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1.
Klin Lab Diagn ; 66(3): 154-159, 2021 Mar 30.
Artículo en Inglés | MEDLINE | ID: mdl-33793114

RESUMEN

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.


Asunto(s)
Leucocitos , Telómero , ADN , Citometría de Flujo , Humanos , Hibridación Fluorescente in Situ , Telómero/genética
2.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 29(2): 348-325, 2021 Apr.
Artículo en Chino | MEDLINE | ID: mdl-33812398

RESUMEN

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.


Asunto(s)
Leucemia Mieloide Aguda , Citometría de Flujo , Humanos , Leucocitos , Neoplasia Residual , Pronóstico
3.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 29(2): 581-585, 2021 Apr.
Artículo en Chino | MEDLINE | ID: mdl-33812434

RESUMEN

OBJECTIVE: To investigate the effect of expression level changes of monocytic myeloid-derived suppressor cells (M-MDSC) to related immune function in the patients with primary immune thrombocytopenia (ITP). METHODS: Peripheral blood samples were collected from 53 newly diagnosed ITP patients and 30 healthy volunteers. The quantity of M-MDSC, mRNA levels of Arg-1 and iNOS were detected. CD4+T, M-MDSC and CD14+HLA-DR+ cells were sorted. CD4+T cells were marked by CFSE, and the immunosuppressive mechanism of M-MDSC was analyzed. RESULTS: The count of M-MDSC in peripheral blood of newly diagnosed ITP patients was significantly higher than that in the control group (P < 0.01). However, the expression level of Arg-1 in peripheral blood was not significantly different between the newly diagnosed ITP group and the control group. But the expression level of iNOS in the newly diagnosed ITP patients was significantly higher than that in the control group (P < 0.01). After treatment, the count of M-MDSC in the patients with ITP was significantly lower than before treatment (P < 0.01), which showed that M-MDSC could significantly inhibit the proliferation and secretion of IFN-γ in CD4+T cells. CONCLUSION: M-MDSC may be related to the disorder of immune tolerance in the patients with ITP, and may become a new index to monitor the curative efficacy of ITP patients.


Asunto(s)
Células Supresoras de Origen Mieloide , Púrpura Trombocitopénica Idiopática , Citometría de Flujo , Antígenos HLA-DR , Humanos , Inmunidad
4.
J Appl Oral Sci ; 29: e20200770, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33825754

RESUMEN

OBJECTIVE: Neutrophils are key effector cells of the innate immune system. They recognize antigens through membrane receptors, which are expressed during their maturation and activation. Neutrophils express FcγRII (CD32), FcγRIII (CD16), and FcγRI (CD64) after being activated by different factors such as cytokines and bacterial products. These receptors are involved with phagocytosis of IgG-opsonized microbes and enhance defense mechanisms. Based on that, our study seeks to compare the expression of FcγRII, FcγRIII, FcγRI, and CD11b on neutrophils from elderly and young subjects and their expression after in vitro activation with cytokines and LPS. METHODOLOGY: Neutrophils were isolated from human peripheral blood and from mice bone marrow by density gradient. After isolation, FCγRs expression was immediately analyzed by flow cytometry or after in vitro stimulation. RESULTS: In freshly isolated cells, the percentage of FcγRIIIb+ and CD11b+ neutrophils were higher in samples from young individuals; FcγRIIIa expression was more prominent on aged neutrophils; FcγRIA expression was similar in all samples analyzed. Exposure to CXCL8 and LPS resulted in a higher percentage of FcγRIa+ neutrophils on elderly individuals' samples but lower when compared with neutrophils from young donors. We observed that LPS caused an increase in FcγRIIa expression on aging human neutrophils. In contrast, FcγRIIIb expression in response to CXCL8 and LPS stimulation was not altered in the four groups. CD11b expression was lower in neutrophils from elderly individuals even in response to LPS and CXCL8. In mice, we observed differences only regarding CD11b expression, which was increased on aged neutrophils. LPS exposure caused an increase in all FcγRs. CONCLUSIONS: Our results suggest that, in humans, the overall pattern of FcγR expression and integrin CD11b are altered during aging and immunosenescence might contribute to age-related infection.


