OBJECTIVE: The aim of this study was to evaluate changes in the relative counts of different leukocyte subsets in peripheral and umbilical cord blood in pregnancies complicated by preterm prelabor rupture of membranes (PPROM) with respect to the presence of intraamniotic inflammation (IAI) and fetal inflammatory response syndrome (FIRS). METHODS: Fifty-two women with singleton pregnancies complicated by PPROM were included in this study. From samples of peripheral and umbilical cord blood, relative counts of these leukocyte subpopulations were determined using multicolor flow cytometry: granulocytes, monocytes, lymphocytes, T cells and their subpopulations, B cells and their subpopulations, and NK cells and their subpopulations. IAI was defined as increased concentrations of interleukin 6 in the amniotic fluid. Amniotic fluid samples were obtained by transabdominal amniocentesis. RESULTS: Women with IAI had higher relative counts of monocytes (p = 0.04) in peripheral blood. There was an increased relative number of granulocytes (p = 0.003) and a decreased number of lymphocytes (p = 0.0048), helper CD4+ T cells (p = 0.019), NK cells (p = 0.0001) within leukocytes, NK cells within lymphocytes (p = 0.003) and CD16+ NK cells within NK cells (p = 0.005) in umbilical cord blood samples of women with FIRS. However, after adjusting the results for gestational age at sampling, all differences disappeared. CONCLUSIONS: The presence of IAI or FIRS is not accompanied by significant changes in the relative counts of immune cells in peripheral blood or umbilical cord blood in pregnancies complicated by PPROM.
- MeSH
- Chorioamnionitis immunology blood MeSH
- Adult MeSH
- Fetal Blood * immunology cytology MeSH
- Interleukin-6 blood metabolism MeSH
- Leukocytes immunology MeSH
- Humans MeSH
- Amniotic Fluid immunology metabolism MeSH
- Leukocyte Count MeSH
- Fetal Membranes, Premature Rupture * immunology blood MeSH
- Flow Cytometry MeSH
- Systemic Inflammatory Response Syndrome immunology blood MeSH
- Pregnancy MeSH
- Inflammation immunology MeSH
- Check Tag
- Adult MeSH
- Humans MeSH
- Pregnancy MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
Multicolor flow cytometry allows for analysis of tens of cellular parameters in millions of cells at a single-cell resolution within minutes. The lack of technologies that would facilitate feasible and relatively cheap profiling of such a number of cells with an antibody-based approach led us to the development of a high-throughput cytometry-based platform for surface profiling. We coupled the fluorescent cell barcoding with preexisting, commercially available screening tools to analyze cell surface fingerprint at a large scale. This powerful approach will help to identify novel biomarkers and druggable targets and facilitate the discovery of new concepts in immunology, oncology, and developmental biology.
With the development of light microscopy, it is becoming increasingly easy to obtain detailed multicolor fluorescence volumetric data. The need for their appropriate visualization has become an integral part of fluorescence imaging. Virtual reality (VR) technology provides a new way of visualizing multidimensional image data or models so that the entire 3D structure can be intuitively observed, together with different object features or details on or within the object. With the need for imaging advanced volumetric data, demands for the control of virtual object properties are increasing; this happens especially for multicolor objects obtained by fluorescent microscopy. Existing solutions with universal VR controllers or software-based controllers with the need to define sufficient space for the user to manipulate data in VR are not usable in many practical applications. Therefore, we developed a custom gesture-based VR control system with a custom controller connected to the FluoRender visualization environment. A multitouch sensor disk was used for this purpose. Our control system may be a good choice for easier and more comfortable manipulation of virtual objects and their properties, especially using confocal microscopy, which is the most widely used technique for acquiring volumetric fluorescence data so far.
