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1.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 29(2): 348-325, 2021 Apr.
Artigo em Chinês | MEDLINE | ID: mdl-33812398

RESUMO

OBJECTIVE: To detect the relationship between leukocytes derived microparticle (CD45+ MP) and minimal residual disease (MRD) and prognosis of acute myeloid leukemia (AML). METHODS: The expression of CD45+ MP, CD44+ MP and CD24+ MP in peripheral blood of 47 AML patients at the time after induction chemotherapy were detected by using flow cytometry, and the relationship between MP, MRD and prognosis were analyzed. RESULTS: The percentages of CD45+ MP, CD44+ MP and CD24+ MP in MRD positive group were significantly higher than those in MRD negative group. In MRD positive group, there were positive correlation between CD45+ MP, CD44+ MP, CD24+ MP and MRD level. The AUC of CD45+ MP, CD44+ MP, CD24+ MP in predicting positive MRD was 0.949, 0.782, and 0.817, respectively. The EFS and OS in HCD45+ MP, HCD44+ MP and HCD24+ MP groups were significantly shorter than low level group. CONCLUSION: High level of CD45+ MP, CD44+ MP, CD24+ MP can be used to predict high level MRD and poor prognosis.


Assuntos
Leucemia Mieloide Aguda , Citometria de Fluxo , Humanos , Leucócitos , Neoplasia Residual , Prognóstico
2.
Klin Lab Diagn ; 66(3): 154-159, 2021 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-33793114

RESUMO

Telomere length can be measured by polymerase chain reaction (PCR), allowing to obtain the absolute length of telomeres (ALT) in base pair, and by flow cytometry, which can only estimate the relative telomere length. The aim of the study was to compare the results of the two methods and to develop an accurate and reliable way of converting the relative telomere length to absolute. The peripheral blood from 21 donors was analyzed. Measurement of leukocyte telomere length by flow cytometry was carried out using a commercial Telomere PNA Kit / FITC (Dako, Denmark) with two CytoFLEX flow cytometers (Beckman Coulter, China) and BD FACSCanto II (Becton Dickinson, USA), obtaining the molecular equivalent of fluorescence (MEF). To measure telomere length by real-time PCR, calibrators with a known number of telomeric repeats were prepared. Two quantitative PCRs were carried out: one for telomeric repeats, the other for determining the number of genome-equivalents of DNA, three times for each sample, which made it possible to calculate ALT. A strong direct relationship was found between the MEF obtained with BD FACSCanto II and CytoFLEX (r = 0.97). Analysis of PCR and flow cytometry results showed a significant correlation between ALT and MEF. We calculated the regression equations of ALT and MEF for CytoFLEX - y = 0.0043x (r = 0.84) and for BD FACSCanto II - y = 0.0051x (r = 0.82). Correlation analysis showed a high comparability of telomere lengths measured by two methods. The obtained regression equations allow converting the results of flow cytometry into absolute values, allowing the comparison of the results of different research groups and the use of this method in clinical trials.


Assuntos
Leucócitos , Telômero , DNA , Citometria de Fluxo , Humanos , Hibridização in Situ Fluorescente , Telômero/genética
3.
Nat Commun ; 12(1): 1507, 2021 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-33686069

RESUMO

ATAC-seq is a widely-applied assay used to measure genome-wide chromatin accessibility; however, its ability to detect active regulatory regions can depend on the depth of sequencing coverage and the signal-to-noise ratio. Here we introduce AtacWorks, a deep learning toolkit to denoise sequencing coverage and identify regulatory peaks at base-pair resolution from low cell count, low-coverage, or low-quality ATAC-seq data. Models trained by AtacWorks can detect peaks from cell types not seen in the training data, and are generalizable across diverse sample preparations and experimental platforms. We demonstrate that AtacWorks enhances the sensitivity of single-cell experiments by producing results on par with those of conventional methods using ~10 times as many cells, and further show that this framework can be adapted to enable cross-modality inference of protein-DNA interactions. Finally, we establish that AtacWorks can enable new biological discoveries by identifying active regulatory regions associated with lineage priming in rare subpopulations of hematopoietic stem cells.


