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Mass cytometry defines distinct immune profile in germinal center B-cell lymphomas.
Roussel M, Lhomme F, Roe CE, Bartkowiak T, Gravelle P, Laurent C, Fest T, Irish JM
(2020) Cancer Immunol Immunother 69: 407-420
MeSH Terms: Adult, Aged, Female, Flow Cytometry, Germinal Center, Humans, Lymphoma, Large B-Cell, Diffuse, Macrophages, Male, Middle Aged, Young Adult
Show Abstract · Added January 14, 2020
Tumor-associated macrophage and T-cell subsets are implicated in the pathogenesis of diffuse large B-cell lymphoma, follicular lymphoma, and classical Hodgkin lymphoma. Macrophages provide essential mechanisms of tumor immune evasion through checkpoint ligand expression and secretion of suppressive cytokines. However, normal and tumor-associated macrophage phenotypes are less well characterized than those of tumor-infiltrating T-cell subsets, and it would be especially valuable to know whether the polarization state of macrophages differs across lymphoma tumor microenvironments. Here, an established mass cytometry panel designed to characterize myeloid-derived suppressor cells and known macrophage maturation and polarization states was applied to characterize B-lymphoma tumors and non-malignant human tissue. High-dimensional single-cell analyses were performed using dimensionality reduction and clustering tools. Phenotypically distinct intra-tumor macrophage subsets were identified based on abnormal marker expression profiles that were associated with lymphoma tumor types. While it had been proposed that measurement of CD163 and CD68 might be sufficient to reveal macrophage subsets in tumors, results here indicated that S100A9, CCR2, CD36, Slan, and CD32 should also be measured to effectively characterize lymphoma-specific tumor macrophages. Additionally, the presence of phenotypically distinct, abnormal macrophage populations was closely linked to the phenotype of intra-tumor T-cell populations, including PD-1 expressing T cells. These results further support the close links between macrophage polarization and T-cell functional state, as well as the rationale for targeting tumor-associated macrophages in cancer immunotherapies.
3 Communities
1 Members
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11 MeSH Terms
Cyt-Geist: Current and Future Challenges in Cytometry: Reports of the CYTO 2019 Conference Workshops.
Czechowska K, Lannigan J, Aghaeepour N, Back JB, Begum J, Behbehani G, Bispo C, Bitoun D, Fernández AB, Boova ST, Brinkman RR, Ciccolella CO, Cotleur B, Davies D, Dela Cruz GV, Del Rio-Guerra R, Des Lauriers-Cox AM, Douagi I, Dumrese C, Bonilla Escobar DL, Estevam J, Ewald C, Fossum A, Gaudillière B, Green C, Groves C, Hall C, Haque Y, Hedrick MN, Hogg K, Hsieh EWY, Irish J, Lederer J, Leipold M, Lewis-Tuffin LJ, Litwin V, Lopez P, Nasdala I, Nedbal J, Ohlsson-Wilhelm BM, Price KM, Rahman AH, Rayanki R, Rieger AM, Robinson JP, Shapiro H, Sun YS, Tang VA, Tesfa L, Telford WG, Walker R, Welsh JA, Wheeler P, Tárnok A
(2019) Cytometry A 95: 1236-1274
MeSH Terms: Animals, Clinical Trials as Topic, Flow Cytometry, Fluorescence, Guidelines as Topic, Humans, Inventions, Reproducibility of Results, Stem Cells, Surveys and Questionnaires
Added June 8, 2020
3 Communities
1 Members
0 Resources
10 MeSH Terms
Heterogeneity within Stratified Epithelial Stem Cell Populations Maintains the Oral Mucosa in Response to Physiological Stress.
