs/sangue , Síndrome de Linfonodos Mucocutâneos/diagnóstico , Análise de Sequência com Séries de Oligonucleotídeos , Biomarcadores/sangue , Estudos de Casos e Controles , Criança , Pré-Escolar , Aneurisma Coronário/sangue , Aneurisma Coronário/genética , Feminino , Regulação da Expressão Gênica , Humanos , Imunoglobulinas Intravenosas/uso terapêutico , Fatores Imunológicos/uso terapêutico , Masculino , MicroRNAs/genética , Síndrome de Linfonodos Mucocutâneos/sangue , Síndrome de Linfonodos Mucocutâneos/tratamento farmacológico , Síndrome de Linfonodos Mucocutâneos/genética , Valor Preditivo dos Testes , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos Testes
15.
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
16.
Mol Biol Rep ; 46(6): 6513-6524, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31637621

RESUMO

It is generally believed that due to evolutionary differences and adaptation to tropical conditions, Indian native cattle has superior heat tolerant ability than Bos taurus cattle. In the present study, 3'-UTR of two most important heat responsive genes i.e., heat shock protein 70.1 (HSP70.1) and heat shock factor- 1 (HSF-1) were sequence characterized in different breeds of Indian native cattle to identify the variations and miRNA binding sites. In addition, the impact of heat stress was assessed in a total of 57 PBMCs samples of native Sahiwal cows (Bos indicus), exotic Holstein cows (Bos taurus) and Murrah buffaloes (Bubalus bubalis) using various cellular parameters like cell viability, cytotoxicity and apoptosis. Further, expression profile of 12 heat responsive miRNAs were also evaluated in unstressed and stressed PBMCs to understand post transcriptional changes in native cows, exotic cows and Murrah buffaloes. The sequence data showed 3'-UTR of HSP70.1 gene of Indian cattle to be exactly similar to Bos taurus with no miRNA binding site. Whereas, sequencing of 3'-UTR of HSF-1 gene revealed 3 SNPs at positions G1762T; C1811T and C1983T with 7 well conserved miRNA binding sites. The impact of heat stress on various cellular parameters in terms of cell viability, cytotoxicity and apoptosis was highest in PBMCs of Holstein cows followed by Murrah buffaloes and Sahiwal cows. Further, in contrast to Holstein Frisian cows and Murrah buffaloes, the expression pattern of 12 heat responsive miRNAs, in heat stressed PBMCs of Sahiwal cows were quite distinct. There was a significant (p < 0.05) induction in expression of most of the miRNAs after heat stress in PBMCs of Sahiwal cows followed by a rapid decline. The distinct cellular response and pattern of miRNA expression across cattle types and buffaloes might be influencing their PBMCs tolerance level to heat stress.


Assuntos
Perfilação da Expressão Gênica/veterinária , Fatores de Transcrição de Choque Térmico/química , Leucócitos Mononucleares/química , MicroRNAs/genética , Análise de Sequência de DNA/veterinária , Regiões 3' não Traduzidas , Animais , Sequência de Bases , Sítios de Ligação , Búfalos , Bovinos , Sequência Conservada , Regulação da Expressão Gênica , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP70/genética , Fatores de Transcrição de Choque Térmico/genética , Resposta ao Choque Térmico , Polimorfismo de Nucleotídeo Único
17.
PLoS One ; 14(9): e0221811, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31532776

