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2.
Ann Work Expo Health ; 63(6): 689-700, 2019 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-31211837

RESUMO

OBJECTIVES: Previous epidemiological evidence for the association of shift work exposure and increased leukocyte count is cross-sectional in nature, thus limiting cause-effect inference. We therefore used a longitudinal design to: (i) compare leukocyte counts at baseline between shift and day workers and (ii) examine the time trend of leukocyte counts over the follow-up period for these workers. METHODS: A retrospective cohort study was conducted among 6737 workers aged <60 years at two large organizations (a humanitarian organization and a university) in Bangkok, Thailand who had participated in at least two annual health check-ups during the period 2005-2016. Shift work exposure history was assessed by a self-administered questionnaire and categorized into day, former, and current shift workers. Data on leukocyte count were collected annually as part of worksite health screening during the observation period. Association of shift work exposure and increased leukocyte count was then examined cross-sectionally and longitudinally by using multiple linear regression and multilevel analysis of repeated measures data, respectively. In addition, trends for leukocyte count over the follow-up period and work years were examined using LOWESS smooth curves. RESULTS: Compared to day work, the current shift work was associated with increased leukocyte counts. The magnitude of percentage increase was the highest for basophil counts, followed by eosinophil and lymphocyte counts. Both cross-sectional and longitudinal evidence revealed this association, although it was less pronounced longitudinally. For total leukocyte count, the magnitude of difference was constant across the 11-year follow-up period. However, for lymphocyte and basophil counts, these discrepancies tapered over the work years until they no longer differed (for lymphocyte count) or even differed in the opposite direction (for basophil count) in later work years. CONCLUSION: This study confirmed previous cross-sectional evidence that shift work exposure-increased leukocyte counts and that this was reversible. Whether this increase in immune cell count also results in an increased immune cell activity and serves as the intermediary in the association between shift work exposure and subsequent chronic disease development needs further investigation.


Assuntos
Contagem de Leucócitos , Exposição Ocupacional/efeitos adversos , Jornada de Trabalho em Turnos/efeitos adversos , Adulto , Basófilos/citologia , Estudos Transversais , Feminino , Humanos , Modelos Lineares , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Tailândia
3.
Lab Chip ; 19(11): 1916-1921, 2019 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-31070645

RESUMO

We have developed a highly integrated lab-on-a-chip containing embedded electrical microsensors, µdegassers and pneumatically-actuated micropumps to monitor allergic hypersensitivity. Rapid antigen-mediated histamine release (e.g. s to min) and resulting muscle contraction (<30 min) is detected by connecting an immune compartment containing sensitized basophile cells to a vascular co-culture model.


Assuntos
Comunicação Celular , Hipersensibilidade/diagnóstico , Hipersensibilidade/imunologia , Dispositivos Lab-On-A-Chip , Basófilos/citologia , Basófilos/imunologia , Desenho de Equipamento , Fatores de Tempo
4.
Cell Rep ; 26(6): 1627-1640.e7, 2019 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-30726743

RESUMO

The molecular characterization of immune subsets is important for designing effective strategies to understand and treat diseases. We characterized 29 immune cell types within the peripheral blood mononuclear cell (PBMC) fraction of healthy donors using RNA-seq (RNA sequencing) and flow cytometry. Our dataset was used, first, to identify sets of genes that are specific, are co-expressed, and have housekeeping roles across the 29 cell types. Then, we examined differences in mRNA heterogeneity and mRNA abundance revealing cell type specificity. Last, we performed absolute deconvolution on a suitable set of immune cell types using transcriptomics signatures normalized by mRNA abundance. Absolute deconvolution is ready to use for PBMC transcriptomic data using our Shiny app (https://github.com/giannimonaco/ABIS). We benchmarked different deconvolution and normalization methods and validated the resources in independent cohorts. Our work has research, clinical, and diagnostic value by making it possible to effectively associate observations in bulk transcriptomics data to specific immune subsets.


