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1.
Talanta ; 233: 122549, 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-34215052

RESUMO

Characterization of protein-protein interactions (PPIs) is essential for understanding cellular signal transduction pathways. However, quantitative measurement of the binding strength remains challenging. Building upon the classical bacterial adenylate cyclase two-hybrid (BACTH) system, we previously demonstrated that the relative reporter protein expression (RRPE), defined as the level of reporter expression normalized to that of the interacting protein, is an intrinsic characteristic associated with the binding strength between the two interacting proteins. In this study, we inserted fluorescent protein tdTomato in the chromosome as the reporter protein by CRISPR/Cas9 technology and employed a 12-amino acid tetracysteine (TC) to tag one of the interacting proteins, which can be further labeled by a membrane-permeable biarsenical dye. The combined use of tdTomato and TC-tag offers rapid and high-throughput analysis of the expression levels of both the reporter protein and one of the interacting proteins at the single-cell level by multicolor flow cytometry, which simplifies the quantitative measurement of PPI. The use of the as-developed RRPE-tdTomato-TC-BACTH approach was demonstrated in three demanding applications. First, binding affinities could be correctly ranked for discriminating interaction strengths with a tenfold difference or of the same order of magnitude. We demonstrate that the method is sensitive enough to discriminate affinities with a small difference of 1.4-fold. Moreover, residues involved in PPI can be easily mapped and ranked. Lastly, protein interaction inhibitors can be rapidly screened.


Assuntos
Bactérias , Corantes , Citometria de Fluxo
2.
Talanta ; 233: 122571, 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-34215067

RESUMO

Single-cell analysis has gained considerable attention for disease diagnosis, drug screening, and differentiation monitoring. Compared to the well-established flow cytometry, which uses fluorescent-labeled antibodies, microfluidic impedance cytometry (MIC) offers a simple, label-free, and noninvasive method for counting, classifying, and monitoring cells. Superior features including a small footprint, low reagent consumption, and ease of use have also been reported. The MIC device detects changes in the impedance signal caused by cells passing through the sensing/electric field zone, which can extract information regarding the size, shape, and dielectric properties of these cells. According to recent studies, electrode configuration has a remarkable effect on detection accuracy, sensitivity, and throughput. With the improvement in microfabrication technology, various electrode configurations have been reported for improving detection accuracy and throughput. However, the various electrode configurations of MIC devices have not been reviewed. In this review, the theoretical background of the impedance technique for single-cell analysis is introduced. Then, two-dimensional, three-dimensional, and liquid electrode configurations are discussed separately; their sensing mechanisms, fabrication processes, advantages, disadvantages, and applications are also described in detail. Finally, the current limitations and future perspectives of these electrode configurations are summarized. The main aim of this review is to offer a guide for researchers on the ongoing advancement in electrode configuration designs.


Assuntos
Técnicas Analíticas Microfluídicas , Microfluídica , Impedância Elétrica , Eletrodos , Citometria de Fluxo , Dispositivos Lab-On-A-Chip
3.
Int J Mol Sci ; 22(11)2021 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-34205175

RESUMO

The FcγRIIA/CD32A is mainly expressed on platelets, myeloid and several endothelial cells. Its affinity is considered insufficient for allowing significant binding of monomeric IgG, while its H131R polymorphism (histidine > arginine at position 131) influences affinity for multimeric IgG2. Platelet FcγRIIA has been reported to contribute to IgG-containing immune-complexe clearance. Given our finding that platelet FcγRIIA actually binds monomeric IgG, we investigated the role of platelets and FcγRIIA in IgG antibody elimination. We used pharmacokinetics analysis of infliximab (IgG1) in individuals with controlled Crohn's disease. The influence of platelet count and FcγRIIA polymorphism was quantified by multivariate linear modelling. The infliximab half-life increased with R allele number (13.2, 14.4 and 15.6 days for HH, HR and RR patients, respectively). It decreased with increasing platelet count in R carriers: from ≈20 days (RR) and ≈17 days (HR) at 150 × 109/L, respectively, to ≈13 days (both HR and RR) at 350 × 109/L. Moreover, a flow cytometry assay showed that infliximab and monomeric IgG1 bound efficiently to platelet FcγRIIA H and R allotypes, whereas panitumumab and IgG2 bound poorly to the latter. We propose that infliximab (and presumably any IgG1 antibody) elimination is partly due to an unappreciated mechanism dependent on binding to platelet FcγRIIA, which is probably tuned by its affinity for IgG2.


