Background: The Human Cell Differentiation Molecules (HCDM) organizes Human Leukocyte Differentiation Antigen (HLDA) workshops to test and name clusters of antibodies that react with a specific antigen. These cluster of differentiation (CD) markers have provided the scientific community with validated antibody clones, consistent naming of targets and reproducible identification of leukocyte subsets. Still, quantitative CD marker expression profiles and benchmarking of reagents at the single-cell level are currently lacking. Objective: To develop a flow cytometric procedure for quantitative expression profiling of surface antigens on blood leukocyte subsets that is standardized across multiple research laboratories. Methods: A high content framework to evaluate the titration and reactivity of Phycoerythrin (PE)-conjugated monoclonal antibodies (mAbs) was created. Two flow cytometry panels were designed: an innate cell tube for granulocytes, dendritic cells, monocytes, NK cells and innate lymphoid cells (12-color) and an adaptive lymphocyte tube for naive and memory B and T cells, including TCRγδ+, regulatory-T and follicular helper T cells (11-color). The potential of these 2 panels was demonstrated via expression profiling of selected CD markers detected by PE-conjugated antibodies and evaluated using 561 nm excitation. Results: Using automated data annotation and dried backbone reagents, we reached a robust workflow amenable to processing hundreds of measurements in each experiment in a 96-well plate format. The immunophenotyping panels enabled discrimination of 27 leukocyte subsets and quantitative detection of the expression of PE-conjugated CD markers of interest that could quantify protein expression above 400 units of antibody binding capacity. Expression profiling of 4 selected CD markers (CD11b, CD31, CD38, CD40) showed high reproducibility across centers, as well as the capacity to benchmark unique clones directed toward the same CD3 antigen. Conclusion: We optimized a procedure for quantitative expression profiling of surface antigens on blood leukocyte subsets. The workflow, bioinformatics pipeline and optimized flow panels enable the following: 1) mapping the expression patterns of HLDA-approved mAb clones to CD markers; 2) benchmarking new antibody clones to established CD markers; 3) defining new clusters of differentiation in future HLDA workshops.

• MeSH
• antigeny povrchové * metabolismus   MeSH
• buňky NK metabolismus   MeSH
• CD antigeny metabolismus   MeSH
• leukocyty MeSH
• lidé MeSH
• monoklonální protilátky MeSH
• přirozená imunita * MeSH
• průběh práce MeSH
• průtoková cytometrie metody   MeSH
• referenční standardy MeSH
• reprodukovatelnost výsledků MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Fusion of the ZNF384 gene as the 3' partner to several different 5' partner genes occurs recurrently in B-cell precursor acute lymphoblastic and mixed phenotype B/myeloid leukemia. These canonical fusions (ZNF384r) contain the complete ZNF384 coding sequence and are associated with a specific gene expression signature. Cases with this signature, but without canonical ZNF384 fusions (ZNF384r-like cases), have been described previously. Although some have been shown to harbor ZNF362 fusions, the primary aberrations remain unknown in a major proportion. We studied 3 patients with the ZNF384r signature and unknown primary genetic background and identified a previously unknown class of genetic aberration affecting the last exon of ZNF384 and resulting in disruption of the C-terminal portion of the ZNF384 protein. Importantly, in 2 cases, the ZNF384 aberration, indel, was missed during the bioinformatic analysis but revealed by the manual, targeted reanalysis. Two cases with the novel aberrations had a mixed (B/myeloid) immunophenotype commonly associated with canonical ZNF384 fusions. In conclusion, we present leukemia cases with a novel class of ZNF384 aberrations that phenocopy leukemia with ZNF384r. Therefore, we show that part of the so-called ZNF384r-like cases represent the same genetic subtype as leukemia with canonical ZNF384 fusions.

• MeSH
• akutní myeloidní leukemie * genetika   MeSH
• imunofenotypizace MeSH
• lidé MeSH
• trans-aktivátory * genetika   MeSH
• transkripční faktory MeSH
• transkriptom MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

We collected a multi-centric retrospective dataset of patients (N = 213) who were admitted to ten hospitals in Czech Republic and tested positive for SARS-CoV-2 during the early phases of the pandemic in March-October 2020. The dataset contains baseline patient characteristics, breathing support required, pharmacological treatment received and multiple markers on daily resolution. Patients in the dataset were treated with hydroxychloroquine (N = 108), azithromycin (N = 72), favipiravir (N = 9), convalescent plasma (N = 7), dexamethasone (N = 4) and remdesivir (N = 3), often in combination. To explore association between treatments and patient outcomes we performed multiverse analysis, observing how the conclusions change between defensible choices of statistical model, predictors included in the model and other analytical degrees of freedom. Weak evidence to constrain the potential efficacy of azithromycin and favipiravir can be extracted from the data. Additionally, we performed external validation of several proposed prognostic models for Covid-19 severity showing that they mostly perform unsatisfactorily on our dataset.