RESUMO

OBJECTIVE: Moyamoya disease (MMD) is a chronic occlusive cerebrovascular disease with unknown etiology, sharing many similar clinical symptoms with other vascular disorders. This study aimed to investigate gene dysregulation in peripheral blood of MMD and compare it with other vascular disorders. METHODS: Transcriptomic profiles of 12 MMD patients and 8 healthy controls were obtained using RNA sequencing. Differentially expressed genes (DEGs) were identified and several were validated by quantitative real-time PCR in independent samples. Biological pathway enrichment analysis of DEGs and deconvolution of leukocyte subsets in peripheral blood were performed. Expression profiles for other vascular diseases were downloaded from public database and consistent DEGs were calculated. Gene set enrichment analysis (GSEA) was conducted to compare gene dysregulation pattern between MMD and other vascular diseases. RESULTS: A total of 533 DEGs were identified for MMD. Up-regulated genes were mainly involved in extracellular matrix (ECM) organization, whereas down-regulated genes were primarily associated with inflammatory and immune responses. As for cell populations, significantly increased naïve B cells and naïve CD4 cells as well as obviously decreased resting natural killer cells were observed in peripheral blood of MMD patients. GSEA analysis indicated that only up-regulated genes of ischemic stroke and down-regulated genes of coronary artery disease and myocardial infarction were enriched in up-regulated and down-regulated genes of MMD, respectively. CONCLUSION: Dysregulated genes in peripheral blood of MMD mainly played key roles in ECM organization, inflammatory and immune responses. This gene dysregulation pattern was specific compared with other vascular diseases. Besides, naïve B cells, naïve CD4 cells and resting natural killer cells were aberrantly disrupted in peripheral blood of MMD patients. These results will help elucidate the complicated pathogenic mechanism of MMD.


Assuntos
Perfilação da Expressão Gênica/métodos , Leucócitos Mononucleares/química , Doença de Moyamoya/genética , Doenças Vasculares/genética , Adulto , Estudos de Casos e Controles , Feminino , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Sequência de RNA
18.
Drug Dev Res ; 80(8): 1128-1135, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31498915

RESUMO

Bipolar disorder (BD) is a complex neuropsychiatric disorder characterized by recurrent mania and depression episodes and requiring lifelong treatment with mood stabilizing drugs. Several lines of evidence, including with BD patient iPSC-derived neurons, suggest that neuronal hyperexcitability may underlie the key clinical symptoms of BD. Indeed, higher mRNA levels of SCN11A, coding for the voltage-gated sodium channel NaV 1.9 implicated in nociception, were detected in iPSC-derived neurons from BD patients, and were normalized by in vitro lithium. Here we studied SCN11A expression in peripheral blood mononuclear cells (PBMCs) from well-phenotyped female BD patients and controls and evaluated their association with several clinical sub-phenotypes. We observed higher mRNA levels of SCN11A in PBMCs from female BD patients with no records of alcohol dependence (p = .0050), no records of psychosis (p = .0097), or no records of suicide attempts (p = .0409). A trend was observed for higher SCN11A expression (FD = 1.91; p = .052) in BD PBMCs compared with controls. Datamining of published postmortem gene expression datasets indicated higher SCN11A expression in dorsolateral prefrontal cortex and orbitofrontal cortex tissues from BD patients compared with controls. Higher phenotype-associated expression levels in PBMC from BD patients were also observed for ID2 (alcohol dependence, suicide attempts) and HDGFRP3 (seasonal BD pattern). Our findings suggest that higher PBMC SCN11A expression levels may be associated with certain behavioral BD sub-phenotypes, including lack of alcohol dependence and psychosis, among BD patients. The NaV 1.9 voltage-gated sodium channel thus deserves consideration as a tentative phenotype modifier in BD.


Assuntos
Transtorno Bipolar/genética , Marcadores Genéticos , Leucócitos Mononucleares/química , Regulação para Cima , Adulto , Transtorno Bipolar/sangue , Estudos de Casos e Controles , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteína 2 Inibidora de Diferenciação/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Pessoa de Meia-Idade , Canal de Sódio Disparado por Voltagem NAV1.9/genética , Fenótipo , Estudos Retrospectivos , Adulto Jovem
19.
PLoS One ; 14(8): e0220902, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31419243