Asunto(s)
Neutrófilos , Receptores de IgG , Animales , Recuento de Células , Citometría de Flujo , Ratones , Fagocitosis
5.
Int J Nanomedicine ; 16: 1977-1992, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33727810

RESUMEN

Background: Phytostanols are naturally occurring compounds that reduce blood cholesterol levels significantly. However, their aqueous insolubility poses formulation challenges. Aim: To formulate and characterize solid lipid nanoparticle carriers for phytostanol esters to enhance the bioavailability of phytostanols. Methods: Phytostanol ester solid lipid nanoparticles were formulated by the microemulsion method. They were characterized for particle size distribution, polydispersity index, shape, surface charge, entrapment efficiency, stability, chemical structure, and thermal properties. The uptake of the formulation by cell lines, HepG2 and HT-29, and its effect on cell viability were evaluated. Results: The formulation of solid lipid nanoparticles was successfully optimised by varying the type of lipids and their concentration relative to that of surfactants in the present study. The optimised formulation had an average diameter of (171 ± 9) nm, a negative surface charge of (-23.0 ± 0.8) mV and was generally spherical in shape. We report high levels of drug entrapment at (89 ± 5)% in amorphous form, drug loading of (9.1 ± 0.5)%, nanoparticle yield of (67 ± 4)% and drug excipient compatibility. The biological safety and uptake of the formulations were demonstrated on hepatic and intestinal cell lines. Conclusion: Phytostanol ester solid lipid nanoparticles were successfully formulated and characterized. The formulation has the potential to provide an innovative drug delivery system for phytostanols which reduce cholesterol and have a potentially ideal safety profile. This can contribute to better management of one of the main risk factors of cardiovascular diseases.


Asunto(s)
Composición de Medicamentos , Ésteres/química , Hipercolesterolemia/tratamiento farmacológico , Lípidos/química , Nanopartículas/química , Fitosteroles/uso terapéutico , Muerte Celular , Emulsiones/química , Endocitosis , Citometría de Flujo , Células HT29 , Células Hep G2 , Humanos , Tamaño de la Partícula , Polvos , Espectroscopía Infrarroja por Transformada de Fourier , Electricidad Estática , Temperatura
6.
Nat Commun ; 12(1): 1792, 2021 03 19.
Artículo en Inglés | MEDLINE | ID: mdl-33741926

RESUMEN

In both sickle cell disease and malaria, red blood cells (RBCs) are phagocytosed in the spleen, but receptor-ligand pairs mediating uptake have not been identified. Here, we report that patches of high mannose N-glycans (Man5-9GlcNAc2), expressed on diseased or oxidized RBC surfaces, bind the mannose receptor (CD206) on phagocytes to mediate clearance. We find that extravascular hemolysis in sickle cell disease correlates with high mannose glycan levels on RBCs. Furthermore, Plasmodium falciparum-infected RBCs expose surface mannose N-glycans, which occur at significantly higher levels on infected RBCs from sickle cell trait subjects compared to those lacking hemoglobin S. The glycans are associated with high molecular weight complexes and protease-resistant, lower molecular weight fragments containing spectrin. Recognition of surface N-linked high mannose glycans as a response to cellular stress is a molecular mechanism common to both the pathogenesis of sickle cell disease and resistance to severe malaria in sickle cell trait.


