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T cell facs phenotyping

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https://www.readbyqxmd.com/read/28465541/transcriptional-landscape-of-epithelial-and-immune-cell-populations-revealed-through-facs-seq-of-healthy-human-skin
#1
Richard S Ahn, Keyon Taravati, Kevin Lai, Kristina M Lee, Joanne Nititham, Rashmi Gupta, David S Chang, Sarah T Arron, Michael Rosenblum, Wilson Liao
Human skin consists of multiple cell types, including epithelial, immune, and stromal cells. Transcriptomic analyses have previously been performed from bulk skin samples or from epithelial and immune cells expanded in cell culture. However, transcriptomic analysis of bulk skin tends to drown out expression signals from relatively rare cells while cell culture methods may significantly alter cellular phenotypes and gene expression profiles. To identify distinct transcriptomic profiles of multiple cell populations without substantially altering cell phenotypes, we employed a fluorescence activated cell sorting method to isolate keratinocytes, dendritic cells, CD4+ T effector cells, and CD8+ T effector cells from healthy skin samples, followed by RNA-seq of each cell population...
May 2, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28453768/%C3%AE-e%C3%AE-7-integrin-identifies-subsets-of-pro-inflammatory-colonic-cd4-t-lymphocytes-in-ulcerative-colitis
#2
Christopher A Lamb, John C Mansfield, Gaik W Tew, Deena Gibbons, Anna K Long, Peter Irving, Lauri Diehl, Jeff Eastham-Anderson, Maria B Price, Graeme O'Boyle, David E J Jones, Sharon O'Byrne, Adrian Hayday, Mary E Keir, Jackson G Egen, John A Kirby
Background and Aims: The αEβ7 integrin is crucial for retention of T lymphocytes at mucosal surfaces through its interaction with E-cadherin. Pathogenic or protective functions of these cells during human intestinal inflammation, such as ulcerative colitis [UC], have not previously been defined, with understanding largely derived from animal model data. Defining this phenotype in human samples is important for understanding UC pathogenesis and is of translational importance for therapeutic targeting of αEβ7-E-cadherin interactions...
May 1, 2017: Journal of Crohn's & Colitis
https://www.readbyqxmd.com/read/28446665/dense-array-of-spikes-on-hiv-1-virion-particles
#3
Armando Stano, Daniel P Leaman, Arthur S Kim, Lei Zhang, Ludovic Autin, Jidnyasa Ingale, Syna K Gift, Jared Truong, Richard T Wyatt, Arthur J Olson, Michael B Zwick
HIV-1 is rare among viruses for having a low number of envelope glycoprotein (Env) spikes per virion, i.e. ∼7-14. This exceptional feature has been associated with avoidance of humoral immunity, i.e. B cell activation and antibody neutralization. Virus-like particles (VLPs) with increased density of Env are being pursued for vaccine development; however these typically require protein engineering that alters Env structure. Here, we used instead a strategy that targets the producer cell. We employed fluorescence activated cell sorting (FACS) to sort for cells that are recognized by trimer crossreactive broadly neutralizing antibody (bnAb) and not by non-neutralizing antibodies...
April 26, 2017: Journal of Virology
https://www.readbyqxmd.com/read/28446142/differences-in-peripheral-myelin-antigen-specific-t-cell-responses-and-t-memory-subsets-in-atypical-versus-typical-cidp
#4
M Staudt, J M Diederich, C Meisel, A Meisel, J Klehmet
BACKGROUND: Chronic inflammatory demyelinating polyneuropathy (CIDP) is presented by a large heterogeneity of clinical phenotypes. Around 50% of patients suffer from typical CIDP and show better therapy response than atypical variants. The goal of our study was to search for cellular immunological differences in typical versus atypical CIDP in comparison to controls. METHODS: We evaluated 26 (9 typical, 17 atypical) patients with mainly active-unstable CIDP using clinical and immunological examinations (enzyme-linked immunospot assay ELISPOT, fluorescence-activated cell sorting FACS) in comparison to 28 healthy, age-matched controls (HC)...