Assuntos
Sequenciamento de Cromatina por Imunoprecipitação/métodos , Aprendizado Profundo , Epigenômica/métodos , Animais , Encéfalo , Cromatina , Humanos , Leucócitos , Camundongos , Sequências Reguladoras de Ácido Nucleico
4.
Nat Commun ; 12(1): 1503, 2021 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-33686071

RESUMO

Brain metastases are the most common tumor of the brain with a dismal prognosis. A fraction of patients with brain metastasis benefit from treatment with immune checkpoint inhibitors (ICI) and the degree and phenotype of the immune cell infiltration has been used to predict response to ICI. However, the anatomical location of brain lesions limits access to tumor material to characterize the immune phenotype. Here, we characterize immune cells present in brain lesions and matched cerebrospinal fluid (CSF) using single-cell RNA sequencing combined with T cell receptor genotyping. Tumor immune infiltration and specifically CD8+ T cell infiltration can be discerned through the analysis of the CSF. Consistently, identical T cell receptor clonotypes are detected in brain lesions and CSF, confirming cell exchange between these compartments. The analysis of immune cells of the CSF can provide a non-invasive alternative to predict the response to ICI, as well as identify the T cell receptor clonotypes present in brain metastasis.


Assuntos
Neoplasias Encefálicas/imunologia , Líquido Cefalorraquidiano/imunologia , Leucócitos , Microambiente Tumoral/imunologia , Adenocarcinoma de Pulmão , Encéfalo/patologia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Linfócitos T CD8-Positivos/imunologia , Humanos , Neoplasias Pulmonares , Prognóstico
5.
Ter Arkh ; 93(1): 20-24, 2021 Jan 10.
Artigo em Russo | MEDLINE | ID: mdl-33720621

RESUMO

AIM: To study the effect of oxidative stress and telomere length in the chromosomes of blood leukocytes in patients with coronary heart disease (CHD) on the development of cardiovascular complications. MATERIALS AND METHODS: In 119 patients with CHD, the level of oxidatively modified low-density lipoproteins (ox-LDL) in blood plasma and the length of telomeres in nuclear blood cells were determined during the examination. After 5 years, a telephone survey of patients (or their relatives) was conducted to obtain data on the presence of cardiovascular complications. Telomere length was determined using quantitative real-time PCR, and the level of ox-LDL was determined by immunochemical method. RESULTS: It was found that reducing the length of telomeres in patients with CHD increases the risk of subsequent development of cardiovascular complications. A strong negative correlation was found between the level of ox-LDL and telomere length in the group of examined CHD patients who had cardiovascular complications after 5 years. CONCLUSION: CHD patients with short telomere length and high levels of ox-LDL have an increased risk of cardiovascular complications during 5 years.


Assuntos
Doença das Coronárias , Telômero , Biomarcadores , Doença das Coronárias/diagnóstico , Doença das Coronárias/epidemiologia , Doença das Coronárias/genética , Humanos , Leucócitos , Lipoproteínas LDL , Telômero/genética
6.
Signal Transduct Target Ther ; 6(1): 110, 2021 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-33677468

RESUMO

The 2019 coronavirus disease (COVID-19) outbreak caused by the SARS-CoV-2 virus is an ongoing global health emergency. However, the virus' pathogenesis remains unclear, and there is no cure for the disease. We investigated the dynamic changes of blood immune response in patients with COVID-19 at different stages by using 5' gene expression, T cell receptor (TCR), and B cell receptors (BCR) V(D)J transcriptome analysis at a single-cell resolution. We obtained single-cell mRNA sequencing (scRNA-seq) data of 341,420 peripheral blood mononuclear cells (PBMCs) and 185,430 clonotypic T cells and 28,802 clonotypic B cells from 25 samples of 16 patients with COVID-19 for dynamic studies. In addition, we used three control samples. We found expansion of dendritic cells (DCs), CD14+ monocytes, and megakaryocytes progenitor cells (MP)/platelets and a reduction of naïve CD4+ T lymphocytes in patients with COVID-19, along with a significant decrease of CD8+ T lymphocytes, and natural killer cells (NKs) in patients in critical condition. The type I interferon (IFN-I), mitogen-activated protein kinase (MAPK), and ferroptosis pathways were activated while the disease was active, and recovered gradually after patient conditions improved. Consistent with this finding, the mRNA level of IFN-I signal-induced gene IFI27 was significantly increased in patients with COVID-19 compared with that of the controls in a validation cohort that included 38 patients and 35 controls. The concentration of interferon-α (IFN-α) in the serum of patients with COVID-19 increased significantly compared with that of the controls in an additional cohort of 215 patients with COVID-19 and 106 controls, further suggesting the important role of the IFN-I pathway in the immune response of COVID-19. TCR and BCR sequences analyses indicated that patients with COVID-19 developed specific immune responses against SARS-CoV-2 antigens. Our study reveals a dynamic landscape of human blood immune responses to SARS-CoV-2 infection, providing clues for therapeutic potentials in treating COVID-19.