Byrd KM, Piehl NC, Patel JH, Huh WJ, Sequeira I, Lough KJ, Wagner BL, Marangoni P, Watt FM, Klein OD, Coffey RJ, Williams SE
(2019) Cell Stem Cell 25: 814-829.e6
MeSH Terms: Animals, Cell Division, Cell Lineage, Cells, Cultured, Female, Flow Cytometry, Fluorescence, Immunohistochemistry, Male, Membrane Glycoproteins, Mice, Mouth Mucosa, Nerve Tissue Proteins, Stem Cells, Wound Healing
Show Abstract · Added March 3, 2020
Stem cells in stratified epithelia are generally believed to adhere to a non-hierarchical single-progenitor model. Using lineage tracing and genetic label-retention assays, we show that the hard palatal epithelium of the oral cavity is unique in displaying marked proliferative heterogeneity. We identify a previously uncharacterized, infrequently-dividing stem cell population that resides within a candidate niche, the junctional zone (JZ). JZ stem cells tend to self-renew by planar symmetric divisions, respond to masticatory stresses, and promote wound healing, whereas frequently-dividing cells reside outside the JZ, preferentially renew through perpendicular asymmetric divisions, and are less responsive to injury. LRIG1 is enriched in the infrequently-dividing population in homeostasis, dynamically changes expression in response to tissue stresses, and promotes quiescence, whereas Igfbp5 preferentially labels a rapidly-growing, differentiation-prone population. These studies establish the oral mucosa as an important model system to study epithelial stem cell populations and how they respond to tissue stresses.
Copyright © 2019 Elsevier Inc. All rights reserved.
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1 Members
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15 MeSH Terms
Central EP3 (E Prostanoid 3) Receptors Mediate Salt-Sensitive Hypertension and Immune Activation.
Xiao L, Itani HA, do Carmo LS, Carver LS, Breyer RM, Harrison DG
(2019) Hypertension 74: 1507-1515
MeSH Terms: Adaptive Immunity, Analysis of Variance, Animals, Biomarkers, Biopsy, Needle, Brain, Dinoprostone, Disease Models, Animal, Female, Flow Cytometry, Hypertension, Immunohistochemistry, Male, Mice, Mice, Inbred C57BL, NG-Nitroarginine Methyl Ester, Random Allocation, Real-Time Polymerase Chain Reaction, Receptors, Prostaglandin E, EP3 Subtype, Sodium, Dietary
Show Abstract · Added December 3, 2019
We recently identified a pathway underlying immune activation in hypertension. Proteins oxidatively modified by reactive isoLG (isolevuglandin) accumulate in dendritic cells (DCs). PGE (Prostaglandin E2) has been implicated in the inflammation associated with hypertension. We hypothesized that PGE via its EP (E prostanoid) 3 receptor contributes to DC activation in hypertension. EP3 mice and wild-type littermates were exposed to sequential hypertensive stimuli involving an initial 2-week exposure to the nitric oxide synthase inhibitor N-nitro-L-arginine methyl ester hydrochloride in drinking water, followed by a 2-week washout period, and a subsequent 4% high-salt diet for 3 weeks. In wild-type mice, this protocol increased systolic pressure from 123±2 to 148±8 mm Hg (<0.05). This was associated with marked renal inflammation and a striking accumulation of isoLG adducts in splenic DCs. However, the increases in blood pressure, renal T-cell infiltration, and DC isoLG formation were completely prevented in EP3 mice. Similar protective effects were also observed in wild-type mice that received intracerebroventricular injection of a lentiviral vector encoding shRNA targeting the EP3 receptor. Further, in vitro experiments indicated that PGE also acts directly on DCs via its EP1 receptors to stimulate intracellular isoLG formation. Together, these findings provide new insight into how EP receptors in both the central nervous system and peripherally on DCs promote inflammation in salt-induced hypertension.
1 Communities
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20 MeSH Terms
Picturing Polarized Myeloid Phagocytes and Regulatory Cells by Mass Cytometry.
Roussel M, Bartkowiak T, Irish JM
(2019) Methods Mol Biol 1989: 217-226
MeSH Terms: Cytokines, Dendritic Cells, Flow Cytometry, Humans, Mass Spectrometry, Monocytes, Myeloid-Derived Suppressor Cells, Phagocytes, Phenotype, Single-Cell Analysis
Show Abstract · Added May 13, 2019
The immune monocyte/phagocyte system (MPS) includes numerous cell subsets of the myeloid lineage including monocyte, macrophage, and dendritic cell (DC) populations that are heterogeneous both phenotypically and functionally. Previously, we characterized these diverse MPS phenotypes with multi-parametric mass cytometry (CyTOF). In order to expansively characterize monocytes, macrophages, and dendritic cells, a CyTOF panel was designed to measure 35 identity-, activation-, and polarization-markers. Here we provide a protocol to define a reference map for the myeloid compartment, including sample preparation, to produce reference cell subsets from the monocyte/phagocyte system. In particular, we focused on monocyte-derived macrophages that were further polarized in vitro with cytokine stimulation (i.e., M-CSF, GM-CSF, IL-4, IL-10, IFNγ, and LPS), as well as monocyte-derived DCs, and myeloid-derived suppressor cells (MDSCs), generated in vitro from human bone marrow and/or peripheral blood.