April 26, 2017: BMC Neurology
https://www.readbyqxmd.com/read/28355218/scd163-levels-as-a-biomarker-of-disease-severity-in-leprosy-and-visceral-leishmaniasis
#5
Ricardo Luís Louzada Silva, Marcio B Santos, Priscila L S Almeida, Thayse S Barros, Lucas Magalhães, Rodrigo A Cazzaniga, Patrícia R M Souza, Nívea F Luz, Jaqueline França-Costa, Valeria M Borges, Djalma S Lima-Junior, Michael W Lipscomb, Malcolm S Duthie, Steven G Reed, Roque Pacheco Almeida, Amélia Ribeiro Jesus
BACKGROUND: CD163, receptor for the haptoglobin-hemoglobin complex, is expressed on monocytes/macrophages and neutrophils. A soluble form of CD163 (sCD163) has been associated with the M2 macrophage phenotype, and M2 macrophages have been shown to down-modulate inflammatory responses. In particular, previous studies have shown that M2 is closely associated with the most severe clinical presentation of leprosy (i.e. lepromatous leprosy (LL)), as well as tuberculosis. We hypothesized that sCD163 correlates with severity of diseases caused by intracellular pathogens...
March 2017: PLoS Neglected Tropical Diseases
https://www.readbyqxmd.com/read/28327575/analysis-of-exome-data-for-4293-trios-suggests-gpi-anchor-biogenesis-defects-are-a-rare-cause-of-developmental-disorders
#6
Alistair T Pagnamenta, Yoshiko Murakami, John M Taylor, Consuelo Anzilotti, Malcolm F Howard, Venessa Miller, Diana S Johnson, Shereen Tadros, Sahar Mansour, I Karen Temple, Rachel Firth, Elisabeth Rosser, Rachel E Harrison, Bronwen Kerr, Niko Popitsch, Taroh Kinoshita, Jenny C Taylor, Usha Kini
Over 150 different proteins attach to the plasma membrane using glycosylphosphatidylinositol (GPI) anchors. Mutations in 18 genes that encode components of GPI-anchor biogenesis result in a phenotypic spectrum that includes learning disability, epilepsy, microcephaly, congenital malformations and mild dysmorphic features. To determine the incidence of GPI-anchor defects, we analysed the exome data from 4293 parent-child trios recruited to the Deciphering Developmental Disorders (DDD) study. All probands recruited had a neurodevelopmental disorder...
June 2017: European Journal of Human Genetics: EJHG
https://www.readbyqxmd.com/read/28202731/microfluidic-droplet-platform-for-ultrahigh-throughput-single-cell-screening-of-biodiversity
#7
Stanislav S Terekhov, Ivan V Smirnov, Anastasiya V Stepanova, Tatyana V Bobik, Yuliana A Mokrushina, Natalia A Ponomarenko, Alexey A Belogurov, Maria P Rubtsova, Olga V Kartseva, Marina O Gomzikova, Alexey A Moskovtsev, Anton S Bukatin, Michael V Dubina, Elena S Kostryukova, Vladislav V Babenko, Maria T Vakhitova, Alexander I Manolov, Maja V Malakhova, Maria A Kornienko, Alexander V Tyakht, Anna A Vanyushkina, Elena N Ilina, Patrick Masson, Alexander G Gabibov, Sidney Altman
Ultrahigh-throughput screening (uHTS) techniques can identify unique functionality from millions of variants. To mimic the natural selection mechanisms that occur by compartmentalization in vivo, we developed a technique based on single-cell encapsulation in droplets of a monodisperse microfluidic double water-in-oil-in-water emulsion (MDE). Biocompatible MDE enables in-droplet cultivation of different living species. The combination of droplet-generating machinery with FACS followed by next-generation sequencing and liquid chromatography-mass spectrometry analysis of the secretomes of encapsulated organisms yielded detailed genotype/phenotype descriptions...