Assuntos
Linfócitos B/imunologia , Linhagem da Célula/genética , Células Dendríticas/imunologia , RNA Mensageiro/genética , Linfócitos T/imunologia , Transcriptoma , Adulto , Linfócitos B/classificação , Linfócitos B/citologia , Basófilos/classificação , Basófilos/citologia , Basófilos/imunologia , Benchmarking , Linhagem da Célula/imunologia , Células Dendríticas/classificação , Células Dendríticas/citologia , Feminino , Citometria de Fluxo , Voluntários Saudáveis , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imunofenotipagem , Células Matadoras Naturais/classificação , Células Matadoras Naturais/citologia , Células Matadoras Naturais/imunologia , Masculino , Monócitos/classificação , Monócitos/citologia , Monócitos/imunologia , Neutrófilos/classificação , Neutrófilos/citologia , Neutrófilos/imunologia , Especificidade de Órgãos , RNA Mensageiro/imunologia , Células-Tronco/classificação , Células-Tronco/citologia , Células-Tronco/imunologia , Linfócitos T/classificação , Linfócitos T/citologia
5.
PLoS One ; 14(2): e0211943, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30735559

RESUMO

The initial steps in the synthesis of leukotrienes are the translocation of 5-lipoxygenase (5-LO) to the nuclear envelope and its subsequent association with its scaffold protein 5-lipoxygenase-activating protein (FLAP). A major gap in our understanding of this process is the knowledge of how the organization of 5-LO and FLAP on the nuclear envelope regulates leukotriene synthesis. We combined single molecule localization microscopy with Clus-DoC cluster analysis, and also a novel unbiased cluster analysis to analyze changes in the relationships between 5-LO and FLAP in response to activation of RBL-2H3 cells to generate leukotriene C4. We identified the time-dependent reorganization of both 5-LO and FLAP into higher-order assemblies or clusters in response to cell activation via the IgE receptor. Clus-DoC analysis identified a subset of these clusters with a high degree of interaction between 5-LO and FLAP that specifically correlates with the time course of LTC4 synthesis, strongly suggesting their role in the initiation of leukotriene biosynthesis.


Assuntos
Proteínas Ativadoras de 5-Lipoxigenase/metabolismo , Araquidonato 5-Lipoxigenase/metabolismo , Basófilos/metabolismo , Leucotrieno C4/biossíntese , Membrana Nuclear/metabolismo , Proteínas Ativadoras de 5-Lipoxigenase/química , Proteínas Ativadoras de 5-Lipoxigenase/genética , Animais , Araquidonato 5-Lipoxigenase/química , Araquidonato 5-Lipoxigenase/genética , Basófilos/citologia , Basófilos/efeitos dos fármacos , Linhagem Celular Tumoral , Análise por Conglomerados , Regulação da Expressão Gênica , Imunoglobulina E/genética , Imunoglobulina E/metabolismo , Imunoglobulina E/farmacologia , Membrana Nuclear/efeitos dos fármacos , Membrana Nuclear/genética , Membrana Nuclear/ultraestrutura , Ligação Proteica , Ratos , Receptores de IgE/genética , Receptores de IgE/metabolismo , Transdução de Sinais , Imagem Individual de Molécula
6.
Cytometry A ; 95(3): 279-289, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30536810

RESUMO

Daratumumab is a CD38-targeted human monoclonal antibody with direct anti-myeloma cell mechanisms of action. Flow cytometry in relapsed and/or refractory multiple myeloma (RRMM) patients treated with daratumumab revealed cytotoxic T-cell expansion and reduction of immune-suppressive populations, suggesting immune modulation as an additional mechanism of action. Here, we performed an in-depth analysis of the effects of daratumumab on immune-cell subpopulations using high-dimensional mass cytometry. Whole-blood and bone-marrow baseline and on-treatment samples from RRMM patients who participated in daratumumab monotherapy studies (SIRIUS and GEN501) were evaluated with high-throughput immunophenotyping. In daratumumab-treated patients, the intensity of CD38 marker expression decreased on many immune cells in SIRIUS whole-blood samples. Natural killer (NK) cells were depleted with daratumumab, with remaining NK cells showing increased CD69 and CD127, decreased CD45RA, and trends for increased CD25, CD27, and CD137 and decreased granzyme B. Immune-suppressive population depletion paralleled previous findings, and a newly observed reduction in CD38+ basophils was seen in patients who received monotherapy. After 2 months of daratumumab, the T-cell population in whole-blood samples from responders shifted to a CD8 prevalence with higher granzyme B positivity (P = 0.017), suggesting increased killing capacity and supporting monotherapy-induced CD8+ T-cell activation. High-throughput cytometry immune profiling confirms and builds upon previous flow cytometry data, including comparable CD38 marker intensity on plasma cells, NK cells, monocytes, and B/T cells. Interestingly, a shift toward cytolytic granzyme B+ T cells was also observed and supports adaptive responses in patients that may contribute to depth of response. © 2018 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.