Assuntos
Doença de Crohn/tratamento farmacológico , Imunoglobulina G/genética , Infliximab/administração & dosagem , Receptores de IgG/genética , Adulto , Complexo Antígeno-Anticorpo/genética , Complexo Antígeno-Anticorpo/imunologia , Plaquetas/efeitos dos fármacos , Plaquetas/imunologia , Doença de Crohn/sangue , Doença de Crohn/genética , Doença de Crohn/imunologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/imunologia , Feminino , Citometria de Fluxo , Humanos , Imunoglobulina G/imunologia , Infliximab/farmacocinética , Masculino , Ativação Plaquetária/efeitos dos fármacos , Contagem de Plaquetas , Polimorfismo Genético/genética
4.
BMC Infect Dis ; 21(1): 631, 2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-34210280

RESUMO

BACKGROUND: COVID-19 continuously threated public health heavily. Present study aimed to investigate the lymphocyte subset alterations with disease severity, imaging manifestation, and delayed hospitalization in COVID-19 patients. METHODS: Lymphocyte subsets was classified using flow cytometry with peripheral blood collected from 106 patients. RESULTS: Multivariate logistic regression showed that chest tightness, lymphocyte count, and γ-glutamyl transpeptidase were the independent predictors for severe COVID-19. The T cell, CD4+ T cell and B cell counts in severe patients were significantly lower than that in mild patients (p = 0.004, 0.003 and 0.046, respectively). Only the T cell count was gradually decreased with the increase of infiltrated quadrants of lesions in computed tomography (CT) (p = 0.043). The T cell, CD4+ T cell, and CD8+ T cell counts were gradually decreased with the increase of infiltrated area of the maximum lesion in CT (p = 0.002, 0.003, 0.028; respectively). For severe patients, the counts of T cell, CD4+ T cell, CD8+ T cell gradually decreased with the increased delayed hospitalization (p = 0.001, 0.03, and <  0.001, respectively). The proportions of T cell, CD8+ T cell gradually decreased with the increased delayed hospitalization (both p <  0.001), but the proportions of NK cell, B cell gradually increased with the increased delayed hospitalization (p = 0.007, and 0.002, respectively). For mild patients, only the NK cell count was gradually decreased with the increased delayed hospitalization (p = 0.012). CONCLUSION: T lymphocyte and its subset negatively correlated with disease severity, CT manifestation and delayed hospitalization. The counts of lymphocyte subset were changed more profound than their proportions.


Assuntos
COVID-19/diagnóstico por imagem , COVID-19/patologia , Subpopulações de Linfócitos , SARS-CoV-2 , Adulto , Linfócitos B , Testes Diagnósticos de Rotina , Citometria de Fluxo , Hospitalização , Humanos , Células Matadoras Naturais , Modelos Logísticos , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Índice de Gravidade de Doença
5.
Front Immunol ; 12: 684014, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34194438

RESUMO

T cells play a fundamental role in the early control and clearance of many viral infections of the respiratory system. In SARS-CoV-2-infected individuals, lymphopenia with drastically reduced CD4+ and CD8+ T cells correlates with Coronavirus disease 2019 (COVID-19)-associated disease severity and mortality. In this study, we characterized cellular and humoral immune responses induced in patients with mild, severe and critical COVID-19. Peripheral blood mononuclear cells of 37 patients with mild, severe and critical COVID-19 and 10 healthy individuals were analyzed by IFNγ ELISpot and multi-color flow cytometry upon stimulation with peptide pools covering complete immunodominant SARS-CoV-2 matrix, nucleocapsid and spike proteins. In addition SARS-CoV-2 antibody levels, neutralization abilities and anaphylatoxin levels were evaluated by various commercially available ELISA platforms. Our data clearly demonstrates a significantly stronger induction of SARS-CoV-2 specific CD8+ T lymphocytes and higher IFNγ production in patients with mild compared to patients with severe or critical COVID-19. In all patients SARS-CoV-2-specific antibodies with similar neutralizing activity were detected, but highest titers of total IgGs were observed in critical patients. Finally, elevated anaphylatoxin C3a and C5a levels were identified in severe and critical COVID-19 patients probably caused by aberrant immune complex formation due to elevated antibody titers in these patients. Crucially, we provide a full picture of cellular and humoral immune responses of COVID-19 patients and prove that robust polyfunctional CD8+ T cell responses concomitant with low anaphylatoxin levels correlate with mild infections. In addition, our data indicates that high SARS-CoV-2 antibody titers are associated with severe disease progression.


Assuntos
Anafilatoxinas/metabolismo , Linfócitos T CD8-Positivos/imunologia , COVID-19/imunologia , SARS-CoV-2/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , COVID-19/fisiopatologia , Progressão da Doença , ELISPOT , Feminino , Citometria de Fluxo , Humanos , Imunidade Humoral , Interferon gama/sangue , Masculino , Pessoa de Meia-Idade , Gravidade do Paciente
6.
BMC Infect Dis ; 21(1): 631, 2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: covidwho-1295446