- MeSH
- Gestures * MeSH
- Microscopy, Confocal MeSH
- Software MeSH
- Virtual Reality * MeSH
- Publication type
- Journal Article MeSH
Background: Chronic hepatitis C (CHC) is associated with altered cell-mediated immune response. Objective: The aim of the study was to characterize functional alterations in CD4+ T cell subsets and myeloid-derived suppressor cells (MDSCs) during chronic hepatitis C virus (HCV) infection. Methodology. The expression levels of the lineage-defining transcriptional factors (TFs) T-bet, Gata3, Rorγt, and Foxp3 in circulating CD4+ T cells and percentages of MDSCs in peripheral blood were evaluated in 33 patients with CHC, 31 persons, who had spontaneously cleared the HCV infection, and 30 healthy subjects. Analysis. The CD4+ T cells TFs T-bet (T-box expressed in T cells), Foxp3 (Forkhead box P3 transcription factor), Gata3 (Gata-binding protein 3), and Rorγt (retinoic-acid-related orphan receptor gamma) and activation of CD8+ T cells, as well as percentages of MDSCs, were measured by multicolor flow cytometry after intracellular and surface staining of peripheral blood mononuclear cells with fluorescent monoclonal antibodies. Result: The patients with CHC had significantly lower percentages of CD4+ T cells expressing Rorγt and Gata3 and higher percentages of Foxp3-expressing CD4+ T cells than healthy controls and persons who spontaneously cleared HCV infection. The ratios of T-bet+/Gata3+ and Foxp3+/Rorγt+ CD4+ T cells were the highest in the patients with CHC. In the patients with CHC, the percentages of Gata3+ and Rorγt+ CD4+ T cells and the percentages of T-bet+ CD4+ T cells and CD38+/HLA-DR+ CD8+ T cells demonstrated significant positive correlations. In addition, the percentage of CD38+/HLA-DR+ CD8+ T cells correlated negatively with the percentage of MDSCs. Conclusion: Chronic HCV infection is associated with downregulation of TFs Gata3 and Rorγt polarizing CD4+ T cells into Th2 and Th17 phenotypes together with upregulation of Foxp3 responsible for induction of regulatory T cells suppressing immune response.
- Publication type
- Journal Article MeSH
There is an agreement in the field that interlaboratory reproducibility of flow cytometry measurements as well as the whole studies might be improved by a consensual use of methodological approach. Typically, a consensus is made on a crucial markers needed in the immunostaining panel, sometimes on the particular fluorochrome conjugates and rarely on a complete set of methods for sample preparation. The term "standardization" is used to describe the complete set of methodical steps, while "harmonization" is used for partial agreement on the method. Standardization can provide a platform for improved reproducibility of cytometry results over prolonged periods of time, across different sites and across different instruments. For the purpose of structured discussion, several desired aims are described: common interpretation of the immunophenotype definition of a target subset, accurate quantification, reproducible pattern of a multicolor immunophenotype, and reproducible intensity of all measured parameters. An overview of how standardization was approached by several large consortia is provided: EuroFlow, The ONE Study, Human Immunology Project Consortium (HIPC), and several other groups. Their particular aims and the tools adopted to reach those aims are noted. How those standardization efforts were adopted in the field and how the resulting outcome was evaluated is reviewed. Multiple challenges in the instrument hardware design, instrument setup tools, reagent design, and quality features need to be addressed to achieve optimal standardization. Furthermore, the aims of different studies vary, and thus, the reasonable requirements for standardization differ. A framework of reference for the reasonable outcomes of different approaches is offered. Finally, it is argued that complete standardization is important not only for the reproducibility of measurements but also for education, for quality assessment and for algorithmic data analysis. The different standardized approaches can and in fact should serve as benchmarking reference tools for the development of future flow cytometry studies. © 2019 International Society for Advancement of Cytometry.