Assuntos
ADP-Ribosil Ciclase 1/imunologia , Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/uso terapêutico , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/imunologia , Antígenos de Diferenciação de Linfócitos T/metabolismo , Basófilos/citologia , Basófilos/efeitos dos fármacos , Basófilos/imunologia , Células da Medula Óssea/citologia , Células da Medula Óssea/imunologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Citometria de Fluxo , Granzimas/metabolismo , Humanos , Imunofenotipagem , Células Matadoras Naturais/citologia , Mieloma Múltiplo/sangue , Mieloma Múltiplo/metabolismo , Recidiva
7.
Comput Methods Programs Biomed ; 168: 69-80, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29173802

RESUMO

BACKGROUND AND OBJECTIVES: White blood cells (WBCs) differential counting yields valued information about human health and disease. The current developed automated cell morphology equipments perform differential count which is based on blood smear image analysis. Previous identification systems for WBCs consist of successive dependent stages; pre-processing, segmentation, feature extraction, feature selection, and classification. There is a real need to employ deep learning methodologies so that the performance of previous WBCs identification systems can be increased. Classifying small limited datasets through deep learning systems is a major challenge and should be investigated. METHODS: In this paper, we propose a novel identification system for WBCs based on deep convolutional neural networks. Two methodologies based on transfer learning are followed: transfer learning based on deep activation features and fine-tuning of existed deep networks. Deep acrivation featues are extracted from several pre-trained networks and employed in a traditional identification system. Moreover, a novel end-to-end convolutional deep architecture called "WBCsNet" is proposed and built from scratch. Finally, a limited balanced WBCs dataset classification is performed through the WBCsNet as a pre-trained network. RESULTS: During our experiments, three different public WBCs datasets (2551 images) have been used which contain 5 healthy WBCs types. The overall system accuracy achieved by the proposed WBCsNet is (96.1%) which is more than different transfer learning approaches or even the previous traditional identification system. We also present features visualization for the WBCsNet activation which reflects higher response than the pre-trained activated one. CONCLUSION: a novel WBCs identification system based on deep learning theory is proposed and a high performance WBCsNet can be employed as a pre-trained network.


Assuntos
Aprendizado Profundo , Processamento de Imagem Assistida por Computador , Leucócitos/citologia , Redes Neurais de Computação , Basófilos/citologia , Eosinófilos/citologia , Humanos , Linfócitos/citologia , Informática Médica , Monócitos/citologia , Neutrófilos/citologia , Reprodutibilidade dos Testes
8.
Clin Immunol ; 197: 224-230, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30290225

RESUMO

Leukocyte populations quickly respond to tissue damage, but most leukocyte kinetic studies are not based on multiparameter flow cytometry. We systematically investigated several blood leukocyte populations after controlled tissue damage. 48 patients were assigned to either an anterior or posterolateral total hip arthroplasty. Peripheral blood was collected pre-operatively and at 2 h, 24 h, 48 h, 2 and 6 weeks postoperatively and assessed by flow cytometry for absolute counts of multiple leukocyte populations using standardized EuroFlow protocols. Absolute counts of leukocyte subsets differed significantly between consecutive time points. Neutrophils increased instantly after surgery, while most leukocyte subsets initially decreased, followed by increasing cell counts until 48 h. At two weeks all leukocyte counts were restored to pre-operative counts. Immune cell kinetics upon acute tissue damage exhibit reproducible patterns, which differ between the leukocyte subsets and with "opposite kinetics" among monocyte subsets. Flow cytometric leukocyte monitoring can be used to minimally invasively monitor tissue damage.