Asunto(s)
Anemia de Células Falciformes/metabolismo , Eritrocitos/metabolismo , Manosa/metabolismo , Fagocitos/metabolismo , Polisacáridos/metabolismo , Membrana Eritrocítica/metabolismo , Membrana Eritrocítica/parasitología , Eritrocitos/parasitología , Citometría de Flujo/métodos , Hemólisis , Humanos , Ligandos , Malaria Falciparum/metabolismo , Malaria Falciparum/parasitología , Glicoproteínas de Membrana/metabolismo , Fagocitosis , Plasmodium falciparum/fisiología , Unión Proteica , Receptores Inmunológicos/metabolismo
7.
Int Heart J ; 62(2): 396-406, 2021 Mar 30.
Artículo en Inglés | MEDLINE | ID: mdl-33731537

RESUMEN

Endothelial injury and inflammation have been found to be essential in the pathogenesis of coronary artery disease (CAD). Circulating exosomes are of great value as novel biomarkers for CAD. However, the role of circulating exosomes in the pathogenesis of CAD remains unclear. Thus, in this study, we aimed to examine whether circulating exosomes from CAD are involved in the endothelial injury and inflammation. The serum-derived exosomes were isolated from CAD and controls using an ExoQuick reagent, and these were then quantified by measuring the protein levels using BCA methods. The uptake of exosomes by human umbilical vein endothelial cells (HUVECs) was observed by laser scanning microscope and analyzed via flow cytometry. Then, HUVECs were treated with vehicle, exosomes from CAD (CAD-exo), and controls (ctrl-exo) in the absence and presence of vascular endothelial growth factor (VEGF). Cell viability, migration, and angiogenesis were evaluated using CCK-8 assay, scratch assay, and tube formation assay. Inflammatory factors including IL-1ß, IL-6, TNF-α, ICAM-1, and VCAM-1 levels were detected via qPCR. As per our findings, no significant differences were noted in uptake of ctrl-exo and CAD-exo by HUVECs. CAD-exo suppressed cell viability in a dose-dependent manner. Compared with ctrl-exo, CAD-exo-treated HUVECs significantly suppressed migration and angiogenesis. However, CAD-exo had a stronger inhibitory effect on VEGF-induced migration and angiogenesis compared with ctrl-exo. Moreover, IL-1ß, TNF-α, and ICAM-1 were determined to be significantly upregulated in HUVECs treated with CAD-exo, but IL-6 and VCAM-1 expressions were not affected. Overall, our results suggest that CAD-exo are involved in endothelial injury and inflammation, which may, in turn, cause endothelial dysfunction and potentially promote the development of CAD.


Asunto(s)
Enfermedad de la Arteria Coronaria/sangre , Exosomas/metabolismo , Movimiento Celular , Proliferación Celular , Células Cultivadas , Enfermedad de la Arteria Coronaria/patología , Femenino , Citometría de Flujo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células Endoteliales de la Vena Umbilical Humana/patología , Humanos , Inflamación/metabolismo , Inflamación/patología , Masculino , Persona de Mediana Edad
8.
Gene ; 782: 145542, 2021 May 25.
Artículo en Inglés | MEDLINE | ID: mdl-33675953

RESUMEN

Squamous cell carcinoma (SCC) is a relatively common cancer with a low survival rate, poor prognosis and no effective treatment strategy. The use of cell-free conditioned media derived from mesenchymal stem cells (CM-MSCs) has shown promising results in treating various diseases. This study aimed to evaluate the effects of CM-MSCs on proliferation and apoptosis of CAL-27 and FaDu SCC cell lines. CM derived from human bone marrow and human amniotic membrane MSCs (BM-MSCs and AM-MSCs) was used in this investigation. MTT assay demonstrated that CM-BMMSC decreased the viability of CAL-27 and FaDu cell lines, 24, 48, and 72 h after treatment. Quantitative real-time PCR indicated that mRNA expression of PCNA as a proliferative marker, and BCL-2 as an anti-apoptotic protein, decreased in both cell lines treated with CM-BMMSC. Based on the flow cytometry results, the number of positive proliferative Ki67 cells and apoptotic Annexin-V cells decreased and increased in both cell lines treated with CM-BMMSC, respectively. However, CM-AMMSC treatment had both pro-and anti-neoplastic effects in our samples and showed considerable differences between the two cell lines. Taken together, our findings demonstrated that CM-BMMSC and, to a lesser degree, CM-AMMSC decrease cell viability and proliferation and increase cell apoptosis in SCC cell lines in a time-dependent manner. However, further studies are needed, especially to evaluate the anti-tumor potential of CM-BMMSC in vivo.