March 7, 2017: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/28138512/aging-affects-bone-marrow-macrophage-polarization-relevance-to-bone-healing
#8
E Gibon, F Loi, Luis A Córdova, J Pajarinen, T Lin, L Lu, A Nabeshima, Z Yao, Stuart B Goodman
Macrophages are an important component of the inflammatory cascade by initiating and modulating the processes leading to tissue regeneration and bone healing. Depending on the local environment, macrophages can be polarized into M1 (pro-inflammatory) or M2 (anti-inflammatory) phenotypes. In order to assess the effects of aging on macrophage function, bone marrow macrophage polarization using primary bone marrow macrophages (BMMs) from young (8 weeks old) and aged (72 weeks old) wild-type male C57BL/6J mice was analyzed...
June 2016: Regenerative Engineering and Translational Medicine
https://www.readbyqxmd.com/read/27976414/low-testosterone-alters-the-activity-of-mouse-prostate-stem-cells
#9
Ye Zhou, Ben Copeland, Maya Otto-Duessel, Miaoling He, Susan Markel, Tim W Synold, Jeremy O Jones
BACKGROUND: Low serum testosterone (low T) has been repeatedly linked to worse outcomes in men with newly diagnosed prostate cancer (PC). How low T contributes to these outcomes is unknown. Here we demonstrate that exposure to low T causes significant changes in the mouse prostate and prostate stem cells. METHODS: Mice were castrated and implanted with capsules to achieve castrate, normal, or sub-physiological levels of T. After 6 weeks of treatment, LC-MS/MS was used to quantify the levels of T and dihydrotestosterone (DHT) in serum and prostate tissue...
December 14, 2016: Prostate
https://www.readbyqxmd.com/read/27942419/immune-suppression-mediated-by-myeloid-and-lymphoid-derived-immune-cells-in-the-tumor-microenvironment-facilitates-progression-of-thyroid-cancers-driven-by-hras-g12v-and-pten-loss
#10
Lee Ann Jolly, Nicole Massoll, Aime T Franco
Thyroid cancer is the most common endocrine malignancy and is predicted to be the 4(th) most commonly diagnosed cancer by 2030. Approximately one-half of follicular thyroid carcinomas (FTC) contain genetic alterations in RAS family members. Furthermore, Cowden's disease, which is characterized by loss of PTEN, predisposes for the development of FTC in humans. We have shown that thyroid specific expression of Hras(G12V) at endogenous levels and Pten inactivation (Hras(G12V)/Pten(-/-)/TPO-cre mice) leads to the development of FTCs that closely recapitulate human disease, with complete penetrance at one year...
October 2016: Journal of Clinical & Cellular Immunology
https://www.readbyqxmd.com/read/27797919/%C3%AE-e%C3%AE-7-integrin-identifies-subsets-of-pro-inflammatory-colonic-cd4-t-lymphocytes-in-ulcerative-colitis
#11
Christopher A Lamb, John C Mansfield, Gaik W Tew, Deena Gibbons, Anna K Long, Peter Irving, Lauri Diehl, Jeff Eastham-Anderson, Maria B Price, Graeme O'Boyle, David E J Jones, Sharon O'Byrne, Adrian Hayday, Mary E Keir, Jackson G Egen, John A Kirby
BACKGROUND AND AIMS: The αEβ7 integrin is crucial for retention of T lymphocytes at mucosal surfaces through its interaction with E-cadherin. Pathogenic or protective functions of these cells during human intestinal inflammation, such as ulcerative colitis [UC], have not previously been defined, with understanding largely derived from animal model data. Defining this phenotype in human samples is important for understanding UC pathogenesis and is of translational importance for therapeutic targeting of αEβ7-E-cadherin interactions...