3 Communities
1 Members
0 Resources
10 MeSH Terms
Myocardial differentiation is dependent upon endocardial signaling during early cardiogenesis .
Saint-Jean L, Barkas N, Harmelink C, Tompkins KL, Oakey RJ, Baldwin HS
(2019) Development 146:
MeSH Terms: Animals, Cell Differentiation, Endocardium, Female, Flow Cytometry, Male, Mice, Mice, Inbred C57BL, Microscopy, Fluorescence, Myocardium, NFATC Transcription Factors, Organogenesis, Reverse Transcriptase Polymerase Chain Reaction, Signal Transduction
Show Abstract · Added November 25, 2019
The endocardium interacts with the myocardium to promote proliferation and morphogenesis during the later stages of heart development. However, the role of the endocardium in early cardiac ontogeny remains under-explored. Given the shared origin, subsequent juxtaposition, and essential cell-cell interactions of endocardial and myocardial cells throughout heart development, we hypothesized that paracrine signaling from the endocardium to the myocardium is crucial for initiating early differentiation of myocardial cells. To test this, we generated an , endocardial-specific ablation model using the diphtheria toxin receptor under the regulatory elements of the genomic locus (). Early treatment of mouse embryoid bodies with diphtheria toxin efficiently ablated endocardial cells, which significantly attenuated the percentage of beating EBs in culture and expression of early and late myocardial differentiation markers. The addition of Bmp2 during endocardial ablation partially rescued myocyte differentiation, maturation and function. Therefore, we conclude that early stages of myocardial differentiation rely on endocardial paracrine signaling mediated in part by Bmp2. Our findings provide novel insight into early endocardial-myocardial interactions that can be explored to promote early myocardial development and growth.
© 2019. Published by The Company of Biologists Ltd.
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14 MeSH Terms
p120ctn-Mediated Organ Patterning Precedes and Determines Pancreatic Progenitor Fate.
Nyeng P, Heilmann S, Löf-Öhlin ZM, Pettersson NF, Hermann FM, Reynolds AB, Semb H
(2019) Dev Cell 49: 31-47.e9
MeSH Terms: Animals, Body Patterning, Cadherins, Catenins, Cell Differentiation, Cell Lineage, Cell Movement, Embryonic Development, Flow Cytometry, Gene Expression Regulation, Developmental, Humans, Islets of Langerhans, Mice, Pancreas, Pancreatic Ducts, Receptors, Notch, Signal Transduction, Stem Cells
Show Abstract · Added March 29, 2019
The mechanism of how organ shape emerges and specifies cell fate is not understood. Pancreatic duct and endocrine lineages arise in a spatially distinct domain from the acinar lineage. Whether these lineages are pre-determined or settle once these niches have been established remains unknown. Here, we reconcile these two apparently opposing models, demonstrating that pancreatic progenitors re-localize to establish the niche that will determine their ultimate fate. We identify a p120ctn-regulated mechanism for coordination of organ architecture and cellular fate mediated by differential E-cadherin based cell sorting. Reduced p120ctn expression is necessary and sufficient to re-localize a subset of progenitors to the peripheral tip domain, where they acquire an acinar fate. The same mechanism is used re-iteratively during endocrine specification, where it balances the choice between the alpha and beta cell fates. In conclusion, organ patterning is regulated by p120ctn-mediated cellular positioning, which precedes and determines pancreatic progenitor fate.
Copyright © 2019 Elsevier Inc. All rights reserved.
1 Communities
0 Members
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18 MeSH Terms
Assessing Acinetobacter baumannii Virulence and Persistence in a Murine Model of Lung Infection.
Palmer LD, Green ER, Sheldon JR, Skaar EP
(2019) Methods Mol Biol 1946: 289-305
MeSH Terms: Acinetobacter Infections, Acinetobacter baumannii, Acute Disease, Animals, Bacterial Load, Biopsy, Disease Models, Animal, Flow Cytometry, Immunity, Immunohistochemistry, Mice, Pneumonia, Bacterial, Virulence
Show Abstract · Added April 2, 2019
Acinetobacter baumannii is a Gram-negative opportunistic pathogen and a leading cause of ventilator-associated pneumonia. Murine models of A. baumannii lung infection allow researchers to experimentally assess A. baumannii virulence and host response. Intranasal administration of A. baumannii models acute lung infection. This chapter describes the methods to test A. baumannii virulence in a murine model of lung infection, including assessing the competitive index of a bacterial mutant and the associated inflammatory responses.