The role of the immune system as an integral component of the inflammatory response in the pathophysiology of migraine remains unclear. The aim of this study was to evaluate the differences in immune system parameters (acquired immunity parameters) in patients with episodic migraine (EM) and in healthy controls. In EM patients, we aimed to determine whether the changes found in peripheral blood parameters were related to migraine severity according to the standardised MIDAS and HIT-6 tests. Forty-nine patients with EM and 50 healthy controls were included in this study. The authors compared different lymphocyte parameters obtained by multicolor flow cytometry in the EM and control groups by performing statistical tests. The relationship between the changes in peripheral blood parameters and migraine severity in EM patients was investigated using correlation and regression analysis. EM patients showed higher values than healthy controls, especially in nine parameters: relative count of lymphocytes, relative and absolute counts of CD3 T cells, relative and absolute counts of CD8 suppressor cytotoxic T cells, relative and absolute counts of CD4 + TEMRA (terminally differentiated helper T lymphocytes), absolute count of CD8 naïve T cells, and absolute count of CD19 switched memory B cells. Among the lymphocyte parameters, CD4 + TEM (effector memory helper T lymphocytes) and CD8 + TEMRA (terminally differentiated cytotoxic T lymphocytes) were statistically significantly associated with HIT-6. Patients with a CD4 + TEM value below 15 had a high probability (90%) that the HIT-6 value would be higher than 60. The results of this study show that EM patients have changes in immune system parameters measured in the peripheral blood. Changes in the abundance of CD4 + TEM could be used as a biomarker for disease severity.
- MeSH
- Biomarkers MeSH
- Adult MeSH
- Immune System immunology metabolism MeSH
- Immunophenotyping MeSH
- Comorbidity MeSH
- Middle Aged MeSH
- Humans MeSH
- Migraine Disorders diagnosis etiology metabolism MeSH
- Adolescent MeSH
- Young Adult MeSH
- Disease Susceptibility * MeSH
- Flow Cytometry MeSH
- Aged MeSH
- Case-Control Studies MeSH
- Severity of Illness Index MeSH
- T-Lymphocyte Subsets immunology metabolism MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Adolescent MeSH
- Young Adult MeSH
- Male MeSH
- Aged MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
BACKGROUND: EGFP is a fluorescent tag extensively used in biological and biomedical research. Over the years many researches have gathered collections of cell lines bearing specific EGFP-tagged proteins. Despite its popularity some photochemical properties of EGFP remain undocumented and unused. We report on so far unexplored lifetime photoconversion of EGFP usable in FLIM. METHODS: Fluorescence lifetime imaging and spectral FLIM has been used for characterization of the EGFP photoconversion and protein tracking. RESULT: Our data suggest that EGFP can be permanently photoconverted to a short-fluorescence-lifetime form (PC-EGFP) by intense blue irradiation. PC-EGFP cannot be reverted back by 405 nm light and exhibits the same spectral emission properties with blue-shifted absorption compared to the unconverted EGFP. Fluorescence of PC-EGFP is pH-independent and the photoconversion efficiency decreases with the solvent viscosity. Utilization of the EGFP photoconversion was demonstrated by tracking of a nucleophosmin mutant in live HEK-293 T cells during its cytoplasm-nuclear relocalization induced by Leptomycin B. CONCLUSIONS: Besides potential FLIM artifacts caused by an unintended EGFP photoconversion, the controlled photoconversion turns EGFP to an excellent tool for kinetic FLIM applications. Since the photoconversion occurs in the lifetime domain, PC-EGFP can be easily distinguished from the unconverted tag by time-resolved detection while all other spectral channels stay free for multicolor labeling. GENERAL SIGNIFICANCE: The reported lifetime photoconversion lines up EGFP with other photoconvertible fluorescent proteins with special advantage for fluorescence lifetime imaging where lifetime-photoconvertible labels are scarce.