Assuntos
Artroplastia de Quadril/métodos , Contagem de Leucócitos , Leucócitos/citologia , Músculos/cirurgia , Tendões/cirurgia , Idoso , Linfócitos B/citologia , Basófilos/citologia , Eosinófilos/citologia , Feminino , Citometria de Fluxo , Humanos , Células Matadoras Naturais/citologia , Cinética , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Monócitos/citologia , Músculos/lesões , Neutrófilos/citologia , Período Pós-Operatório , Período Pré-Operatório , Linfócitos T/citologia , Traumatismos dos Tendões
9.
Methods Mol Biol ; 1799: 81-92, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29956146

RESUMO

Mast cells and basophils are important innate immune cells involved in resistance to parasitic infection and are critical orchestrators of allergic disease. The relative ease with which they are cultured from mouse or human tissues allows one to work with primary cells that maintain a differentiated and functional phenotype. In this chapter, we describe the methods by which mouse mast cells and basophils can be cultured from bone marrow. We also provide methods for isolating and expanding mouse peritoneal mast cells and human skin mast cells.


Assuntos
Basófilos/imunologia , Basófilos/metabolismo , Inflamação/imunologia , Inflamação/metabolismo , Mastócitos/imunologia , Mastócitos/metabolismo , Animais , Basófilos/citologia , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Diferenciação Celular , Separação Celular , Humanos , Imunoglobulina E/imunologia , Interleucina-3/metabolismo , Mastócitos/citologia , Camundongos , Lavagem Peritoneal , Pele/citologia , Pele/imunologia , Pele/metabolismo
10.
Food Chem ; 263: 307-314, 2018 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-29784322

RESUMO

This study investigates the anti-allergic properties of peanut skin polyphenols (PSP)-enriched peanut (PN) protein aggregates. PSP was blended with PN flour at concentrations of 5, 10, 15, 30, and 40% (w/w). Rat basophil leukemia cells (RBL-2H3) were sensitized with either anti-DNP-IgE or PN-allergic plasma followed by co-exposure to unmodified PN flour (control) or PSP-PN protein aggregates and Ca2+ ionophore, ionomycin. Immunoblotting and staining were performed to measure the IgE binding capacity of PSP-PN aggregates. Results showed that 30% PSP-PN aggregate significantly reduced ß-hexosaminidase and histamine levels by 54.2% and 49.2%, respectively compared with control. Immunoblotting results revealed 40% PSP-PN aggregates significantly decreased IgE binding by 19%. The phosphorylation of p44/42 MAPK was significantly reduced while phosphorylation of p38 MAPK and SAPK/JNK increased upon PSP-PN protein aggregate exposure to the cells. Our results show that aggregation of PSP to PN proteins reduces allergic response by inhibiting Ca2+-induced MAPK-dependent cell degranulation.


Assuntos
Arachis/química , Degranulação Celular/efeitos dos fármacos , Imunoglobulina E/imunologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Polifenóis/farmacologia , Alérgenos/imunologia , Alérgenos/metabolismo , Animais , Arachis/metabolismo , Basófilos/citologia , Basófilos/efeitos dos fármacos , Basófilos/metabolismo , Linhagem Celular Tumoral , Ensaio de Imunoadsorção Enzimática , Histamina/metabolismo , Imunoglobulina E/análise , Proteínas de Plantas/imunologia , Proteínas de Plantas/metabolismo , Polifenóis/química , Ratos
11.
A A Pract ; 11(7): 181-183, 2018 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-29672323

RESUMO

We describe 2 patients who developed anaphylactic shock after sugammadex administration during anesthesia. Both had no history of prior sugammadex administration. The serum tryptase concentrations were elevated after the allergic reaction. Basophil activation testing 1 month after the events was positive for sugammadex in 1 patient, and negative in the other. However, it was positive for light-exposed sugammadex solution in both patients, suggesting a possible allergic reaction to a denatured compound of sugammadex generated by light exposure of the sugammadex solution.