RESUMO

BACKGROUND: COVID-19 continuously threated public health heavily. Present study aimed to investigate the lymphocyte subset alterations with disease severity, imaging manifestation, and delayed hospitalization in COVID-19 patients. METHODS: Lymphocyte subsets was classified using flow cytometry with peripheral blood collected from 106 patients. RESULTS: Multivariate logistic regression showed that chest tightness, lymphocyte count, and γ-glutamyl transpeptidase were the independent predictors for severe COVID-19. The T cell, CD4+ T cell and B cell counts in severe patients were significantly lower than that in mild patients (p = 0.004, 0.003 and 0.046, respectively). Only the T cell count was gradually decreased with the increase of infiltrated quadrants of lesions in computed tomography (CT) (p = 0.043). The T cell, CD4+ T cell, and CD8+ T cell counts were gradually decreased with the increase of infiltrated area of the maximum lesion in CT (p = 0.002, 0.003, 0.028; respectively). For severe patients, the counts of T cell, CD4+ T cell, CD8+ T cell gradually decreased with the increased delayed hospitalization (p = 0.001, 0.03, and <  0.001, respectively). The proportions of T cell, CD8+ T cell gradually decreased with the increased delayed hospitalization (both p <  0.001), but the proportions of NK cell, B cell gradually increased with the increased delayed hospitalization (p = 0.007, and 0.002, respectively). For mild patients, only the NK cell count was gradually decreased with the increased delayed hospitalization (p = 0.012). CONCLUSION: T lymphocyte and its subset negatively correlated with disease severity, CT manifestation and delayed hospitalization. The counts of lymphocyte subset were changed more profound than their proportions.


Assuntos
COVID-19/diagnóstico por imagem , COVID-19/patologia , Subpopulações de Linfócitos , SARS-CoV-2 , Adulto , Linfócitos B , Testes Diagnósticos de Rotina , Citometria de Fluxo , Hospitalização , Humanos , Células Matadoras Naturais , Modelos Logísticos , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Índice de Gravidade de Doença
7.
Int J Mol Sci ; 22(12)2021 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-34208617

RESUMO

Retinitis pigmentosa (RP) is an inherited form of retinal degeneration characterized by primary rod photoreceptor cell death followed by cone loss. Mutations in several genes linked to the disease cause increased levels of cyclic guanosine monophosphate (cGMP) and calcium ion influxes. The purpose of this project was to develop a new in vitro photoreceptor degeneration model for molecular studies of RP. 661W cells were genetically modified to stably express the neural retina leucine zipper (NRL) transcription factor. One clone (661W-A11) was selected based on the expression of Nrl target genes. 661W-A11 showed a significant increase in expression of rod-specific genes but not of cone-specific genes, compared with 661W cells. Zaprinast was used to inhibit phosphodiesterase 6 (PDE6) activity to mimic photoreceptor degeneration in vitro. The activation of cell death pathways resulting from PDE6 inhibition was confirmed by detection of decreased viability and increased intracellular cGMP and calcium, as well as activation of protein kinase G (PKG) and calpains. In this new in vitro system, we validated the effects of previously published neuroprotective drugs. The 661W-A11 cells may serve as a new model for molecular studies of RP and for high-throughput drug screening.


Assuntos
Retinite Pigmentosa/etiologia , Retinite Pigmentosa/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Biomarcadores , Linhagem Celular , Células Cultivadas , Clonagem Molecular , Suscetibilidade a Doenças , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Citometria de Fluxo , Imunofluorescência , Expressão Gênica , Humanos , Camundongos , Degeneração Retiniana/tratamento farmacológico , Degeneração Retiniana/etiologia , Degeneração Retiniana/metabolismo , Degeneração Retiniana/patologia , Células Fotorreceptoras Retinianas Bastonetes/efeitos dos fármacos , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/patologia , Retinite Pigmentosa/tratamento farmacológico , Retinite Pigmentosa/patologia
8.
Zhonghua Yi Xue Za Zhi ; 101(27): 2133-2139, 2021 Jul 20.
Artigo em Chinês | MEDLINE | ID: mdl-34275248