Assuntos
/imunologia , Leucócitos/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Análise de Célula Única , Adulto , Feminino , Ferroptose/genética , Ferroptose/imunologia , Humanos , Sistema de Sinalização das MAP Quinases/genética , Sistema de Sinalização das MAP Quinases/imunologia , Masculino , Pessoa de Meia-Idade , RNA-Seq , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos T/genética , /genética
7.
Biomed Environ Sci ; 34(1): 19-28, 2021 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-33531104

RESUMO

Objective: In the present study, the ABCA1 was used as a label to capture specific exosomes, the level of ABCA1-labeled exosomal microRNA-135a (miR-135a) was evaluated for the diagnosis of Alzheimer's disease (AD), especially in patients with early stages of AD. Methods: This is a preliminary research focused on the levels of ABCA1 in WBCs, RBCs, HT-22 cells, and neuron cells. The diagnostic value of ABCA1-labeled exosomal miR-135a was examined using the CSF and serum of APP/PS1 double transgenic mice, and 152 patients with SCD, 131 patients with MCI, 198 patients with DAT, and 30 control subjects. Results: The level of ABCA1 exosomes harvested from HT-22 cells and neuron culture medium was significantly higher compared to that of RBCs and WBCs ( P < 0.05). The levels of ABCA1-labeled exosomal miR-135a increased in the CSF of MCI and DAT group compared to those of control group ( P < 0.05), slightly increased ( P > 0.05) in the serum of SCD patient group, and significantly increased in MCI and DAT patient groups compared to those of the control group ( P < 0.05). Conclusion: This study outlines a method to capture specific exosomes and detect them using immunological methods, which is more efficient for early diagnosis of AD.


Assuntos
Transportador 1 de Cassete de Ligação de ATP , Disfunção Cognitiva/sangue , Exossomos , MicroRNAs/sangue , Transportador 1 de Cassete de Ligação de ATP/sangue , Transportador 1 de Cassete de Ligação de ATP/líquido cefalorraquidiano , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/sangue , Doença de Alzheimer/líquido cefalorraquidiano , Animais , Biomarcadores/sangue , Biomarcadores/líquido cefalorraquidiano , Linhagem Celular , Disfunção Cognitiva/líquido cefalorraquidiano , Eritrócitos/metabolismo , Feminino , Humanos , Leucócitos/metabolismo , Masculino , Camundongos Transgênicos , Neurônios/metabolismo
8.
Aging Cell ; 20(2): e13316, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33524238

RESUMO

The ageing of the global population brings about unprecedented challenges. Chronic age-related diseases in an increasing number of people represent an enormous burden for health and social care. The immune system deteriorates during ageing and contributes to many of these age-associated diseases due to its pivotal role in pathogen clearance, tissue homeostasis and maintenance. Moreover, in order to develop treatments for COVID-19, we urgently need to acquire more knowledge about the aged immune system, as older adults are disproportionally and more severely affected. Changes with age lead to impaired responses to infections, malignancies and vaccination, and are accompanied by chronic, low-degree inflammation, which together is termed immunosenescence. However, the molecular and cellular mechanisms that underlie immunosenescence, termed immune cell senescence, are mostly unknown. Cellular senescence, characterised by an irreversible cell cycle arrest, is thought to be the cause of tissue and organismal ageing. Thus, better understanding of cellular senescence in immune populations at single-cell level may provide us with insight into how immune cell senescence develops over the life time of an individual. In this review, we will briefly introduce the phenotypic characterisation of aged innate and adaptive immune cells, which also contributes to overall immunosenescence, including subsets and function. Next, we will focus on the different hallmarks of cellular senescence and cellular ageing, and the detection techniques most suitable for immune cells. Applying these techniques will deepen our understanding of immune cell senescence and to discover potential druggable pathways, which can be modulated to reverse immune ageing.