Assuntos
Anafilaxia/imunologia , Luz/efeitos adversos , Sugammadex/efeitos adversos , Idoso , Anafilaxia/induzido quimicamente , Anafilaxia/tratamento farmacológico , Anestesia/efeitos adversos , Basófilos/citologia , Clorfeniramina/administração & dosagem , Clorfeniramina/uso terapêutico , Feminino , Humanos , Masculino , Metilprednisolona/administração & dosagem , Metilprednisolona/uso terapêutico , Adulto Jovem
12.
Am J Rhinol Allergy ; 32(3): 194-201, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29676185

RESUMO

Background Blood eosinophil and basophil levels have recently been considered for the purpose of endotyping chronic rhinosinusitis with nasal polyps (CRSwNP). Histologically, eosinophilic-type CRSwNPs have been associated with high recurrence rates after treatment. Objective The present study was the first to compare blood eosinophil and basophil counts in eosinophilic-type CRSwNP patients before and after endoscopic sinus surgery. Methods The study concerned 79 consecutive patients with histologically confirmed eosinophilic-type CRSwNP treated with endoscopic sinus surgery. Results A significant drop in mean blood eosinophil counts and percentages occurred from before to after endoscopic sinus surgery in the cohort as a whole. Mean blood eosinophil counts and percentages were also reduced after surgery in the subcohorts of CRSwNP patients with (i) asthma, (ii) aspirin-exacerbated respiratory disease (AERD), and (iii) no allergy. Although blood eosinophil and basophil counts correlated directly before and after surgery, a statistical reduction in blood basophil counts and percentages after surgery emerged only in the subcohort of nonallergic CRSwNP patients. Conclusion Endoscopic sinus surgery can clear polyps, remove inflammatory tissue, and reduce inflammatory cytokine levels. Consistently with the biological mechanism described, endoscopic sinus surgery could coincide with a reduction in blood eosinophils in eosinophilic-type CRSwNP.


Assuntos
Basófilos/citologia , Eosinofilia/sangue , Eosinófilos/citologia , Pólipos Nasais/sangue , Rinite/sangue , Sinusite/sangue , Adulto , Endoscopia , Eosinofilia/cirurgia , Feminino , Hospitais Universitários , Humanos , Contagem de Leucócitos , Masculino , Pessoa de Meia-Idade , Pólipos Nasais/cirurgia , Período Pós-Operatório , Estudos Retrospectivos , Rinite/cirurgia , Sinusite/cirurgia
13.
Nature ; 555(7694): 54-60, 2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29466336

RESUMO

The formation of red blood cells begins with the differentiation of multipotent haematopoietic progenitors. Reconstructing the steps of this differentiation represents a general challenge in stem-cell biology. Here we used single-cell transcriptomics, fate assays and a theory that allows the prediction of cell fates from population snapshots to demonstrate that mouse haematopoietic progenitors differentiate through a continuous, hierarchical structure into seven blood lineages. We uncovered coupling between the erythroid and the basophil or mast cell fates, a global haematopoietic response to erythroid stress and novel growth factor receptors that regulate erythropoiesis. We defined a flow cytometry sorting strategy to purify early stages of erythroid differentiation, completely isolating classically defined burst-forming and colony-forming progenitors. We also found that the cell cycle is progressively remodelled during erythroid development and during a sharp transcriptional switch that ends the colony-forming progenitor stage and activates terminal differentiation. Our work showcases the utility of linking transcriptomic data to predictive fate models, and provides insights into lineage development in vivo.


Assuntos
Eritrócitos/citologia , Células Precursoras Eritroides/citologia , Eritropoese , Animais , Basófilos/citologia , Ciclo Celular/genética , Ciclo Celular/fisiologia , Linhagem da Célula/efeitos dos fármacos , Linhagem da Célula/genética , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Células Precursoras Eritroides/efeitos dos fármacos , Células Precursoras Eritroides/metabolismo , Eritropoese/efeitos dos fármacos , Feminino , Citometria de Fluxo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Mastócitos/citologia , Camundongos , Proteínas Proto-Oncogênicas c-kit/metabolismo , RNA Citoplasmático Pequeno/análise , RNA Citoplasmático Pequeno/genética , Análise de Célula Única , Transcriptoma
14.
Immunol Rev ; 282(1): 47-57, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29431208