October 20, 2016: Journal of Crohn's & Colitis
https://www.readbyqxmd.com/read/27787803/single-cell-rna-sequencing-of-human-t-cells
#12
Alexandra-Chloé Villani, Karthik Shekhar
Understanding how populations of human T cells leverage cellular heterogeneity, plasticity, and diversity to achieve a wide range of functional flexibility, particularly during dynamic processes such as development, differentiation, and antigenic response, is a core challenge that is well suited for single-cell analysis. Hypothesis-free evaluation of cellular states and subpopulations by transcriptional profiling of single T cells can identify relationships that may be obscured by targeted approaches such as FACS sorting on cell-surface antigens, or bulk expression analysis...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/27787790/facs-analysis-of-memory-t-lymphocytes
#13
Enrico Lugli, Veronica Zanon, Domenico Mavilio, Alessandra Roberto
Flow cytometry is a powerful and robust technology for detecting and monitoring multiple markers at the level of single cells. Since its early development, flow cytometry has been used to assess heterogeneity in a cell suspension. Over the years, the increasing number of parameters that could be included in a single assay combined with physical separation by fluorescence-activated cell sorting (FACS) revealed that the T cell compartment is extremely heterogenous in terms of phenotypic diversity, functional capacity, and transcriptional regulation...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/27783636/online-haemodiafiltration-improves-inflammatory-state-in-dialysis-patients-a-longitudinal-study
#14
Ines Rama, Ines Llaudó, Pere Fontova, Gema Cerezo, Carlos Soto, Casimiro Javierre, Miguel Hueso, Nuria Montero, Alberto Martínez-Castelao, Juan Torras, Josep M Grinyó, Josep M Cruzado, Nuria Lloberas
BACKGROUND: Patients undergoing conventional hemodialysis (C-HD) present a greater immuno-inflammatory state probably related to uremia, sympathetic nervous system (SNS) activation and /or membrane bioincompatibility, which could improve with a technique-switching to online hemodiafiltration (OL-HD). The antigen-independent pathway activation of this modified immunologic state turns dendritic cells (DC) into an accurate cell model to study these patients. The aim of this study is to further evaluate the immune-inflammatory state of patients in C-HD assessed by DC maturation...
2016: PloS One
https://www.readbyqxmd.com/read/27685012/fluorescence-activated-cell-sorting-facs-and-gene-expression-analysis-of-fos-expressing-neurons-from-fresh-and-frozen-rat-brain-tissue
#15
F Javier Rubio, Xuan Li, Qing-Rong Liu, Raffaello Cimbro, Bruce T Hope
The study of neuroplasticity and molecular alterations in learned behaviors is switching from the study of whole brain regions to the study of specific sets of sparsely distributed activated neurons called neuronal ensembles that mediate learned associations. Fluorescence Activated Cell Sorting (FACS) has recently been optimized for adult rat brain tissue and allowed isolation of activated neurons using antibodies against the neuronal marker NeuN and Fos protein, a marker of strongly activated neurons. Until now, Fos-expressing neurons and other cell types were isolated from fresh tissue, which entailed long processing days and allowed very limited numbers of brain samples to be assessed after lengthy and complex behavioral procedures...
August 27, 2016: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/27622939/altered-b-cell-homeostasis-and-toll-like-receptor-9-driven-response-in-patients-affected-by-autoimmune-polyglandular-syndrome-type-1-altered-b-cell-phenotype-and-dysregulation-of-the-b-cell-function-in-apeced-patients
#16
Valentina Perri, Elena Gianchecchi, Riccardo Scarpa, Mariella Valenzise, Maria Manuela Rosado, Ezio Giorda, Antonino Crinò, Marco Cappa, Susi Barollo, Silvia Garelli, Corrado Betterle, Alessandra Fierabracci
APECED is a T-cell mediated disease with increased frequencies of CD8+ effector and reduction of FoxP3+ T regulatory cells. Antibodies against affected organs and neutralizing to cytokines are found in the peripheral blood. The contribution of B cells to multiorgan autoimmunity in Aire-/- mice was reported opening perspectives on the utility of anti-B cell therapy. We aimed to analyse the B cell phenotype of APECED patients compared to age-matched controls. FACS analysis was conducted on PBMC in basal conditions and following CpG stimulation...