- MeSH
- Cell Adhesion MeSH
- Fluorescence MeSH
- Microscopy, Fluorescence methods MeSH
- Photochemistry methods MeSH
- HEK293 Cells MeSH
- HeLa Cells MeSH
- Nuclear Proteins chemistry MeSH
- Kinetics MeSH
- Hydrogen-Ion Concentration MeSH
- Humans MeSH
- Mutation MeSH
- Fatty Acids, Unsaturated chemistry MeSH
- Solvents chemistry MeSH
- Viscosity MeSH
- Green Fluorescent Proteins chemistry MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
... Beads to Visualize Antibody-Bound Antigens 775 -- Immunofluorescence-Based Imaging Techniques • Fluorescence ... ... Can Be Used to Visualize Cells and Molecules • Confocal Fluorescence Microscopy Provides Three-Dimensional ... ... Images of Extraordinary Clarity -- 775 -- 778 -- 779 -- Multiphoton Fluorescence Microscopy Is a Variation ... ... -- 781 -- Analysis of Chromatin Structure -- 793 • The Flow Cytometer Measures Scattered and Fluorescent ... ... Cell Sorters Have Important Clinical Applications • The Analysis of Multicolor Fluorescence Data Has ...
Eight edition různé stránkování : barevné ilustrace ; 28 cm
- MeSH
- Allergy and Immunology MeSH
- Immune System MeSH
- Publication type
- Textbook MeSH
- Conspectus
- Patologie. Klinická medicína
- Učební osnovy. Vyučovací předměty. Učebnice
- NML Fields
- alergologie a imunologie
The BMP signaling pathway has been shown to be involved in different aspects of embryonic development across diverse metazoan phyla. Comparative studies on the roles of the BMP signaling pathway provide crucial insights into the evolution of the animal body plans. In this chapter, we present the general workflow on how to investigate the roles of BMP signaling pathway during amphioxus embryonic development. As amphioxus are basal invertebrate chordates, studies on the BMP signaling pathway in amphioxus could elucidate the functional evolution of BMP pathway in the chordate group. Here, we describe methods for animal husbandry, spawning induction, and manipulation of the BMP signaling pathway during embryonic development through drug inhibitors and recombinant proteins. We also introduce an efficient method of using mesh baskets to handle amphioxus embryos for fluorescence immunostaining and multicolor fluorescence in situ hybridization and to assay the effects of manipulating BMP signaling pathway during amphioxus embryogenesis.
- MeSH
- Models, Biological * MeSH
- Embryo, Nonmammalian MeSH
- Embryonic Development MeSH
- In Situ Hybridization, Fluorescence MeSH
- Lancelets metabolism MeSH
- Bone Morphogenetic Proteins metabolism pharmacology MeSH
- Recombinant Proteins pharmacology MeSH
- Signal Transduction * drug effects MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
BACKGROUND/AIMS: Endothelial progenitor cells (EPCs) and circulating endothelial cells (CECs) have been described as markers of endothelial damage and dysfunction in several diseases, including deep venous thrombosis. Their role in patients with known thrombophilia has not yet been evaluated. Both EPCs and CECs represent extremely rare cell populations. Therefore, it is essential to use standardized methods for their identification and quantification. METHODS: In this study, we used multicolor flow cytometry to analyze the number of EPCs and CECs in patients with thrombophilia with or without a history of thrombosis. Patients with hematological malignancies after high-dose chemotherapy and patients with acute myocardial infarction were used as positive controls. RESULTS: EPC and CEC immunophenotypes were determined as CD45dim/-CD34+CD146+CD133+ and CD45dim/-CD34+CD146+CD133-, respectively. Increased levels of endothelial cells were observed in positive control groups. No significant changes in the number of EPCs or CECs were detected in patients with thrombophilia compared to healthy controls. CONCLUSION: Our optimized multicolor flow cytometry method allows unambiguous identification and quantification of endothelial cells in the peripheral blood. Our results support previous studies showing that elevated levels of CECs could serve as an indicator of endothelial injury or dysfunction. Normal levels of CECs or EPCs were found in patients with thrombophilia.
- MeSH
- Antigens, CD blood MeSH
- Adult MeSH
- Endothelial Cells * metabolism pathology MeSH
- Endothelial Progenitor Cells * metabolism pathology MeSH
- Middle Aged MeSH
- Humans MeSH
- Adolescent MeSH
- Flow Cytometry * MeSH
- Aged, 80 and over MeSH
- Aged MeSH
- Thrombophilia * blood pathology MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Adolescent MeSH
- Male MeSH
- Aged, 80 and over MeSH
- Aged MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