• MeSH
• COVID-19 farmakoterapie   epidemiologie   patologie   terapie   MeSH
• dospělí MeSH
• hospitalizace * MeSH
• lidé středního věku MeSH
• lidé MeSH
• progrese nemoci * MeSH
• senioři MeSH
• statistické modely MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Česká republika MeSH

Recently, we described B-cell precursor acute lymphoblastic leukemia (BCP-ALL) subtype with early switch to the monocytic lineage and loss of the B-cell immunophenotype, including CD19 expression. Thus far, the genetic background has remained unknown. Among 726 children consecutively diagnosed with BCP-ALL, 8% patients experienced switch detectable by flow cytometry (FC). Using exome and RNA sequencing, switch was found to positively correlate with three different genetic subtypes: PAX5-P80R mutation (5 cases with switch out of 5), rearranged DUX4 (DUX4r; 30 cases of 41) and rearranged ZNF384 (ZNF384r; 4 cases of 10). Expression profiles or phenotypic patterns correlated with genotypes, but within each genotype they could not identify cases who subsequently switched. If switching was not taken into account, the B-cell-oriented FC assessment underestimated the minimal residual disease level. For patients with PAX5-P80R, a discordance between FC-determined and PCR-determined MRD was found on day 15, resulting from a rapid loss of the B-cell phenotype. Discordance on day 33 was observed in all the DUX4r, PAX5-P80R and ZNF384r subtypes. Importantly, despite the substantial phenotypic changes, possibly even challenging the appropriateness of BCP-ALL therapy, the monocytic switch was not associated with a higher incidence of relapse and poorer prognosis in patients undergoing standard ALL treatment.

• MeSH
• aktivátorový protein specifický pro B-buňky genetika   MeSH
• akutní lymfatická leukemie * MeSH
• B-lymfocyty MeSH
• imunofenotypizace MeSH
• lidé MeSH
• mutace MeSH
• pre-B-buněčná leukemie * diagnóza   genetika   MeSH
• reziduální nádor MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The nuclear pore complex (NPC) has emerged as a hub for the transcriptional regulation of a subset of genes, and this type of regulation plays an important role during differentiation. Nucleoporin TPR forms the nuclear basket of the NPC and is crucial for the enrichment of open chromatin around NPCs. TPR has been implicated in the regulation of transcription; however, the role of TPR in gene expression and cell differentiation has not been described. Here we show that depletion of TPR results in an aberrant morphology of murine proliferating C2C12 myoblasts (MBs) and differentiated C2C12 myotubes (MTs). The ChIP-Seq data revealed that TPR binds to genes linked to muscle formation and function, such as myosin heavy chain (Myh4), myocyte enhancer factor 2C (Mef2C) and a majority of olfactory receptor (Olfr) genes. We further show that TPR, possibly via lysine-specific demethylase 1 (LSD1), promotes the expression of Myh4 and Olfr376, but not Mef2C. This provides a novel insight into the mechanism of myogenesis; however, more evidence is needed to fully elucidate the mechanism by which TPR affects specific myogenic genes.

• MeSH
• buněčná diferenciace MeSH
• buněčné linie MeSH
• exprese genu MeSH
• komplex proteinů jaderného póru metabolismus   MeSH
• kosterní svalová vlákna * cytologie   metabolismus   MeSH
• myoblasty kosterní * cytologie   metabolismus   MeSH
• myši MeSH
• protoonkogenní proteiny metabolismus   MeSH
• regulace genové exprese MeSH
• těžké řetězce myosinu metabolismus   MeSH
• vývoj svalů MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

OBJECTIVE:  Blood monocyte subsets are emerging as biomarkers of cardiovascular inflammation. However, our understanding of human monocyte heterogeneity and their immunophenotypic features under healthy and inflammatory conditions is still evolving. RATIONALE:  In this study, we sought to investigate the immunophenome of circulating human monocyte subsets. METHODS:  Multiplexed, high-throughput flow cytometry screening arrays and computational data analysis were used to analyze the expression and hierarchical relationships of 242 specific surface markers on circulating classical (CD14++CD16-), intermediate (CD14++CD16+), and nonclassical (CD14+CD16++) monocytes in healthy adults. RESULTS:  Using generalized linear models and hierarchical cluster analysis, we selected and clustered epitopes that most reliably differentiate between monocyte subsets. We validated existing transcriptional profiling data and revealed potential new surface markers that uniquely define the classical (e.g., BLTR1, CD35, CD38, CD49e, CD89, CD96), intermediate (e.g., CD39, CD275, CD305, CDw328), and nonclassical (e.g., CD29, CD132) subsets. In addition, our analysis revealed phenotypic cell clusters, identified by dendritic markers CMRF-44 and CMRF-56, independent of the traditional monocyte classification. CONCLUSION:  These results reveal an advancement of the clinically applicable multiplexed screening arrays that may facilitate monocyte subset characterization and cytometry-based biomarker selection in various inflammatory disorders.