RESUMO

BACKGROUND: Vascular endothelial growth factor (VEGF) is a signal protein, implicated in various physiological and pathophysiological processes together with other common inflammatory biomarkers. However, their associations have not yet been fully elucidated. In the present study, we investigated associations between VEGF and four specific VEGF mRNA isoforms with levels of 11 inflammation molecules, derived from peripheral blood mononuclear cells (PBMCs) extracts. METHODS: Healthy participants from the STANISLAS Family Study (n = 285) were included. Levels of VEGF (four mRNA isoforms and protein levels) and inflammatory molecules (IL-1α, IL-1ß, IL-2, IL-4, IL-6, IL-8, IL-10, INF-γ, TNF-α, MCP-1, EGF) were measured in PBMCs extracts. Multiple regression analyses were performed, adjusted for age and gender. RESULTS: The analyses revealed significant associations between VEGF protein levels and levels of IL-4 (ß = 0.028, P = 0.013), MCP-1 (ß = 0.015, P<0.0001) and EGF (ß = 0.017, P<0.0001). Furthermore, mRNA isoform VEGF165 was associated with MCP-1 and IL-1α (P = 0.002 and P = 0.008, respectively); and mRNA isoform VEGF189 was associated with IL-4 and IL-6 (P = 0.019 and P = 0.034, respectively). CONCLUSIONS: To our knowledge, the present study represents the first investigation that successfully demonstrates links between VEGF protein levels and inflammatory molecules levels derived from PBMCs extracts and identifies associations between specific VEGF mRNA isoforms and inflammatory molecules. IMPACT: These findings provide novel insights that may assist in the development of new tissue and mRNA isoform specific measurements of VEGF levels, which may positively contribute to predicting the risk of common complex diseases and response of currently used anti-VEGF agents, and developing of novel targeted therapies for VEGF-related pathophysiology.


Assuntos
Inflamação/imunologia , Leucócitos Mononucleares/imunologia , Fator A de Crescimento do Endotélio Vascular/imunologia , Adulto , Células Cultivadas , Quimiocina CCL2/análise , Quimiocina CCL2/imunologia , Feminino , Humanos , Inflamação/genética , Interleucinas/análise , Interleucinas/imunologia , Leucócitos Mononucleares/química , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/análise , RNA Mensageiro/genética , Fator de Necrose Tumoral alfa/análise , Fator de Necrose Tumoral alfa/imunologia , Fator A de Crescimento do Endotélio Vascular/análise , Fator A de Crescimento do Endotélio Vascular/genética
20.
Mutat Res ; 843: 11-17, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31421731

RESUMO

This study was designed within the frame of the COST Action hCOMET 15132 (Working Group 6), with the aim of comparing different peripheral blood cell preparations for their feasibility in human biomonitoring studies, using the comet assay for the evaluation of DNA damage. Basal levels of strand breaks/ALS and formamidopyrimidine DNA glycosylase (Fpg) - sites, and H2O2 (500 µM)-induced strand breaks, were measured in whole blood, peripheral blood mononuclear cells - lymphocytes and monocytes - and buffy coat; in fresh and 1, 4 and 12 weeks-frozen samples. The comparison among the fresh preparations showed that the basal levels of DNA damage were all very low and similar in the three samples. Frozen whole blood samples stored in cryostraws without cryoprotection showed similar basal levels of DNA damage as fresh samples, indicating that this preparation, often chosen for biobanks, resists efficiently freezing/thawing artifacts. However, long-term storage of frozen buffy coat samples in cryostraws and with no cryopreservative did not appear feasible. Storage up to 3 months of frozen cryoprotected peripheral blood mononuclear cells induced small increases in basal strand breaks and no other statistically significant modification. Altogether, this study suggests that whole blood could be the most suitable sample to be used to perform comet assay in human epidemiological biomonitoring for genotoxicity assessment in frozen samples, such as those stored in biobanks.


Assuntos
Monitoramento Biológico/métodos , Preservação de Sangue , Ensaio Cometa , Criopreservação , Crioprotetores/farmacologia , Dano ao DNA , Leucócitos Mononucleares/efeitos dos fármacos , Linfócitos/efeitos dos fármacos , Monócitos/efeitos dos fármacos , Adulto , Artefatos , Buffy Coat/citologia , Quebras de DNA/efeitos dos fármacos , DNA-Formamidopirimidina Glicosilase , Estudos de Viabilidade , Feminino , Raios gama , Guanina/análogos & derivados , Guanina/sangue , Humanos , Peróxido de Hidrogênio/farmacologia , Leucócitos Mononucleares/química , Leucócitos Mononucleares/efeitos da radiação , Linfócitos/química , Linfócitos/efeitos da radiação , Pessoa de Meia-Idade , Monócitos/química , Monócitos/efeitos da radiação , Fatores de Tempo , Adulto Jovem
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