RESUMO

Objective: To investigate the changes of natural killer(NK) cell function, and clarify the effect of granulocytic myeloid derived suppressor cells (G-MDSCs) on NK cell functionality in patients with treatment-naive chronic hepatitis C (CHC) who were cured by direct-acting antiviral agents (DAAs). Methods: Thirteen treatment-naive CHC patients and 13 healthy controls were prospectively included in this study from March 2016 to January 2017. They were divided into case group and control group, respectively. The patients of case group,6 males and 7 females aged 21-65 years old with an average of (37±14),were treated with daclatasvir and asunaprevir combination (DCV/ASV) at the Department of Infectious Diseases, Fifth Medical Center of Chinese PLA General Hospital. While 13 healthy individuals, 6 males and 7 females aged 21-57 (36±11) years old, were enrolled as healthy controls(control group). Flow cytometry was used to determine the immunological characteristics of peripheral blood NK cells subset, and detect the frequencies of gMDSCs in peripheral blood of people in two groups. It was specifically notes that CHC patients of case group would be detected before, during and after treatment. The correlations between gMDSCs and each NK cell subset function were also examined. The impact of gMDSCs on NK cell functionalities and the relevant regulatory mechanisms were explored using co-culture experiments of sorted NK cells and gMDSCs in vitro. Results: Compared with healthy controls, the decreased IFN-γ production[M(Q1,Q3)] [3.182 (2.757, 4.237) vs 6.675 (4.476, 8.280),1.434 (1.127, 2.434) vs 3.045 (1.680, 4.856), 2.611 (1.749, 3.498) vs 5.160 (4.232, 7.683)] and increased CD107a degranulation [9.314 (7.838, 13.543) vs 3.480 (2.938, 6.824), 2.544 (1.366, 4.768) vs 0.552 (0.408, 1.560), 10.339 (9.145, 12.534) vs 3.488 (3.117, 5.651)] (all P<0.05) were found on NK cell and its subsets. The frequencies of gMDSCs and plasma concentration of arginase-1 in CHC patients was significantly higher than that in healthy controls [7.050 (4.180, 12.538) vs 1.440 (0.444, 2.261), 114.278 (68.492, 163.724) vs 64.753 (50.809, 93.278)](all P<0.05). The production of IFN-γ was increased and the secretion of CD107a was decreased in NK cell and its subsets after DAAs treatment (P<0.05). The frequencies of gMDSCs and plasma arignase I levels were also decreased in CHC patients treated with DAAs (P<0.05).The results of the study indicated that the frequencies of G-MDSCs were inversely associated with the levels of IFN-γproduction of NK cells and CD56dim NK cells in CHC patients (r=0.668, -0.750, respectively, both P<0.05). In addition, the frequencies of gMDSCs were positively associated with the expression of CD107a in the CD56bright NK cell subset (r=0.711, P=0.021). In vitro, the inhibition of gMDSCs on the IFN-γ production of NK cells was demonstrated in the co-culture experiments of sorted NK cells and gMDSCs, and blocking arginase I can significantly increase the ability of NK cells to produce IFN-γ, restore NK cell IFN-γ production. Conclusions: gMDSCs in peripheral blood of CHC patients has been shown to suppress NK cell IFN-γ production in an arginase I-dependent manner. Direct-acting antiviral-mediated clearance of HCV is associated with the normalization of NK cell function and gMDSCs frequency.


Assuntos
Hepatite C Crônica , Células Supressoras Mieloides , Adulto , Idoso , Antivirais/uso terapêutico , Feminino , Citometria de Fluxo , Hepatite C Crônica/tratamento farmacológico , Humanos , Células Matadoras Naturais , Masculino , Pessoa de Meia-Idade , Adulto Jovem
9.
Int J Mol Sci ; 22(11)2021 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-34072360

RESUMO

For many years, oncological clinical trials have taken advantage of dendritic cells (DC) for the design of DC-based cellular therapies. This has required the design of suitable quality control assays to evaluate the potency of these products. The purpose of our work was to develop and validate a novel bioassay that uses flow cytometry as a read-out measurement. In this method, CD3+ cells are labeled with a fluorescent dye and the DC costimulatory activity is measured by the degree of T cell proliferation caused by the DC-T cell interaction. The validation of the method was achieved by the evaluation of essential analytical parameters defined by international guidelines. Our results demonstrated that the method could be considered specific, selective, and robust. The comparison between measured values and estimated true values confirmed a high level of accuracy and a lack of systematic error. Repeated experiments have shown the reproducibility of the assay and the proportionality between the potency and the DC amount has proven its linearity. Our results suggest that the method is compliant with the guidelines and could be adopted as a quality control assay or batch-release testing within GMP facilities.


Assuntos
Vacinas Anticâncer/imunologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Biomarcadores , Vacinas Anticâncer/uso terapêutico , Citometria de Fluxo/métodos , Humanos , Imunofenotipagem , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Linfócitos T/imunologia , Linfócitos T/metabolismo
10.
Medicine (Baltimore) ; 100(25): e26214, 2021 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-34160385

RESUMO

ABSTRACT: To investigate the relationship between the changes in circulating CD45RO+T lymphocyte subsets following neoadjuvant therapy for rectal cancer in patients with locally advanced rectal cancer.The clinicopathological data of 185 patients with rectal cancer who received neoadjuvant therapy in the General Surgery Department of Beijing Chaoyang Hospital affiliated to Capital Medical University from June 2015 to June 2017 were analyzed. Venous blood samples were collected 1 week before neoadjuvant therapy and 1 week before surgery, and the expression of CD45RO+T was detected by flow cytometry. The receiver operating characteristic curve analysis was used to determine the optimal cut-off point of CD45RO+ratio. Log-rank test and multivariate Cox regression were used to analyze the overall survival rate (OS) and disease-free survival rate (DFS) associated with CD45RO+ratio.Circulating CD45RO+ratio of 1.07 was determined as the optimal cut-off point and CD45RO+ratio-high was associated with lower tumor regression grade grading (P = .031), T stage (P = .001), and tumor node metastasis (TNM) stage (P = .012). The 3-year DFS and OS rate in the CD45RO+ratio-high group was significantly higher than that in the CD45RO+ratio-low group (89.2% vs 60.1%, P<.001; 94.4% vs 73.2%, P<.001). The multivariate Cox analysis revealed that elevated CD45RO+ratio was an independent factor for better DFS (OR, 0.339; 95% CI, 0.153-0.752; P = .008) and OS (OR, 0.244; 95% CI,0.082-0.726; P = .011).Circulating CD45RO+ratio could predict the tumor regression grade of neoadjuvant therapy for rectal cancer, as well as long-term prognosis. These findings could be used to stratify patients and develop alternative strategies for adjuvant therapy.