Asunto(s)
Apoptosis/efectos de los fármacos , Carcinoma de Células Escamosas/tratamiento farmacológico , Proliferación Celular/efectos de los fármacos , Medios de Cultivo Condicionados/farmacología , Células Madre Mesenquimatosas/metabolismo , Amnios/metabolismo , Células de la Médula Ósea , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Citometría de Flujo , Humanos , Neoplasias Hepáticas
9.
BMC Bioinformatics ; 22(1): 137, 2021 Mar 22.
Artículo en Inglés | MEDLINE | ID: mdl-33752595

RESUMEN

BACKGROUND: Flow and mass cytometry are important modern immunology tools for measuring expression levels of multiple proteins on single cells. The goal is to better understand the mechanisms of responses on a single cell basis by studying differential expression of proteins. Most current data analysis tools compare expressions across many computationally discovered cell types. Our goal is to focus on just one cell type. Our narrower field of application allows us to define a more specific statistical model with easier to control statistical guarantees. RESULTS: Differential analysis of marker expressions can be difficult due to marker correlations and inter-subject heterogeneity, particularly for studies of human immunology. We address these challenges with two multiple regression strategies: a bootstrapped generalized linear model and a generalized linear mixed model. On simulated datasets, we compare the robustness towards marker correlations and heterogeneity of both strategies. For paired experiments, we find that both strategies maintain the target false discovery rate under medium correlations and that mixed models are statistically more powerful under the correct model specification. For unpaired experiments, our results indicate that much larger patient sample sizes are required to detect differences. We illustrate the CytoGLMM R package and workflow for both strategies on a pregnancy dataset. CONCLUSION: Our approach to finding differential proteins in flow and mass cytometry data reduces biases arising from marker correlations and safeguards against false discoveries induced by patient heterogeneity.


Asunto(s)
Citometría de Flujo , Modelos Estadísticos , Humanos , Modelos Lineales , Tamaño de la Muestra
10.
BMC Bioinformatics ; 22(1): 138, 2021 Mar 22.
Artículo en Inglés | MEDLINE | ID: mdl-33752602

RESUMEN

BACKGROUND: The rapidly increasing dimensionality and throughput of flow and mass cytometry data necessitate new bioinformatics tools for analysis and interpretation, and the recently emerging single-cell-based algorithms provide a powerful strategy to meet this challenge. RESULTS: Here, we present CytoTree, an R/Bioconductor package designed to analyze and interpret multidimensional flow and mass cytometry data. CytoTree provides multiple computational functionalities that integrate most of the commonly used techniques in unsupervised clustering and dimensionality reduction and, more importantly, support the construction of a tree-shaped trajectory based on the minimum spanning tree algorithm. A graph-based algorithm is also implemented to estimate the pseudotime and infer intermediate-state cells. We apply CytoTree to several examples of mass cytometry and time-course flow cytometry data on heterogeneity-based cytology and differentiation/reprogramming experiments to illustrate the practical utility achieved in a fast and convenient manner. CONCLUSIONS: CytoTree represents a versatile tool for analyzing multidimensional flow and mass cytometry data and to producing heuristic results for trajectory construction and pseudotime estimation in an integrated workflow.