Assuntos
Senescência Celular , Imunossenescência , Leucócitos/fisiologia , Animais , Biomarcadores/metabolismo , Humanos , Estresse Oxidativo , Proteostase
9.
Nat Commun ; 12(1): 1178, 2021 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-33633114

RESUMO

Enzyme and chaperone therapies are used to treat Fabry disease. Such treatments are expensive and require intrusive biweekly infusions; they are also not particularly efficacious. In this pilot, single-arm study (NCT02800070), five adult males with Type 1 (classical) phenotype Fabry disease were infused with autologous lentivirus-transduced, CD34+-selected, hematopoietic stem/progenitor cells engineered to express alpha-galactosidase A (α-gal A). Safety and toxicity are the primary endpoints. The non-myeloablative preparative regimen consisted of intravenous melphalan. No serious adverse events (AEs) are attributable to the investigational product. All patients produced α-gal A to near normal levels within one week. Vector is detected in peripheral blood and bone marrow cells, plasma and leukocytes demonstrate α-gal A activity within or above the reference range, and reductions in plasma and urine globotriaosylceramide (Gb3) and globotriaosylsphingosine (lyso-Gb3) are seen. While the study and evaluations are still ongoing, the first patient is nearly three years post-infusion. Three patients have elected to discontinue enzyme therapy.


Assuntos
Doença de Fabry/enzimologia , Doença de Fabry/terapia , Terapia Genética/métodos , Lentivirus/genética , alfa-Galactosidase/genética , alfa-Galactosidase/uso terapêutico , Adulto , Antígenos CD34 , Células da Medula Óssea , Doença de Fabry/genética , Vetores Genéticos , Células-Tronco Hematopoéticas , Humanos , Leucócitos , Masculino , Pessoa de Meia-Idade , Triexosilceramidas/sangue , Triexosilceramidas/urina
10.
BMC Med ; 19(1): 37, 2021 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-33568158

RESUMO

BACKGROUND: Chronic inflammation, which can be modulated by diet, is linked to high white blood cell counts and correlates with higher cardiometabolic risk and risk of more severe infections, as in the case of COVID-19. METHODS: Here, we assessed the association between white blood cell profile (lymphocytes, basophils, eosinophils, neutrophils, monocytes and total white blood cells) as markers of chronic inflammation, habitual diet and gut microbiome composition (determined by sequencing of the 16S RNA) in 986 healthy individuals from the PREDICT-1 nutritional intervention study. We then investigated whether the gut microbiome mediates part of the benefits of vegetable intake on lymphocyte counts. RESULTS: Higher levels of white blood cells, lymphocytes and basophils were all significantly correlated with lower habitual intake of vegetables, with vegetable intake explaining between 3.59 and 6.58% of variation in white blood cells after adjusting for covariates and multiple testing using false discovery rate (q < 0.1). No such association was seen with fruit intake. A mediation analysis found that 20.00% of the effect of vegetable intake on lymphocyte counts was mediated by one bacterial genus, Collinsella, known to increase with the intake of processed foods and previously associated with fatty liver disease. We further correlated white blood cells to other inflammatory markers including IL6 and GlycA, fasting and post-prandial glucose levels and found a significant relationship between inflammation and diet. CONCLUSION: A habitual diet high in vegetables, but not fruits, is linked to a lower inflammatory profile for white blood cells, and a fifth of the effect is mediated by the genus Collinsella. TRIAL REGISTRATION: The ClinicalTrials.gov registration identifier is NCT03479866 .


Assuntos
Dieta , Frutas , Microbioma Gastrointestinal/genética , Leucócitos , Verduras , Actinobacteria , Adulto , Biomarcadores/sangue , Clostridiales , Clostridium , Jejum , Feminino , Humanos , Interleucina-6/sangue , Contagem de Leucócitos , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , RNA Ribossômico 16S/genética , Ruminococcus
11.
Ecotoxicol Environ Saf ; 210: 111825, 2021 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-33412284

RESUMO

To explore the effect of florfenicol (FFC) combined with Salvia miltiorrhiza polysaccharide (SMPs) on immune function of Broilers. One hundred and twenty-one-day-old chicks were chosen and divided into 6 groups. The group A received standard basal diet only, the group B received a basal diet with FFC (0.15 g/L diet), and the group C, D, E received a basal diet with FFC (0.15 g/L diet) and SMPs (1.25 g/L, 2.5 g/L, 5 g/L diet),the group F received a basal diet with SMPs (5 g/L diet). FFC can significantly inhibit the growth performance of broilers, but has no significant damage to the immune function of broilers. The combination of FFC and SMPs can improve the growth performance of broilers, increase the number of leukocyte subtypes in blood (P < 0.05), increase the number of Newcastle disease (ND) and avian influenza (AI) antibodies in blood, the number of immunoglobulins, and the content of cytokines (P < 0.05). In addition, it significantly improve the lymphocyte conversion rate of broiler peripheral blood (P < 0.05). So that, synergistic use of FFC and SMPs can enhance immune responses in Broilers.