RESUMO

Since their establishment in 1981, RBL-2H3 cells have been widely used as a mast cell (MC) model. Their ability to be easily grown in culture in large amounts, their responsiveness to FcεRI-mediated triggers and the fact that they can be genetically manipulated, have provided advantages over primary MCs, in particular for molecular studies relying on genetic screening. Furthermore, the ability to generate clones that stably express proteins of interest, for example, a human receptor, have marked the RBL cells as an attractive MC model for drug screening. Indeed, 3 RBL reporter cell lines (RS-ATL8, NFAT-DsRed, and NPY-mRFP) have been generated providing useful models for drug and allergen screening. Similarly, RBL cells stably expressing the human MrgprX2 receptor provide a unique paradigm for analyzing ligand interactions and signaling pathways of the unique human receptor. Finally, transient co-transfections of RBL cells allow functional genomic analyses of MC secretion by combining library screening with simultaneous expression of a reporter for exocytosis. RBL cells thus comprise powerful tools for the study of intracellular membrane trafficking and exocytosis and the detection of allergens, vaccine safety studies and diagnosis of allergic sensitization. Their recent uses as an investigative tool are reviewed here.


Assuntos
Basófilos/fisiologia , Hipersensibilidade/diagnóstico , Mastócitos/fisiologia , Alérgenos/imunologia , Animais , Basófilos/citologia , Degranulação Celular , Linhagem Celular , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Mastócitos/citologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Ratos , Receptores Acoplados a Proteínas-G/genética , Receptores Acoplados a Proteínas-G/metabolismo , Receptores de IgE/metabolismo , Receptores de Neuropeptídeos/genética , Receptores de Neuropeptídeos/metabolismo , Transdução de Sinais
15.
Eur J Immunol ; 48(5): 861-873, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29315532

RESUMO

Basophils have been recently recognized to play important roles in type 2 immune responses during allergies and parasitic infection, largely due to the development of novel tools for the in vivo study of these cells. As such, the genetically-engineered MCPT8DTR mouse line has been used to specifically deplete basophils following treatment with diphtheria toxin (DT). In this study, we showed that DT-injected MCPT8DTR mice exhibited a striking decrease of eosinophils and neutrophils in skin when subjected to a hapten fluorescein isothiocyanate (FITC)-induced allergic contact dermatitis (ACD) experimental protocol. Unexpectedly, we found that loss of skin eosinophils and neutrophils was not due to a lack of basophil-mediated recruitment, as DT injection caused a systemic reduction of eosinophils and neutrophils in MCPT8DTR mice in a time-dependent manner. Furthermore, we found that hematopoietic stem-cell-derived granulocyte-macrophage progenitors (GMPs) expressed MCPT8 gene, and that these cells were depleted upon DT injection. Finally, we optimized a protocol in which a low-dose DT achieved a better specificity for depleting basophils, but not GMPs, in MCPT8DTR mice, and demonstrate that basophils do not play a major role in recruiting eosinophils and neutrophils to ACD skin. These data provide new and valuable information about functional studies of basophils.


Assuntos
Basófilos/imunologia , Dermatite Alérgica de Contato/imunologia , Toxina Diftérica/toxicidade , Eosinófilos/imunologia , Células Progenitoras de Granulócitos e Macrófagos/citologia , Neutrófilos/imunologia , Triptases/metabolismo , Animais , Basófilos/citologia , Eosinófilos/citologia , Feminino , Células Progenitoras de Granulócitos e Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Neutrófilos/citologia , Triptases/genética
16.
Cell Death Differ ; 25(1): 204-216, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28960207