Asunto(s)
Algoritmos , Biología Computacional , Diferenciación Celular , Análisis por Conglomerados , Citometría de Flujo , Programas Informáticos
11.
Sci Immunol ; 6(57)2021 03 02.
Artículo en Inglés | MEDLINE | ID: mdl-33653907

RESUMEN

Pediatric COVID-19 following SARS-CoV-2 infection is associated with fewer hospitalizations and often milder disease than in adults. A subset of children, however, present with Multisystem Inflammatory Syndrome in Children (MIS-C) that can lead to vascular complications and shock, but rarely death. The immune features of MIS-C compared to pediatric COVID-19 or adult disease remain poorly understood. We analyzed peripheral blood immune responses in hospitalized SARS-CoV-2 infected pediatric patients (pediatric COVID-19) and patients with MIS-C. MIS-C patients had patterns of T cell-biased lymphopenia and T cell activation similar to severely ill adults, and all patients with MIS-C had SARS-CoV-2 spike-specific antibodies at admission. A distinct feature of MIS-C patients was robust activation of vascular patrolling CX3CR1+ CD8+ T cells that correlated with the use of vasoactive medication. Finally, whereas pediatric COVID-19 patients with acute respiratory distress syndrome (ARDS) had sustained immune activation, MIS-C patients displayed clinical improvement over time, concomitant with decreasing immune activation. Thus, non-MIS-C versus MIS-C SARS-CoV-2 associated illnesses are characterized by divergent immune signatures that are temporally distinct from one another and implicate CD8+ T cells in the clinical presentation and trajectory of MIS-C.


Asunto(s)
/inmunología , Activación de Linfocitos , Síndrome de Respuesta Inflamatoria Sistémica/inmunología , Linfocitos T/inmunología , Adolescente , Adulto , Envejecimiento/inmunología , Niño , Preescolar , Femenino , Citometría de Flujo , Humanos , Leucopenia/inmunología , Masculino , Adulto Joven
12.
J Vis Exp ; (168)2021 02 21.
Artículo en Inglés | MEDLINE | ID: mdl-33682851

RESUMEN

Within the same patient, absence of NKG2D ligands (NKG2DL) surface expression was shown to distinguish leukemic subpopulations with stem cell properties (so called leukemic stem cells, LSCs) from more differentiated counterpart leukemic cells that lack disease initiation potential although they carry similar leukemia specific genetic mutations. NKG2DL are biochemically highly diverse MHC class I-like self-molecules. Healthy cells in homeostatic conditions generally do not express NKG2DL on the cell surface. Instead, expression of these ligands is induced upon exposure to cellular stress (e.g., oncogenic transformation or infectious stimuli) to trigger elimination of damaged cells via lysis through NKG2D-receptor-expressing immune cells such as natural killer (NK) cells. Interestingly, NKG2DL surface expression is selectively suppressed in LSC subpopulations, allowing these cells to evade NKG2D-mediated immune surveillance. Here, we present a side-by-side analysis of two different flow cytometry methods that allow the investigation of NKG2DL surface expression on cancer cells i.e., a method involving pan-ligand recognition and a method involving staining with multiple antibodies against single ligands. These methods can be used to separate viable NKG2DL negative cellular subpopulations with putative cancer stem cell properties from NKG2DL positive non-LSC.


Asunto(s)
Citometría de Flujo/métodos , Leucemia Mieloide Aguda/patología , Subfamilia K de Receptores Similares a Lectina de Células NK/metabolismo , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Anticuerpos Antineoplásicos/metabolismo , Biotinilación , Recuento de Células , Humanos , Ligandos , Subfamilia K de Receptores Similares a Lectina de Células NK/genética , Subfamilia K de Receptores Similares a Lectina de Células NK/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Coloración y Etiquetado , Células Tumorales Cultivadas
13.
J Vis Exp ; (168)2021 02 23.
Artículo en Inglés | MEDLINE | ID: mdl-33720124

RESUMEN

Cancer stem cells (CSCs) are an important cause of tumor growth, metastasis, and recurrence. Isolation and identification of CSCs are of great significance for tumor research. Currently, several techniques are used for the identification and purification of CSCs from tumor tissues and tumor cell lines. Separation and analysis of side population (SP) cells are two of the commonly used methods. The methods rely on the ability of CSCs to rapidly expel fluorescent dyes, such as Hoechst 33342. The efflux of the dye is associated with the ATP-binding cassette (ABC) transporters and can be inhibited by ABC transporter inhibitors. Methods for staining cultured tumor cells with Hoechst 33342 and analyzing the proportion of their SP cells by flow cytometry are described. This assay is convenient, fast, and cost-effective. Data generated in this assay can contribute to a better understanding of the effect of genes or other extracellular and intracellular signals on the stemness properties of tumor cells.