Assuntos
Antibacterianos/farmacologia , Galinhas/imunologia , Polissacarídeos/farmacologia , Salvia miltiorrhiza , Tianfenicol/análogos & derivados , Ração Animal , Animais , Anticorpos Antivirais/sangue , Galinhas/sangue , Galinhas/crescimento & desenvolvimento , Citocinas/sangue , Dieta , Suplementos Nutricionais , Humanos , Imunidade/efeitos dos fármacos , Imunoglobulinas/sangue , Influenza Humana/imunologia , Leucócitos/efeitos dos fármacos , Vírus da Doença de Newcastle/imunologia , Tianfenicol/farmacologia
12.
Ecotoxicol Environ Saf ; 210: 111859, 2021 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-33429319

RESUMO

Accuracy, sensitivity, simplicity, reproducibility, and low-cost are desirable requirements for genotoxicity assessment techniques. Here we describe a simple electrophoretic assay for genomic DNA lesions quantification (EAsy-GeL) based on subjecting DNA samples to rapid unwinding/renaturation treatments and neutral agarose gel electrophoresis. The experiments performed in this work involved different biological samples exposed to increasing environmental-simulated doses of ultraviolet-B (UVB) radiation, such as Escherichia coli, human leukocytes, and isolated human genomic DNA. DNA extraction was carried out using a universal and low-cost protocol, which takes about 4 h. Before electrophoresis migration, DNA samples were kept into a neutral buffer to detect double-strand breaks (DSBs) or subjected to a 5-min step of alkaline unwinding and neutral renaturation to detect single-strand breaks (SSBs) or incubated with the DNA repair enzyme T4-endonuclease V for the detection of cyclobutane pyrimidine dimers (CPDs) before the 5-min step of DNA unwinding/renaturation. Then, all DNA samples were separated by neutral agarose gel electrophoresis, the DNA average length of each lane was calculated through the use of free software, and the frequency of DNA breaks per kbp was determined by a simple rule of three. Dose-response experiments allowed the quantification of different levels of DNA damage per electrophoretic run, varying from a constant and low amount of DSBs/SSBs to high and dose-dependent levels of CPDs. Compared with other assays based on alkaline unwinding and gel electrophoresis, EAsy-GeL has important advantages for both environmental monitoring and laboratory testing purposes. The simplicity and applicability of this protocol to other types of DNA lesions, biological models, and agents make it ideal for genotoxicity, DNA repair studies, as well as for assessing exposure risks to ecosystems and human health.


Assuntos
Bioensaio/métodos , Dano ao DNA , DNA/efeitos da radiação , Eletroforese , Raios Ultravioleta , DNA/química , DNA/genética , Escherichia coli/genética , Genoma , Genômica , Humanos , Leucócitos
13.
Arterioscler Thromb Vasc Biol ; 41(3): 1047-1061, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33504179

RESUMO

Shortened telomeres have been linked to numerous chronic diseases, most importantly coronary artery disease, but the underlying mechanisms remain ill defined. Loss-of-function mutations and deletions in telomerase both accelerate telomere shortening but do not necessarily lead to a clinical phenotype associated with atherosclerosis, questioning the causal role of telomere length in cardiac pathology. The differential extranuclear functions of the 2 main components of telomerase, telomerase reverse transcriptase and telomerase RNA component, offer important clues about the complex relationship between telomere length and cardiovascular pathology. In this review, we critically discuss relevant preclinical models, genetic disorders, and clinical studies to elucidate the impact of telomerase in cardiovascular disease and its potential role as a therapeutic target. We suggest that the antioxidative function of mitochondrial telomerase reverse transcriptase might be atheroprotective, making it a potential target for clinical trials. Graphic Abstract: A graphic abstract is available for this article.