RESUMO

Basophil granulocytes and mast cells are recognized for their roles in immunity and are central effectors of diverse immunological disorders. Despite their similarities, there is emerging evidence for non-redundant roles of the circulating yet scarce basophils and tissue-resident mast cells, respectively. Because of their importance in allergic pathogenesis, specific induction of apoptosis in basophils and mast cells may represent an interesting novel treatment strategy. The pro-inflammatory cytokine interleukin-3 serves as a key factor for basophil and mouse mast cell survival. Interleukin-3 increases the expression of anti-apoptotic BCL-2 family members, such as BCL-2, BCL-XL or MCL-1; however, little is known how strongly these individual proteins contribute to basophil survival. Here, we were applying small molecule inhibitors called BH3 mimetics, some of which show remarkable success in cancer treatments, to neutralize the function of anti-apoptotic BCL-2 family members. We observed that expression levels of anti-apoptotic BCL-2 proteins do not necessarily correlate with their respective importance for basophil survival. Whereas naive in vitro-differentiated mouse basophils efficiently died upon BCL-2 or BCL-XL inhibition, interleukin-3 priming rendered the cells highly resistant toward apoptosis, and this could only be overcome upon combined targeting of BCL-2 and BCL-XL. Of note, human basophils differed from mouse basophils as they depended on BCL-2 and MCL-1, but not on BCL-XL, for their survival at steady state. On the other hand, and in contrast to mouse basophils, MCL-1 proved critical in mediating survival of interleukin-3 stimulated mouse mast cells, whereas BCL-XL seemed dispensable. Taken together, our results indicate that by choosing the right combination of BH3 mimetic compounds, basophils and mast cells can be efficiently killed, even after stimulation with potent pro-survival cytokines such as interleukin-3. Because of the tolerable side effects of BH3 mimetics, targeting basophils or mast cells for apoptosis opens interesting possibilities for novel treatment approaches.


Assuntos
Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Apoptose , Basófilos/metabolismo , Mastócitos/metabolismo , Compostos de Anilina/farmacologia , Animais , Basófilos/citologia , Basófilos/efeitos dos fármacos , Basófilos/enzimologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Caspase 3/metabolismo , Sobrevivência Celular , Humanos , Interleucina-3/antagonistas & inibidores , Interleucina-3/farmacologia , Mastócitos/citologia , Mastócitos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Proteína de Sequência 1 de Leucemia de Células Mieloides/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Sulfonamidas/farmacologia , Proteína bcl-X/antagonistas & inibidores
17.
PLoS One ; 12(9): e0185509, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28957409

RESUMO

Immature B cells are the first B cell progenitors to express a fully formed B cell receptor and are therefore subject to extensive selection processes that act to mitigate the emergence of autoreactive clones. While it is well appreciated that most B cell generation in the bone marrow is highly dependent on access to molecules present in the local milieu, the existence of extrinsically provided factors that modulate immature B cell biology is ambiguous. Nonetheless, a population of CD49b+CD90lo cells has demonstrated in vitro potential to promote immature B cell survival. Using a mouse basophil reporter strain we confirmed the identity of these CD49b+CD90lo supportive cells as basophils. However, analysis of bone marrow B cell populations following lineage specific basophil depletion demonstrates that basophils do not have a significant role in vivo in modulating immature B cell biology during steady-state conditions.


Assuntos
Basófilos/citologia , Células da Medula Óssea/citologia , Células Precursoras de Linfócitos B/citologia , Animais , Basófilos/metabolismo , Células da Medula Óssea/metabolismo , Linhagem da Célula , Sobrevivência Celular , Técnicas de Cocultura , Citoproteção , Feminino , Proteínas de Homeodomínio/metabolismo , Cadeias lambda de Imunoglobulina/metabolismo , Integrina alfa2/metabolismo , Contagem de Linfócitos , Camundongos , Células Precursoras de Linfócitos B/metabolismo , Antígenos Thy-1/metabolismo
18.
Anat Histol Embryol ; 46(5): 446-455, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28762256

RESUMO

The increasing prevalence of yellow-bellied sliders (Trachemys scripta scripta) as pets in the European Union and also its utilization as animal models for experimental purposes makes crucial an accurate classification of their blood cells. The aim of this work was to provide a morphologic classification based on the cytochemical characteristics of the blood cells of 15 yellow-bellied sliders. Cytochemical stains included benzidine peroxidase, chloroacetate esterase, alpha-naphthyl butyrate esterase (with and without sodium fluoride), acid phosphatase (with and without tartaric acid), Sudan black B, periodic acid-Schiff and toluidine blue. Nuclear and cellular dimensions were also measured based on quick Romanowsky-type stained smears. Besides erythrocytes and thrombocytes, five types of white blood cells were identified: heterophils, eosinophils, basophils, lymphocytes and monocytes. The cytochemical patterns of heterophils, eosinophils and basophils were unique compared to those described for other chelonians. This paper provides a useful guideline for clinical settings and further haematological studies of this species.