Asunto(s)
Neoplasias/patología , Células de Población Lateral/patología , Bencimidazoles/metabolismo , Línea Celular Tumoral , Análisis de Datos , Citometría de Flujo , Colorantes Fluorescentes/metabolismo , Humanos , Proteínas Proto-Oncogénicas c-fos/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-fos/metabolismo , Factor de Transcripción STAT3/metabolismo , Células de Población Lateral/metabolismo , Coloración y Etiquetado
14.
Front Immunol ; 12: 655934, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33777054

RESUMEN

COVID-19 manifests with a wide diversity of clinical phenotypes characterized by dysfunctional and exaggerated host immune responses. Many results have been described on the status of the immune system of patients infected with SARS-CoV-2, but there are still aspects that have not been fully characterized or understood. In this study, we have analyzed a cohort of patients with mild, moderate and severe disease. We performed flow cytometric studies and correlated the data with the clinical characteristics and clinical laboratory values of the patients. Both conventional and unsupervised data analyses concluded that patients with severe disease are characterized, among others, by a higher state of activation in all T cell subsets (CD4, CD8, double negative and T follicular helper cells), higher expression of perforin and granzyme B in cytotoxic cells, expansion of adaptive NK cells and the accumulation of activated and immature dysfunctional monocytes which are identified by a low expression of HLA-DR and an intriguing shift in the expression pattern of CD300 receptors. More importantly, correlation analysis showed a strong association between the alterations in the immune cells and the clinical signs of severity. These results indicate that patients with severe COVID-19 have a broad perturbation of their immune system, and they will help to understand the immunopathogenesis of COVID-19.


Asunto(s)
/inmunología , Citotoxicidad Inmunológica , Células Asesinas Naturales/inmunología , Activación de Linfocitos , Monocitos/inmunología , Receptores Inmunológicos/sangre , Linfocitos T/inmunología , Anciano , /diagnóstico , Estudios de Casos y Controles , Estudios Transversales , Femenino , Citometría de Flujo , Interacciones Huésped-Patógeno , Humanos , Inmunofenotipificación , Células Asesinas Naturales/metabolismo , Células Asesinas Naturales/virología , Masculino , Persona de Mediana Edad , Monocitos/metabolismo , Monocitos/virología , Fenotipo , Índice de Severidad de la Enfermedad , Linfocitos T/metabolismo , Linfocitos T/virología
15.
Sci Signal ; 14(673)2021 03 09.
Artículo en Inglés | MEDLINE | ID: mdl-33688079

RESUMEN

IL-1ß is a key mediator of the cytokine storm linked to high morbidity and mortality from COVID-19, and IL-1ß blockade with anakinra and canakinumab during COVID-19 infection has entered clinical trials. Using mass cytometry of human peripheral blood mononuclear cells, we identified effector memory CD4+ T cells and CD4-CD8low/-CD161+ T cells, specifically those positive for the chemokine receptor CCR6, as the circulating immune subtypes with the greatest response to IL-1ß. This response manifested as increased phosphorylation and, thus, activation of the proinflammatory transcription factor NF-κB and was also seen in other subsets, including CD11c+ myeloid dendritic cells, classical monocytes, two subsets of natural killer cells (CD16-CD56brightCD161- and CD16-CD56dimCD161+), and lineage- (Lin-) cells expressing CD161 and CD25. IL-1ß also induced a rapid but less robust increase in the phosphorylation of the kinase p38 as compared to that of NF-κB in most of these immune cell subsets. Prolonged IL-1ß stimulation increased the phosphorylation of the transcription factor STAT3 and to a lesser extent that of STAT1 and STAT5 across various immune cell types. IL-1ß-induced production of IL-6 likely led to the activation of STAT1 and STAT3 at later time points. Interindividual heterogeneity and inhibition of STAT activation by anakinra raise the possibility that assays measuring NF-κB phosphorylation in response to IL-1ß in CCR6+ T cell subtypes could identify those patients at higher risk of cytokine storm and most likely to benefit from IL-1ß-neutralizing therapies.