Assuntos
Quimiorradioterapia Adjuvante/métodos , Terapia Neoadjuvante/métodos , Recidiva Local de Neoplasia/epidemiologia , Neoplasias Retais/terapia , Subpopulações de Linfócitos T/imunologia , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Separação Celular , Colonoscopia , Ciclofosfamida/uso terapêutico , Intervalo Livre de Doença , Doxorrubicina/uso terapêutico , Citometria de Fluxo , Seguimentos , Humanos , Antígenos Comuns de Leucócito/metabolismo , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/diagnóstico , Recidiva Local de Neoplasia/imunologia , Estadiamento de Neoplasias , Oxaliplatina/uso terapêutico , Prednisona/uso terapêutico , Período Pré-Operatório , Protectomia , Prognóstico , Radioterapia de Intensidade Modulada , Neoplasias Retais/sangue , Neoplasias Retais/imunologia , Neoplasias Retais/mortalidade , Reto/diagnóstico por imagem , Reto/patologia , Reto/cirurgia , Estudos Retrospectivos , Taxa de Sobrevida , Subpopulações de Linfócitos T/metabolismo , Vindesina/uso terapêutico
11.
Methods Mol Biol ; 2276: 203-213, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34060043

RESUMO

To evaluate how a cell responds to the external stimuli, treatment, or alteration of the microenvironment, the quantity and quality of mitochondria are commonly used as readouts. However, it is challenging to apply mitochondrial analysis to the samples that are composed of mixed cell populations originating from tissues or when multiple cell populations are of interest, using methods such as Western blot, electron microscopy, or extracellular flux analysis.Flow cytometry is a technique allowing the detection of individual cell status and its identity simultaneously when used in combination with surface markers. Here we describe how to combine mitochondria-specific dyes or the dyes targeting the superoxide produced by mitochondria with surface marker staining to measure the mitochondrial content and activity in live cells by flow cytometry. This method can be applied to all types of cells in suspension and is particularly useful for analysis of samples composed of heterogeneous cell populations.


Assuntos
Células Sanguíneas/metabolismo , Citometria de Fluxo/métodos , Corantes Fluorescentes/metabolismo , Mitocôndrias/metabolismo , Baço/metabolismo , Superóxidos/metabolismo , Animais , Células Sanguíneas/citologia , Células Sanguíneas/ultraestrutura , Humanos , Baço/citologia , Baço/ultraestrutura
12.
Egypt J Immunol ; 28(1): 12-22, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-34147050

RESUMO

Accumulating evidence has indicated that immune regulatory cells are involved in the establishment of the anti-tumor activity, however; the role of regulatory B cells (B-regs) in breast cancer (BC) remains unclear. This study intended to assess the frequency of peripheral B-regs phenotypes in patients with BC, and to determine the relation between these phenotypes and the patient's clinicopathological characters. The expressions of the immune cell populations were analyzed by four-color flow cytometry in 40 naïve BC patients and 10 age-matched apparently healthy individuals as controls attending the department of Clinical Oncology and Nuclear Medicine at Assiut University Hospitals. The percentages of B-regs phenotypes CD19+IL10+ and CD19+CD24hiCD27+IL10+ were higher in BC patients than in the controls. The percentage of CD19+IL10+ B cells phenotype was significantly associated with the HER-2 expression levels, T, and N stages of BC. In conclusion, high percentage of B-regs phenotypes CD19+IL10+ and CD19+CD24hiCD27+IL10+ in BC patients indicates a possible role in immune suppression during the development of BC.


Assuntos
Linfócitos B Reguladores , Neoplasias da Mama , Antígenos CD19 , Egito/epidemiologia , Feminino , Citometria de Fluxo , Humanos , Fenótipo , Linfócitos T Reguladores
13.
Cells ; 10(5)2021 05 05.
Artigo em Inglês | MEDLINE | ID: covidwho-1223959