Assuntos
Doenças Cardiovasculares/enzimologia , Doenças Cardiovasculares/terapia , Telomerase/metabolismo , Animais , Biomarcadores/sangue , Doenças Cardiovasculares/sangue , Ensaios Clínicos como Assunto , Medicamentos de Ervas Chinesas/uso terapêutico , Exercício Físico , Estudo de Associação Genômica Ampla , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Leucócitos/enzimologia , Camundongos , Modelos Cardiovasculares , Mutação , RNA/genética , Telomerase/sangue , Telomerase/genética , Homeostase do Telômero/fisiologia , Encurtamento do Telômero/fisiologia
14.
Nat Commun ; 12(1): 11, 2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33397889

RESUMO

Circulating tumor DNA (ctDNA) provides a noninvasive approach to elucidate a patient's genomic landscape and actionable information. Here, we design a ctDNA-based study of over 10,000 pan-cancer Chinese patients. Using parallel sequencing between plasma and white blood cells, 14% of plasma cell-free DNA samples contain clonal hematopoiesis (CH) variants, for which detectability increases with age. After eliminating CH variants, ctDNA is detected in 73.5% of plasma samples, with small cell lung cancer (91.1%) and prostate cancer (87.9%) showing the highest detectability. The landscape of putative driver genes revealed by ctDNA profiling is similar to that in a tissue-based database (R2 = 0.87, p < 0.001) but also shows some discrepancies, such as higher EGFR (44.8% versus 25.2%) and lower KRAS (6.8% versus 27.2%) frequencies in non-small cell lung cancer, and a higher TP53 frequency in hepatocellular carcinoma (53.1% versus 28.6%). Up to 41.2% of plasma samples harbor drug-sensitive alterations. These findings may be helpful for identifying therapeutic targets and combined treatment strategies.


Assuntos
Grupo com Ancestrais do Continente Asiático/genética , DNA Tumoral Circulante/análise , Neoplasias/sangue , Neoplasias/genética , DNA Tumoral Circulante/sangue , DNA Tumoral Circulante/genética , Células Clonais , Estudos de Coortes , Genoma Humano , Hematopoese , Humanos , Leucócitos/metabolismo , Mutação/genética , Carga Tumoral/genética
15.
Arterioscler Thromb Vasc Biol ; 41(3): 1167-1178, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33441028

RESUMO

OBJECTIVE: People with diabetes are at a significantly higher risk of cardiovascular disease, in part, due to accelerated atherosclerosis. Diabetic subjects have increased number of platelets that are activated, more reactive, and respond suboptimally to antiplatelet therapies. We hypothesized that reducing platelet numbers by inducing their premature apoptotic death would decrease atherosclerosis. Approach and Results: This was achieved by targeting the antiapoptotic protein Bcl-xL (B-cell lymphoma-extra large; which is essential for platelet viability) via distinct genetic and pharmacological approaches. In the former, we transplanted bone marrow from mice carrying the Tyr15 to Cys loss of function allele of Bcl-x (known as Bcl-xPlt20) or wild-type littermate controls into atherosclerotic-prone Ldlr+/- mice made diabetic with streptozotocin and fed a Western diet. Reduced Bcl-xL function in hematopoietic cells significantly decreased platelet numbers, exclusive of other hematologic changes. This led to a significant reduction in atherosclerotic lesion formation in Bcl-xPlt20 bone marrow transplanted Ldlr+/- mice. To assess the potential therapeutic relevance of reducing platelets in atherosclerosis, we next targeted Bcl-xL with a pharmacological strategy. This was achieved by low-dose administration of the BH3 (B-cell lymphoma-2 homology domain 3) mimetic, ABT-737 triweekly, in diabetic Apoe-/- mice for the final 6 weeks of a 12-week study. ABT-737 normalized platelet numbers along with platelet and leukocyte activation to that of nondiabetic controls, significantly reducing atherosclerosis while promoting a more stable plaque phenotype. CONCLUSIONS: These studies suggest that selectively reducing circulating platelets, by targeting Bcl-xL to promote platelet apoptosis, can reduce atherosclerosis and lower cardiovascular disease risk in diabetes. Graphic Abstract: A graphic abstract is available for this article.


Assuntos
Aterosclerose/sangue , Aterosclerose/complicações , Plaquetas/patologia , Angiopatias Diabéticas/sangue , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Aterosclerose/prevenção & controle , Compostos de Bifenilo/administração & dosagem , Plaquetas/efeitos dos fármacos , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/complicações , Feminino , Humanos , Leucócitos/patologia , Leucócitos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nitrofenóis/administração & dosagem , Piperazinas/administração & dosagem , Contagem de Plaquetas , Receptores de LDL/deficiência , Receptores de LDL/genética , Fatores de Risco , Sulfonamidas/administração & dosagem
16.
Mol Cell ; 81(5): 998-1012.e7, 2021 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-33440169