Asunto(s)
/inmunología , Interleucina-1beta/sangre , Subgrupos de Linfocitos T/inmunología , /sangre , Síndrome de Liberación de Citoquinas/sangre , Síndrome de Liberación de Citoquinas/etiología , Síndrome de Liberación de Citoquinas/inmunología , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Citometría de Flujo , Humanos , Interleucina-1beta/farmacología , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Monocitos/clasificación , Monocitos/inmunología , Monocitos/metabolismo , FN-kappa B/sangre , Pandemias , Fosforilación , Receptores CCR6/sangre , Factores de Transcripción STAT/sangre , Factores de Transcripción STAT/inmunología , Transducción de Señal/inmunología , Subgrupos de Linfocitos T/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/sangre
16.
Nat Commun ; 12(1): 1403, 2021 03 03.
Artículo en Inglés | MEDLINE | ID: mdl-33658497

RESUMEN

SARS-CoV-2 vaccines are advancing into human clinical trials, with emphasis on eliciting high titres of neutralising antibodies against the viral spike (S). However, the merits of broadly targeting S versus focusing antibody onto the smaller receptor binding domain (RBD) are unclear. Here we assess prototypic S and RBD subunit vaccines in homologous or heterologous prime-boost regimens in mice and non-human primates. We find S is highly immunogenic in mice, while the comparatively poor immunogenicity of RBD is associated with limiting germinal centre and T follicular helper cell activity. Boosting S-primed mice with either S or RBD significantly augments neutralising titres, with RBD-focussing driving moderate improvement in serum neutralisation. In contrast, both S and RBD vaccines are comparably immunogenic in macaques, eliciting serological neutralising activity that generally exceed levels in convalescent humans. These studies confirm recombinant S proteins as promising vaccine candidates and highlight multiple pathways to achieving potent serological neutralisation.


Asunto(s)
/uso terapéutico , /patogenicidad , Animales , Anticuerpos Neutralizantes/inmunología , Formación de Anticuerpos/fisiología , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Humanos , Macaca , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos T Colaboradores-Inductores/metabolismo , Vacunas Virales/uso terapéutico
17.
Methods Mol Biol ; 2265: 73-80, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33704706

RESUMEN

Melanoma cells have high glycolytic capacity. Glucose uptake is a key rate-limiting step in glucose utilization. Here we describe a simple protocol for measuring direct glucose uptake in living melanoma cells by flow cytometry.


Asunto(s)
Citometría de Flujo/métodos , Glucosa/metabolismo , Melanoma/metabolismo , 4-Cloro-7-nitrobenzofurazano/análogos & derivados , 4-Cloro-7-nitrobenzofurazano/química , Transporte Biológico , Técnicas de Cultivo de Célula/métodos , Línea Celular Tumoral , Desoxiglucosa/análogos & derivados , Desoxiglucosa/química , Fluorescencia , Colorantes Fluorescentes/química , Humanos
18.
Methods Mol Biol ; 2265: 111-118, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33704709

RESUMEN

Within the adaptive and innate immune system, effector lymphocytes known as cytotoxic T cells (CTLs) or natural killer (NK) cells play an essential role in host defense against tumor cells and virally infected cells. Here we describe a flow cytometry-based method to quantify CTLs or NK cell cytotoxic activity against melanoma cells. In this assay, spleen cells, peripheral blood mononuclear cells (PBMCs), or purified NK cell preparations are co-incubated at different ratios with a target tumor cell line. The target cells are pre-labeled with a fluorescent dye to allow their discrimination from the effector cells. After the incubation period, killed target cells are identified by a nucleic acid stain, which specifically permeates dead cells. This method is amenable to both diagnostic and research applications.