RESUMO

A malfunction of the innate immune response in COVID-19 is associated with eosinopenia, particularly in more severe cases. This study tested the hypothesis that this eosinopenia is COVID-19 specific and is associated with systemic activation of eosinophils. Blood of 15 healthy controls and 75 adult patients with suspected COVID-19 at the ER were included before PCR testing and analyzed by point-of-care automated flow cytometry (CD10, CD11b, CD16, and CD62L) in the absence or presence of a formyl peptide (fNLF). Forty-five SARS-CoV-2 PCR positive patients were grouped based on disease severity. PCR negative patients with proven bacterial (n = 20) or other viral (n = 10) infections were used as disease controls. Eosinophils were identified with the use of the FlowSOM algorithm. Low blood eosinophil numbers (<100 cells/µL; p < 0.005) were found both in patients with COVID-19 and with other infectious diseases, albeit less pronounced. Two discrete eosinophil populations were identified in healthy controls both before and after activation with fNLF based on the expression of CD11b. Before activation, the CD11bbright population consisted of 5.4% (CI95% = 3.8, 13.4) of total eosinophils. After activation, this population of CD11bbright cells comprised nearly half the population (42.21%, CI95% = 35.9, 54.1). Eosinophils in COVID-19 had a similar percentage of CD11bbright cells before activation (7.6%, CI95% = 4.5, 13.6), but were clearly refractory to activation with fNLF as a much lower percentage of cells end up in the CD11bbright fraction after activation (23.7%, CI95% = 18.5, 27.6; p < 0.001). Low eosinophil numbers in COVID-19 are associated with refractoriness in responsiveness to fNLF. This might be caused by migration of fully functional cells to the tissue.


Assuntos
COVID-19/imunologia , Eosinófilos/imunologia , Imunidade Inata , N-Formilmetionina Leucil-Fenilalanina/metabolismo , SARS-CoV-2/imunologia , Adulto , COVID-19/sangue , COVID-19/diagnóstico , COVID-19/virologia , Teste de Ácido Nucleico para COVID-19 , Estudos de Casos e Controles , Separação Celular , Estudos de Coortes , Eosinófilos/metabolismo , Citometria de Fluxo , Voluntários Saudáveis , Humanos , Contagem de Leucócitos , RNA Viral/isolamento & purificação , SARS-CoV-2/isolamento & purificação , Índice de Gravidade de Doença
14.
Lab Chip ; 21(14): 2812-2824, 2021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34109338

RESUMO

Cellular mechanical properties (e.g. compressibility) are important biophysical markers in relation to cellular processes and functionality. Among the methods for cell mechanical measurement, acoustofluidic methods appear to be advantageous due to tunability, biocompatibility and acousto-mechanical nature. However, the previous acoustofluidic methods were limited in throughput and number of measurements. In this study, we developed a high-throughput microfluidic compressibility cytometry approach using multi-tilted-angle surface acoustic wave, which can provide thousands of single-cell compressibility measurements within minutes. The compressibility cytometer was constructed to drag microparticles or cells towards the microfluidic channel sidewall at different segments based on their biophysical properties (such as size and compressibility), as a result of the varied balance between acoustics and flow. Mathematical analysis and computational simulation revealed that the compressibility of a cell could be estimated from the position of collision with the sidewall. Microbeads of different materials and sizes were experimentally tested to validate the simulation and to demonstrate the capability to characterise size and compressibility. MDA MB231 cells, of the triple negative breast cancer subtype, were treated with the microtubule disrupting agent colchicine which increased compressibility and treated with the actin disrupting agent cytochalasin B which increased cell size but did not change compressibility. Moreover, the highly metastatic variant MDA MB231 LNm5 cell line showed increased compressibility compared to the parent MDA MB231 cells, indicating the potential utility of high-throughput mechanophenotyping for tumour cell characterisation.


Assuntos
Técnicas Analíticas Microfluídicas , Microfluídica , Acústica , Linhagem Celular , Citometria de Fluxo , Microesferas , Som
15.
Nat Protoc ; 16(7): 3596-3624, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34172975

RESUMO

Deaminase fused-Cas9 base editing technologies have enabled precise single-nucleotide genomic editing without the need for the introduction of damaging double-stranded breaks and inefficient homology-directed repair. However, current methods to isolate base-edited cell populations are ineffective, especially when utilized with human pluripotent stem cells, a cell type resistant to genome modification. Here, we outline a series of methods that employ transient reporters of editing enrichment (TREE) to facilitate the highly efficient single-base editing of human cells at precise genomic loci. Briefly, these transient reporters of editing enrichment based methods employ a transient episomal fluorescent reporter that allows for the real-time, flow-cytometry-based enrichment of cells that have had single nucleotide changes at precise genomic locations. This protocol details how these approaches can enable the rapid (~3-4 weeks) and efficient (clonal editing efficiencies >80%) generation of biallelic or multiplexed edited isogenic hPSC lines using adenosine and cytosine base editors.