RESUMO

Pre-mRNA processing steps are tightly coordinated with transcription in many organisms. To determine how co-transcriptional splicing is integrated with transcription elongation and 3' end formation in mammalian cells, we performed long-read sequencing of individual nascent RNAs and precision run-on sequencing (PRO-seq) during mouse erythropoiesis. Splicing was not accompanied by transcriptional pausing and was detected when RNA polymerase II (Pol II) was within 75-300 nucleotides of 3' splice sites (3'SSs), often during transcription of the downstream exon. Interestingly, several hundred introns displayed abundant splicing intermediates, suggesting that splicing delays can take place between the two catalytic steps. Overall, splicing efficiencies were correlated among introns within the same transcript, and intron retention was associated with inefficient 3' end cleavage. Remarkably, a thalassemia patient-derived mutation introducing a cryptic 3'SS improved both splicing and 3' end cleavage of individual ß-globin transcripts, demonstrating functional coupling between the two co-transcriptional processes as a determinant of productive gene output.


Assuntos
Células Eritroides/metabolismo , Eritropoese/genética , RNA Polimerase II/genética , Processamento de RNA , Elongação da Transcrição Genética , Globinas beta/genética , Animais , Sequência de Bases , Diferenciação Celular , Linhagem Celular Tumoral , Células Eritroides/citologia , Éxons , Humanos , Íntrons , Leucócitos/citologia , Leucócitos/metabolismo , Camundongos , Mutação , Clivagem do RNA , RNA Polimerase II/metabolismo , Sítios de Splice de RNA , Spliceossomos/genética , Spliceossomos/metabolismo , Globinas beta/deficiência , Talassemia beta/genética , Talassemia beta/metabolismo , Talassemia beta/patologia
17.
Methods Mol Biol ; 2260: 111-119, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33405033

RESUMO

Fluorescence-based techniques enable researchers to monitor physiologic processes, specifically fungal cell viability and death, during cellular encounters with the mammalian immune system with single event resolution. By incorporating two independent fluorescent probes in fungal organisms either prior to, or ensuing experimental infection in mice or in cultured leukocytes, it is possible to distinguish and quantify live and killed fungal cells to interrogate genetic, pharmacologic, and cellular determinants that shape host-fungal cell outcomes. This chapter reviews the techniques and applications of fluorescent fungal reporters of viability, with emphasis on the North American endemic dimorphic fungus, Blastomyces dermatitidis.


Assuntos
Blastomyces/genética , Regulação Fúngica da Expressão Gênica , Genes Reporter , Leucócitos/microbiologia , Proteínas Luminescentes/genética , Pulmão/microbiologia , Microscopia de Fluorescência , Animais , Blastomyces/imunologia , Blastomyces/metabolismo , Citometria de Fluxo , Interações Hospedeiro-Patógeno , Humanos , Leucócitos/imunologia , Leucócitos/metabolismo , Proteínas Luminescentes/biossíntese , Pulmão/imunologia , Pulmão/metabolismo , Viabilidade Microbiana
18.
Methods Mol Biol ; 2260: 121-132, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33405034

RESUMO

Fluorescence-based techniques enable researchers to monitor physiologic processes, specifically fungal cell viability and death, during cellular encounters with the mammalian immune system with single event resolution. By incorporating two independent fluorescent probes in fungal organisms either prior to, or ensuing experimental infection in mice or in cultured leukocytes, it is possible to distinguish and quantify live and killed fungal cells to interrogate genetic, pharmacologic, and cellular determinants that shape host-fungal cell outcomes. This chapter reviews the techniques and applications of fluorescent fungal reporters of viability, with emphasis on the filamentous mold Aspergillus fumigatus.


Assuntos
Aspergillus fumigatus/genética , Regulação Fúngica da Expressão Gênica , Genes Reporter , Leucócitos/microbiologia , Proteínas Luminescentes/genética , Pulmão/microbiologia , Microscopia de Fluorescência , Animais , Aspergillus fumigatus/imunologia , Aspergillus fumigatus/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Leucócitos/imunologia , Leucócitos/metabolismo , Proteínas Luminescentes/biossíntese , Pulmão/imunologia , Pulmão/metabolismo , Camundongos , Viabilidade Microbiana
19.
Crit Care Med ; 49(4): 661-670, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33405410