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13 MeSH Terms
Novel kidney dissociation protocol and image-based flow cytometry facilitate improved analysis of injured proximal tubules.
Manolopoulou M, Matlock BK, Nlandu-Khodo S, Simmons AJ, Lau KS, Phillips-Mignemi M, Ivanova A, Alford CE, Flaherty DK, Gewin LS
(2019) Am J Physiol Renal Physiol 316: F847-F855
MeSH Terms: Acute Kidney Injury, Animals, Aristolochic Acids, Biomarkers, Cell Cycle, Cell Separation, Disease Models, Animal, Epithelial Cells, Flow Cytometry, Genes, Reporter, Kidney Tubules, Proximal, Luminescent Proteins, Male, Mice, Transgenic, Polyploidy, Tissue Fixation
Show Abstract · Added March 26, 2019
Flow cytometry studies on injured kidney tubules are complicated by the low yield of nucleated single cells. Furthermore, cell-specific responses such as cell cycle dynamics in vivo have conventionally relied on indirect immunohistochemistry and proximal tubule markers that may be downregulated in injury. Here, we report a new tissue dissociation protocol for the kidney with an early fixation step that greatly enhances the yield of single cells. Genetic labeling of the proximal tubule with either mT/mG "tomato" or R26Fucci2aR (Fucci) cell cycle reporter mice allows us to follow proximal tubule-specific changes in cell cycle after renal injury. Image-based flow cytometry (FlowSight) enables gating of the cell cycle and concurrent visualization of the cells with bright field and fluorescence. We used the Fucci mouse in conjunction with FlowSight to identify a discrete polyploid population in proximal tubules after aristolochic acid injury. The tissue dissociation protocol in conjunction with genetic labeling and image-based flow cytometry is a tool that can improve our understanding of any discrete cell population after injury.
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16 MeSH Terms
Protease-activated receptor 4 activity promotes platelet granule release and platelet-leukocyte interactions.
Rigg RA, Healy LD, Chu TT, Ngo ATP, Mitrugno A, Zilberman-Rudenko J, Aslan JE, Hinds MT, Vecchiarelli LD, Morgan TK, Gruber A, Temple KJ, Lindsley CW, Duvernay MT, Hamm HE, McCarty OJT
(2019) Platelets 30: 126-135
MeSH Terms: Animals, Biomarkers, Blood Platelets, Cell Communication, Cytoplasmic Granules, Flow Cytometry, Humans, Leukocytes, Male, Papio, Platelet Activation, Platelet Aggregation, Receptors, Thrombin
Show Abstract · Added March 24, 2020
Human platelets express two protease-activated receptors (PARs), PAR1 (F2R) and PAR4 (F2RL3), which are activated by a number of serine proteases that are generated during pathological events and cause platelet activation. Recent interest has focused on PAR4 as a therapeutic target, given PAR4 seems to promote experimental thrombosis and procoagulant microparticle formation, without a broadly apparent role in hemostasis. However, it is not yet known whether PAR4 activity plays a role in platelet-leukocyte interactions, which are thought to contribute to both thrombosis and acute or chronic thrombo-inflammatory processes. We sought to determine whether PAR4 activity contributes to granule secretion from activated platelets and platelet-leukocyte interactions. We performed in vitro and ex vivo studies of platelet granule release and platelet-leukocyte interactions in the presence of PAR4 agonists including PAR4 activating peptide, thrombin, cathepsin G, and plasmin in combination with small-molecule PAR4 antagonists. Activation of human platelets with thrombin, cathepsin G, or plasmin potentiated platelet dense granule secretion that was specifically impaired by PAR4 inhibitors. Platelet-leukocyte interactions and platelet P-selectin exposure the following stimulation with PAR4 agonists were also impaired by activated PAR4 inhibition in either a purified system or in whole blood. These results indicate PAR4-specific promotion of platelet granule release and platelet-leukocyte aggregate formation and suggest that pharmacological control of PAR4 activity could potentially attenuate platelet granule release or platelet-leukocyte interaction-mediated pathological processes.
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MeSH Terms