Assuntos
Adenosina/metabolismo , Citosina/metabolismo , Edição de Genes/métodos , Genes Reporter , Células-Tronco Pluripotentes Induzidas/metabolismo , Sequência de Bases , Células Clonais , Criopreservação , Citometria de Fluxo , Humanos , Plasmídeos/genética , RNA Guia/metabolismo , Reprodutibilidade dos Testes , Análise de Célula Única , Transfecção
16.
Cells ; 10(5)2021 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-34062964

RESUMO

A malfunction of the innate immune response in COVID-19 is associated with eosinopenia, particularly in more severe cases. This study tested the hypothesis that this eosinopenia is COVID-19 specific and is associated with systemic activation of eosinophils. Blood of 15 healthy controls and 75 adult patients with suspected COVID-19 at the ER were included before PCR testing and analyzed by point-of-care automated flow cytometry (CD10, CD11b, CD16, and CD62L) in the absence or presence of a formyl peptide (fNLF). Forty-five SARS-CoV-2 PCR positive patients were grouped based on disease severity. PCR negative patients with proven bacterial (n = 20) or other viral (n = 10) infections were used as disease controls. Eosinophils were identified with the use of the FlowSOM algorithm. Low blood eosinophil numbers (<100 cells/µL; p < 0.005) were found both in patients with COVID-19 and with other infectious diseases, albeit less pronounced. Two discrete eosinophil populations were identified in healthy controls both before and after activation with fNLF based on the expression of CD11b. Before activation, the CD11bbright population consisted of 5.4% (CI95% = 3.8, 13.4) of total eosinophils. After activation, this population of CD11bbright cells comprised nearly half the population (42.21%, CI95% = 35.9, 54.1). Eosinophils in COVID-19 had a similar percentage of CD11bbright cells before activation (7.6%, CI95% = 4.5, 13.6), but were clearly refractory to activation with fNLF as a much lower percentage of cells end up in the CD11bbright fraction after activation (23.7%, CI95% = 18.5, 27.6; p < 0.001). Low eosinophil numbers in COVID-19 are associated with refractoriness in responsiveness to fNLF. This might be caused by migration of fully functional cells to the tissue.


Assuntos
COVID-19/imunologia , Eosinófilos/imunologia , Imunidade Inata , N-Formilmetionina Leucil-Fenilalanina/metabolismo , SARS-CoV-2/imunologia , Adulto , COVID-19/sangue , COVID-19/diagnóstico , COVID-19/virologia , Teste de Ácido Nucleico para COVID-19 , Estudos de Casos e Controles , Separação Celular , Estudos de Coortes , Eosinófilos/metabolismo , Citometria de Fluxo , Voluntários Saudáveis , Humanos , Contagem de Leucócitos , RNA Viral/isolamento & purificação , SARS-CoV-2/isolamento & purificação , Índice de Gravidade de Doença
17.
Molecules ; 26(10)2021 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-34067581

RESUMO

We proposed to perform a comparative analysis of growth factors, cytokines, and chemokine receptors on the salivary cells in the saliva obtained from trigeminal neuralgia (TN) and normal subjects. Saliva was collected from TN and healthy subjects. Salivary cells were isolated by centrifugation. The expression of the cell surface marker was analyzed by flow cytometry. A cytometric bead array was done to measure the levels of cytokines and growth factors on the flow cytometer. Saliva from TN subjects showed lower growth factor levels of Angiopoietin-2, bFGF, HGF, SCF, TGF-α, and VEGF and higher cytokine levels of IL-1ß, TNF-α, CCL2, IL-17A, IL-6, and CXCL8, as well as higher expression levels of chemokine receptors CCR1 (CD191), CR3 (CD11b), CCR2 (CD192), CXCR5 (CD185), and CCR5 (CD196) in the cells from TN saliva. A certain set of cytokines and growth factors in the saliva, as well as chemokine receptors on salivary cells, could be a useful tool in the diagnostics and prognostics of trigeminal neuralgia. Trigeminal neuralgia is one of the significant pathological conditions in the class of chronic diseases around the world. Many targeted approaches are being tried by various research groups to utilize the information of the inflammatory microenvironment to resolve the pathology of chronic TN.


Assuntos
Citocinas/análise , Peptídeos e Proteínas de Sinalização Intercelular/análise , Neuralgia do Trigêmeo/metabolismo , Adulto , Biomarcadores/análise , Quimiocinas/análise , Feminino , Citometria de Fluxo/métodos , Humanos , Masculino , Pessoa de Meia-Idade , Saliva/química , Neuralgia do Trigêmeo/fisiopatologia
18.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 29(3): 715-719, 2021 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-34105462

RESUMO

OBJECTIVE: To investigate the value of CD44+ mononuclear cells (MNC) and spleen stiffness measurement (SSM) in minimal residual disease (MRD) in acute myeloid leukemia (AML) and its prognosis. METHODS: Flow cytometry was used to detected the proportion of CD44+ and CD24+ MNC in 44 AML patients after induction chemotherapy. The SSM was tested by FS. The value of MNC and SSM in MRD and its prognosis was explored. RESULTS: The percentage of CD44+ MNC and SSM in MRD positive group were significantly higher than those in MRD negative group (P<0.05). In MRD positive group, there were positive correlation between CD44+ MNC, SSM and MRD level (r=0.998, r=0.939, P<0.05). The median EFS and OS in HCD44+ MNC and HSSM groups were significantly shorter than those in LCD44+ MNC and LSSM (P<0.05). CD24+ MNC showed no association with MRD and its prognosis. CONCLUSION: HCD44+ MNC and HSSM may be used to predict high level MRD and poor prognosis.