RESUMO

OBJECTIVES: In this study, we hypothesized that coronavirus disease 2019 patients exhibit sublingual microcirculatory alterations caused by inflammation, coagulopathy, and hypoxemia. DESIGN: Multicenter case-controlled study. SETTING: Two ICUs in The Netherlands and one in Switzerland. PATIENTS: Thirty-four critically ill coronavirus disease 2019 patients were compared with 33 healthy volunteers. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: The microcirculatory parameters quantified included total vessel density (mm × mm-2), functional capillary density (mm × mm-2), proportion of perfused vessels (%), capillary hematocrit (%), the ratio of capillary hematocrit to systemic hematocrit, and capillary RBC velocity (µm × s-1). The number of leukocytes in capillary-postcapillary venule units per 4-second image sequence (4 s-1) and capillary RBC microaggregates (4 s-1) was measured. In comparison with healthy volunteers, the microcirculation of coronavirus disease 2019 patients showed increases in total vessel density (22.8 ± sd 5.1 vs 19.9 ± 3.3; p < 0.0001) and functional capillary density (22.2 ± 4.8 vs 18.8 ± 3.1; p < 0.002), proportion of perfused vessel (97.6 ± 2.1 vs 94.6 ± 6.5; p < 0.01), RBC velocity (362 ± 48 vs 306 ± 53; p < 0.0001), capillary hematocrit (5.3 ± 1.3 vs 4.7 ± 0.8; p < 0.01), and capillary-hematocrit-to-systemic-hematocrit ratio (0.18 ± 0.0 vs 0.11 ± 0.0; p < 0.0001). These effects were present in coronavirus disease 2019 patients with Sequential Organ Failure Assessment scores less than 10 but not in patients with Sequential Organ Failure Assessment scores greater than or equal to 10. The numbers of leukocytes (17.6 ± 6.7 vs 5.2 ± 2.3; p < 0.0001) and RBC microaggregates (0.90 ± 1.12 vs 0.06 ± 0.24; p < 0.0001) was higher in the microcirculation of the coronavirus disease 2019 patients. Receiver-operating-characteristics analysis of the microcirculatory parameters identified the number of microcirculatory leukocytes and the capillary-hematocrit-to-systemic-hematocrit ratio as the most sensitive parameters distinguishing coronavirus disease 2019 patients from healthy volunteers. CONCLUSIONS: The response of the microcirculation to coronavirus disease 2019-induced hypoxemia seems to be to increase its oxygen-extraction capacity by increasing RBC availability. Inflammation and hypercoagulation are apparent in the microcirculation by increased numbers of leukocytes and RBC microaggregates.


Assuntos
/mortalidade , Capilares , Hipóxia/etiologia , Leucócitos , Microcirculação/fisiologia , Eritrócitos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
20.
Trop Anim Health Prod ; 53(1): 101, 2021 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-33417070

RESUMO

This study investigated Lippia palmeri Watt (oregano) phytochemical compounds, their antioxidant capacity, and immunological effects on goat peripheral blood leukocytes (PBL), and on the presence of intermediate polar compounds in goat feces fed dietary oregano. The polar and nonpolar fractions of L. palmeri W. were characterized and phytochemical contents and antioxidant capacity were determined. Twelve healthy Anglo-Nubian goats were used for the in vivo trials, which were randomly assigned to control fed with basal diet, or oregano group fed with basal diet + 2.6% (DM basis) dried oregano leaves. Goat peripheral blood leukocytes (PBL) were isolated for the in vitro study, and PBL were stimulated with oregano extracts at 100 and 150 µg/mL after 24 h. For the in vivo trial, dietary oregano (2.6% on DM basis) was evaluated in the goats for 90 days. Relatively high abundance of carvacrol and thymol phytochemical compounds was found in oregano. The highest antioxidant capacity of oregano extracts was detected at 100 and 150 µg/mL. Nitric oxide production, phagocytosis, and superoxide dismutase activities increased (p < 0.05) in stimulated PBL with oregano extracts, whereas the pro-inflammatory (TNF-α and IL-1ß) transcription and antioxidant (CAT and GPX-4) genes downregulated. In the in vivo experiment, dietary oregano enabled the detection of nine compounds found in goat feces, from which caproic (C6) was in a high relative quantity compared with the control group. Oregano has phytochemical compounds with strong antioxidant capacity that protect cells against oxidative stress damage and could modulate immune response and feces composition in goats.


Assuntos
Antioxidantes/farmacologia , Cabras/fisiologia , Intestinos/fisiologia , Leucócitos/imunologia , Lippia/química , Extratos Vegetais/metabolismo , Extratos Vegetais/farmacologia , Ração Animal/análise , Animais , Dieta/veterinária , Suplementos Nutricionais/análise , Feminino , Extratos Vegetais/administração & dosagem , Distribuição Aleatória
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