Asunto(s)
Pruebas Inmunológicas de Citotoxicidad/métodos , Citometría de Flujo/métodos , Células Asesinas Naturales/inmunología , Melanoma Experimental/inmunología , Linfocitos T Citotóxicos/inmunología , Animales , Técnicas de Cultivo de Célula/métodos , Muerte Celular/inmunología , Línea Celular Tumoral , Técnicas de Cocultivo/métodos , Femenino , Colorantes Fluorescentes , Humanos , Leucocitos Mononucleares/inmunología , Ratones , Bazo/citología , Bazo/inmunología
19.
Methods Mol Biol ; 2265: 129-138, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33704711

RESUMEN

Lymph node invasion by tumor cells is an important process in the progression of melanoma and is a poor prognostic factor for patients with this cancer. Before they are able to spread to regional lymph nodes, though, melanoma cells must first adhere to lymphatic endothelium and transmigrate into the lymphatic vasculature. In order to study melanoma cell adhesion to lymphatic endothelial cells and the factors that regulate this process, we have developed an in vitro flow cytometry-based assay to measure melanoma cell attachment to lymphatic endothelial cells. This assay will be a useful tool for investigating the interactions that take place between melanoma cells and lymphatic endothelial cells during the adhesion process.


Asunto(s)
Adhesión Celular , Células Endoteliales/metabolismo , Endotelio Linfático/metabolismo , Citometría de Flujo/métodos , Melanoma/metabolismo , Melanoma/patología , Técnicas de Cultivo de Célula/métodos , Endotelio Linfático/citología , Humanos
20.
Methods Mol Biol ; 2265: 203-212, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33704716

RESUMEN

Early detection of cancer has been a goal of cancer research in general and melanoma research in particular (Birnbaum et al., Lancet Glob Health 6:e885-e893, 2018; Alendar et al., Bosnian J Basic Med Sci 9:77-80, 2009). Early detection of metastasis has been targeted as pivotal to increasing survival rates (Menezes et al., Adv Cancer Res 132:1-44, 2016). Melanoma, though curable in its early stages, has a dramatic decrease in survival rates once metastasis has occurred (Sharma et al., Biotechnol Adv 36:1063-1078, 2018). The transition to metastasis is not well understood and is an area of increasing interest. Metastasis is always premeditated by the shedding of circulating tumor cells (CTCs) from the primary tumor. The ability to isolate rare CTCs from the bloodstream has led to a host of new targets and therapies for cancer (Micalizzi et al., Genes Dev 31:1827-1840, 2017). Detection of CTCs also allows for disease progression to be tracked in real time while eliminating the need to wait for additional tumors to grow. Using a photoacoustic flowmeter, in which we induce ultrasonic responses from circulating melanoma cells (CMCs), we identify and quantify these cells in order to track disease progression. Additionally, these CMCs are captured and isolated allowing for future analysis such as RNA-Seq or microarray analysis.


Asunto(s)
Citometría de Flujo/métodos , Melanoma/diagnóstico , Células Neoplásicas Circulantes , Técnicas Fotoacústicas/instrumentación , Técnicas Fotoacústicas/métodos , Reología/instrumentación , Reología/métodos , Neoplasias Cutáneas/diagnóstico , Progresión de la Enfermedad , Detección Precoz del Cáncer/métodos , Citometría de Flujo/instrumentación , Biblioteca de Genes , Humanos , Inmunohistoquímica/métodos , Melanoma/sangre , Melanoma/genética , Melanoma/patología , Metástasis de la Neoplasia/diagnóstico , Metástasis de la Neoplasia/patología , Células Neoplásicas Circulantes/patología , Reacción en Cadena en Tiempo Real de la Polimerasa , Neoplasias Cutáneas/sangre , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Ultrasonografía/métodos
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