Assuntos
Leucemia Mieloide Aguda , Baço , Citometria de Fluxo , Humanos , Receptores de Hialuronatos , Quimioterapia de Indução , Leucemia Mieloide Aguda/tratamento farmacológico , Neoplasia Residual , Prognóstico
19.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 29(3): 832-839, 2021 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-34105480

RESUMO

OBJECTIVE: To investigate the quantitative expression of immunophenotype of CD34+ myeloid precursor cells in myelodysplastic syndrome (MDS) patients and its correlation with clinical characteristics, and understand the effect of quantitative expression of CD7 and CD117 on the prognosis of low-risk MDS patients. METHODS: Multi-parameter flow cytometry (FCM) was used to detect the proportion and mean fluorescence intensity (MFI) of each antigen of bone marrow CD34+ myeloid precursor cells in 79 MDS patients. The correlation between the expression level of each immune marker and clinical characteristics was compared. The effects of quantitative expressions of CD7 and CD117 on the overall survival rate of low-risk patients were explored. RESULTS: Bone marrow blast cell proportion (P<0.01), RBC level (P<0.01), and Hb level (P<0.05) of high-risk MDS patients were higher, while EPO level (P<0.05) was lower than those of low-risk patients. The proportion of CD34+ blast cells (P<0.01), the proportion of CD117 (P<0.05) and the MFI of CD7 (P<0.05) were higher in high-risk patients than those in low-risk patients, but the MFI of CD123 was lower (P<0.05). In high-risk MDS patients, CD15/CD34 (MFI) and CD19/CD34 (MFI) positively correlated with the proportion of total T cells (r=0.458; r=0.505), while CD19/CD34 (%) and CD19/CD34 (MFI) negatively correlated with WBC levels (r=-0.469; r=-0.503). In low-risk MDS patients, CD34+ (%) positively correlated with bone marrow erythrocyte proportion, PLT level and neutrophil level (r=0.426; r=0.486; r=0.495), but negatively correlated with LDH level (r=-0.421); WT1 expression level was positively correlated with CD10/CD34 (%), CD10/CD34 (MFI) and CD117/CD34 (MFI) (r=0.745; r=0.800; r=0.434), while negatively correlated with CD11b/CD34 (%)(r=-0.457); CD19/CD34 (%) and CD71/CD34 (MFI) negatively correlated with NK cell proportion (r=-0.786; r=-0.514); CD10/CD34 (%) positively correlated with Th/Ts, while CD7/CD34 (MFI) negatively correlated with the proportion of Th cells (r=0.738; r=-0.513); HLADR/CD34 (%) and HLADR/CD34 (MFI) negatively correlated with PLT level (r=-0.461; r=-0.445), while HLADR/CD34 (MFI) positively correlated with bone marrow NAP fraction (r=0.552). The quantitative expression of CD7 and CD117 had no significant effect on the overall survival rate of low-risk MDS patients. CONCLUSION: The immunophenotype of CD34+ myeloid precursor cell in different risk groups in MDS patients is related to clinical characteristics. Bone marrow cell morphology, clinical and laboratory features and immunophenotype will be of great significance to the diagnosis, clinical classification and prognosis evaluation of MDS patients.


Assuntos
Síndromes Mielodisplásicas , Antígenos CD34 , Medula Óssea , Células da Medula Óssea , Citometria de Fluxo , Humanos , Imunofenotipagem
20.
Nat Commun ; 12(1): 3318, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-34083536

RESUMO

Dormancy, a reversible quiescent cellular state characterized by greatly reduced metabolic activity, protects from genetic damage, prolongs survival and is crucial for tissue homeostasis and cellular response to injury or transplantation. Dormant cells have been characterized in many tissues, but their identification, isolation and characterization irrespective of tissue of origin remains elusive. Here, we develop a live cell ratiometric fluorescent Optical Stem Cell Activity Reporter (OSCAR) based on the observation that phosphorylation of RNA Polymerase II (RNApII), a hallmark of active mRNA transcription elongation, is largely absent in dormant stem cells from multiple lineages. Using the small intestinal crypt as a model, OSCAR reveals in real time the dynamics of dormancy induction and cellular differentiation in vitro, and allows the identification and isolation of several populations of transcriptionally diverse OSCARhigh and OSCARlow intestinal epithelial cell states in vivo. In particular, this reporter is able to identify a dormant OSCARhigh cell population in the small intestine. OSCAR therefore provides a tool for a better understanding of dormant stem cell biology.


Assuntos
RNA Polimerase II/metabolismo , Fase de Repouso do Ciclo Celular/fisiologia , Animais , Separação Celular , Quinase 9 Dependente de Ciclina/metabolismo , Citometria de Fluxo , Corantes Fluorescentes/metabolismo , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Intestino Delgado/citologia , Intestino Delgado/metabolismo , Proteínas Luminescentes/metabolismo , Camundongos , Camundongos Transgênicos , RNA Mensageiro/metabolismo , Transcrição Genética
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