February 2017: Immunobiology
https://www.readbyqxmd.com/read/27601573/fluorescence-based-flow-sorting-in-parallel-with-transposon-insertion-site-sequencing-identifies-multidrug-efflux-systems-in-acinetobacter-baumannii
#17
Karl A Hassan, Amy K Cain, TaoTao Huang, Qi Liu, Liam D H Elbourne, Christine J Boinett, Anthony J Brzoska, Liping Li, Martin Ostrowski, Nguyen Thi Khanh Nhu, Tran Do Hoang Nhu, Stephen Baker, Julian Parkhill, Ian T Paulsen
UNLABELLED: Multidrug efflux pumps provide clinically significant levels of drug resistance in a number of Gram-negative hospital-acquired pathogens. These pathogens frequently carry dozens of genes encoding putative multidrug efflux pumps. However, it can be difficult to determine how many of these pumps actually mediate antimicrobial efflux, and it can be even more challenging to identify the regulatory proteins that control expression of these pumps. In this study, we developed an innovative high-throughput screening method, combining transposon insertion sequencing and cell sorting methods (TraDISort), to identify the genes encoding major multidrug efflux pumps, regulators, and other factors that may affect the permeation of antimicrobials, using the nosocomial pathogen Acinetobacter baumannii A dense library of more than 100,000 unique transposon insertion mutants was treated with ethidium bromide, a common substrate of multidrug efflux pumps that is differentially fluorescent inside and outside the bacterial cytoplasm...
September 6, 2016: MBio
https://www.readbyqxmd.com/read/27540594/pd-1-and-foxp3-t-cell-reduction-correlates-with-survival-of-hcc-patients-after-sorafenib-therapy
#18
Suresh Gopi Kalathil, Amit Anand Lugade, Austin Miller, Renuka Iyer, Yasmin Thanavala
BACKGROUND: Sorafenib is an oral antiangiogenic agent administered in advanced-stage hepatocellular carcinoma (HCC). Based on preclinical and human studies, we hypothesized that, in addition to its antiangiogenic properties, sorafenib may beneficially reduce the extent of the immunosuppressive network in HCC patients. To test this hypothesis, we examined whether alterations in the immunosuppressive burden of advanced-stage HCC patients correlated with clinical outcome. METHODS: In before and after sorafenib treatment, blood samples collected from 19 patients with advanced HCC, the frequency of PD-1(+) T cells, Tregs, and myeloid derived suppressor cells (MDSC) were quantified by multiparameter FACS...
July 21, 2016: JCI Insight
https://www.readbyqxmd.com/read/27532024/adenosine-generating-ovarian-cancer-cells-attract-myeloid-cells-which-differentiate-into-adenosine-generating-tumor-associated-macrophages-a-self-amplifying-cd39-and-cd73-dependent-mechanism-for-tumor-immune-escape
#19
Itsaso Montalbán Del Barrio, Cornelia Penski, Laura Schlahsa, Roland G Stein, Joachim Diessner, Achim Wöckel, Johannes Dietl, Manfred B Lutz, Michel Mittelbronn, Jörg Wischhusen, Sebastian F M Häusler
BACKGROUND: Ovarian cancer (OvCA) tissues show abundant expression of the ectonucleotidases CD39 and CD73 which generate immunomodulatory adenosine, thereby inhibiting cytotoxic lymphocytes. Little, however, is known about the effect of adenosine on myeloid cells. Considering that tumor associated macrophages (TAM) and myeloid-derived suppressor cells (MDSC) constitute up to 20 % of OvCA tissue, we investigated the effect of adenosine on myeloid cells and explored a possible contribution of myeloid cells to adenosine generation in vitro and ex vivo...
2016: Journal for Immunotherapy of Cancer
https://www.readbyqxmd.com/read/27525228/overexpression-of-molecular-chaperons-grp78-and-grp94-in-cd44-hi-cd24-lo-breast-cancer-stem-cells
#20
Babak Nami, Armin Ghasemi-Dizgah, Akbar Vaseghi
INTRODUCTION: Breast cancer stem cell with CD44(hi)/CD24(lo) phonotype is described having stem cell properties and represented as the main driving factor in breast cancer initiation, growth, metastasis and low response to anti-cancer agents. Glucoseregulated proteins (GRPs) are heat shock protein family chaperons that are charged with regulation of protein machinery and modulation of endoplasmic reticulum homeostasis whose important roles in stem cell development and invasion of various cancers have been demonstrated...
2016: BioImpacts: BI
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