Assuntos
Células Sanguíneas/química , Células Sanguíneas/citologia , Tartarugas/sangue , Animais , Basófilos/química , Basófilos/citologia , Plaquetas/química , Plaquetas/citologia , Eosinófilos/química , Eosinófilos/citologia , Eritrócitos/química , Eritrócitos/citologia , Feminino , Histocitoquímica/veterinária , Linfócitos/química , Linfócitos/citologia , Masculino , Monócitos/química , Monócitos/citologia , Caracteres Sexuais
19.
Transfusion ; 57(9): 2084-2095, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28656655

RESUMO

BACKGROUND: In previous studies, we demonstrated that the basophil activation test, which is performed using patient blood and the supernatants from transfused blood components, was able to elucidate not only the causative relationship between allergic transfusion reactions and the transfusion but also the mechanisms behind allergic transfusion reactions. However, for a large number of allergic transfusion reactions, patients are in a state of myelosuppression, and the basophil activation test cannot be performed for these patients because there are insufficient numbers of peripheral blood basophils. STUDY DESIGN AND METHODS: To overcome this obstacle, we developed a passive immune basophil activation test, in which patient plasma and residually transfused blood are used as the patient's sources of immunoglobulin E and allergen, respectively, whereas healthy volunteer basophils serve as the responder cell source. The passive immune basophil activation test was performed for two patients who had severe allergic transfusion reactions, using supernatants of the residual platelet concentrates and the patients' own immunoglobulin E. RESULTS: There were no differences in either surface immunoglobulin E or activation in response to allergens between untreated basophils and so-called quasi-basophils, in which immunoglobulin E was replaced by a third party's immunoglobulin E. In these patients, the supernatants of the residual platelet concentrates exclusively activated basophils in response to quasi-basophils onto which the patients' immunoglobulin E, but not a third party's immunoglobulin E, was bound. CONCLUSION: The passive immune basophil activation test may help clarify the causal relationship between allergic transfusion reactions and transfused blood, even when patients experience myelosuppression.


Assuntos
Basófilos/imunologia , Plaquetas/imunologia , Hipersensibilidade Imediata/prevenção & controle , Reação Transfusional , Reação Transfusional/imunologia , Alérgenos/sangue , Basófilos/citologia , Humanos , Hipersensibilidade Imediata/imunologia , Imunoglobulina E , Reação Transfusional/etiologia
20.
Cytometry A ; 91(7): 686-693, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28505391

RESUMO

The soluble form of the transmembrane glycoprotein, FcεRIα which corresponds to the high-affinity receptor for IgE, is found in serum. Growing evidence suggests the pathogenic role of IgE and FcεRI in systemic lupus erythematosus (SLE). The goal of this study is to develop a sensitive and standardized cytometric assay for quantification of sFcεRIα. A membrane emulsification technique was utilized to incorporate CuInS2 /ZnS quantum dots and Fe3 O4 nanoparticles into poly (styrene-co-maleic anhydride) microbeads. The beads were then carboxylated and coated with capture antibody monoclonal anti-human FcεRIα. This antibody binds to FcεRIα but does not block the binding of FcεRIα to IgE. After incubation with standards or serum samples, the microbeads were incubated with excessive native human IgE, followed by incubation with Phycoerythrin (PE) conjugated anti-human IgE. The resulting quantum dot microbeads were gated, and sFcεRIα quantification was analyzed based on PE fluorescence intensity. The method exhibited good linearity (R2 > 0.99), and the limit of detection was established at 0.29 ng/mL with the dynamic range of up to 200 ng/mL. The precision of the assay validated by intra- and inter-assay variability met the acceptance criteria with the mean recovery falling within 80-110% of the theoretical concentration and a corresponding CV < 20%. We tested 149 serum samples which 89 were from SLE patients and 60 were from healthy volunteers. For the first time, we detected an increased sFcεRIα level in the serum of SLE patients, which was confirmed by a commercial ELISA kit. Compared to ELISA, this novel method is more sensitive and efficient. It allows for the simple comparative analysis of sFcεRIα levels in health and disease. © 2017 International Society for Advancement of Cytometry.


Assuntos
Basófilos/citologia , Pontos Quânticos/análise , Receptores de IgE/sangue , Basófilos/imunologia , Citometria de Fluxo/métodos , Humanos , Hipersensibilidade , Imunoglobulina E/sangue , Microesferas
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