• MeSH
• antigeny CD14 metabolismus   MeSH
• ateroskleróza diagnóza   imunologie   MeSH
• biodiverzita MeSH
• biologické markery metabolismus   MeSH
• fenotyp MeSH
• imunofenotypizace metody   MeSH
• krevní oběh MeSH
• lidé MeSH
• monocyty fyziologie   MeSH
• průtoková cytometrie MeSH
• receptory IgG metabolismus   MeSH
• rychlé screeningové testy MeSH
• separace buněk MeSH
• shluková analýza MeSH
• zánět diagnóza   imunologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

BACKGROUND: The immunological microenvironment of primary high-grade serous carcinomas (HGSCs) has a major impact on disease outcome. Conversely, little is known on the microenvironment of metastatic HGSCs and its potential influence on patient survival. Here, we explore the clinical relevance of the immunological configuration of HGSC metastases. METHODS: RNA sequencing was employed on 24 paired primary tumor microenvironment (P-TME) and metastatic tumor microenvironment (M-TME) chemotherapy-naive HGSC samples. Immunohistochemistry was used to evaluate infiltration by CD8+ T cells, CD20+ B cells, DC-LAMP+ (lysosomal-associated membrane protein 3) dendritic cells (DCs), NKp46+ (natural killer) cells and CD68+CD163+ M2-like tumor-associated macrophages (TAMs), abundance of PD-1+ (programmed cell death 1), LAG-3+ (lymphocyte-activating gene 3) cells, and PD-L1 (programmed death ligand 1) expression in 80 samples. Flow cytometry was used for functional assessments on freshly resected HGSC samples. RESULTS: 1468 genes were differentially expressed in the P-TME versus M-TME of HGSCs, the latter displaying signatures of extracellular matrix remodeling and immune infiltration. M-TME infiltration by immune effector cells had little impact on patient survival. Accordingly, M-TME-infiltrating T cells were functionally impaired, but not upon checkpoint activation. Conversely, cytokine signaling in favor of M2-like TAMs activity appeared to underlie inhibited immunity in the M-TME and poor disease outcome. CONCLUSIONS: Immunosuppressive M2-like TAM infiltrating metastatic sites limit clinically relevant immune responses against HGSCs.

• MeSH
• imunosupresivní léčba metody   MeSH
• lidé středního věku MeSH
• lidé MeSH
• makrofágy imunologie   MeSH
• metastázy nádorů MeSH
• nádorové biomarkery metabolismus   MeSH
• nádorové mikroprostředí MeSH
• nádory vaječníků imunologie   MeSH
• retrospektivní studie MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH

• MeSH
• biologické markery krev   MeSH
• buňky Th17 imunologie   metabolismus   MeSH
• chronická nemoc MeSH
• fenotyp MeSH
• fibróza MeSH
• imunofenotypizace MeSH
• interleukin-17 krev   MeSH
• interleukiny krev   MeSH
• lidé středního věku MeSH
• lidé MeSH
• magnetická rezonanční tomografie * MeSH
• myokard patologie   MeSH
• nemoci koronárních tepen krev   diagnostické zobrazování   imunologie   patologie   MeSH
• prediktivní hodnota testů MeSH
• prospektivní studie MeSH
• průtoková cytometrie MeSH
• remodelace komor * MeSH
• senioři MeSH
• studie případů a kontrol MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• dopisy MeSH
• práce podpořená grantem MeSH

SUMMARY: ShinySOM offers a user-friendly interface for reproducible, high-throughput analysis of high-dimensional flow and mass cytometry data guided by self-organizing maps. The software implements a FlowSOM-style workflow, with improvements in performance, visualizations and data dissection possibilities. The outputs of the analysis include precise statistical information about the dissected samples, and R-compatible metadata useful for the batch processing of large sample volumes. AVAILABILITY AND IMPLEMENTATION: ShinySOM is free and open-source, available online at gitlab.com/exaexa/ShinySOM. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

• MeSH
• algoritmy * MeSH
• metadata MeSH
• průběh práce MeSH
• software * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Hematopoiesis in mammalian embryos proceeds through three successive waves of hematopoietic progenitors. Since their emergence spatially and temporally overlap and phenotypic markers are often shared, the specifics regarding their origin, development, lineage restriction and mutual relationships have not been fully determined. The identification of wave-specific markers would aid to resolve these uncertainties. Here, we show that toll-like receptors (TLRs) are expressed during early mouse embryogenesis. We provide phenotypic and functional evidence that the expression of TLR2 on E7.5 c-kit+ cells marks the emergence of precursors of erythro-myeloid progenitors (EMPs) and provides resolution for separate tracking of EMPs from primitive progenitors. Using in vivo fate mapping, we show that at E8.5 the Tlr2 locus is already active in emerging EMPs and in progenitors of adult hematopoietic stem cells (HSC). Together, this data demonstrates that the activation of the Tlr2 locus tracks the earliest events in the process of EMP and HSC specification.

• MeSH
• dospělé kmenové buňky metabolismus   MeSH
• hematopoetické kmenové buňky metabolismus   MeSH
• hematopoéza MeSH
• myši inbrední C57BL MeSH
• myši embryologie   genetika   metabolismus   MeSH
• protoonkogenní proteiny c-kit genetika   metabolismus   MeSH
• toll-like receptor 2 genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši embryologie   genetika   metabolismus   MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH