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T cell facs phenotyping

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https://www.readbyqxmd.com/read/28202731/microfluidic-droplet-platform-for-ultrahigh-throughput-single-cell-screening-of-biodiversity
#1
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...
February 15, 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
#2
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
#3
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
#4
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
#5
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
#6
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
#7
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
#8
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
#9
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
#10
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
#11
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...
2016: MBio
https://www.readbyqxmd.com/read/27540594/pd-1-and-foxp3-t-cell-reduction-correlates-with-survival-of-hcc-patients-after-sorafenib-therapy
#12
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
#13
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
#14
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
https://www.readbyqxmd.com/read/27409668/branched-multipeptide-immunotherapy-for-glioblastoma-using-human-leukocyte-antigen-a-0201-restricted-cytotoxic-t-lymphocyte-epitopes-from-erbb2-birc5-and-cd99
#15
Young-Hee Kim, Thi-Anh-Thuy Tran, Hyun-Ju Lee, Sook-In Jung, Je-Jung Lee, Wool-Youl Jang, Kyung-Sub Moon, In-Young Kim, Shin Jung, Tae-Young Jung
We investigated the use of cytotoxic T-lymphocyte (CTL) epitopes in peptide immunotherapy for glioblastoma. Three peptides (ERBB2, BIRC5 and CD99) were selected based on their peptide-T2 cell binding affinities and combined in a multipeptide cocktail or a branched multipeptide synthesized with mini-polyethylene glycol spacers. Dendritic cells (DCs) pulsed with the multipeptide cocktail or branched multipeptide were compared based on their immunophenotype and cytokine secretion. FACS analysis of alpha-type 1 polarized dendritic cells (αDC1s) revealed that both groups highly expressed CD80, CD83 and CD86, indicating that both treatments efficiently generated mature αDC1s with the expected phenotype...
August 2, 2016: Oncotarget
https://www.readbyqxmd.com/read/27246930/microglia-are-less-pro-inflammatory-than-myeloid-infiltrates-in-the-hippocampus-of-mice-exposed-to-status-epilepticus
#16
Jonathan Vinet, Ilia D Vainchtein, Carlotta Spano, Carmela Giordano, Domenico Bordini, Giulia Curia, Massimo Dominici, Hendrikus W G M Boddeke, Bart J L Eggen, Giuseppe Biagini
Activated microglia, astrogliosis, expression of pro-inflammatory cytokines, blood brain barrier (BBB) leakage and peripheral immune cell infiltration are features of mesial temporal lobe epilepsy. Numerous studies correlated the expression of pro-inflammatory cytokines with the activated morphology of microglia, attributing them a pro-epileptogenic role. However, microglia and myeloid cells such as macrophages have always been difficult to distinguish due to an overlap in expressed cell surface molecules. Thus, the detrimental role in epilepsy that is attributed to microglia might be shared with myeloid infiltrates...
August 2016: Glia
https://www.readbyqxmd.com/read/27182006/a-novel-rabies-virus-lipopeptide-provides-a-better-protection-by-improving-the-magnitude-of-dcs-activation-and-t-cell-responses
#17
Rui Liu, Jingbo Wang, Yan Yang, Inamullah Khan, Yue Dong, Naishuo Zhu
Besides rabies virus neutralizing antibody, non-neutralizing antibody to internal vital proteins, interferon, and possibly cell-mediated immunity also have a critical role in preventing the infection by rabies virus (RV). We identified novel CTL and Th epitopes which could induce lymphocyte proliferation and IFN-γ, IL-4 production, and designed linear and branched lipopeptides with these selected CTL and Th epitopes. Compared to linear construct, branched lipopeptides, especially Lipo C, stimulate stronger phenotypic and functional maturation of DCs, as well as more efficient CD8(+) T cell responses, evaluating by using FACS, G333-341 tetramer staining and specific CTL assay...
August 2, 2016: Virus Research
https://www.readbyqxmd.com/read/27141211/effect-of-chaetocin-on-renal-cell-carcinoma-cells-and-cytokine-induced-killer-cells
#18
COMPARATIVE STUDY
Roman Rombo, Hans Weiher, Ingo G H Schmidt-Wolf
We examined the cytotoxic effects of chaetocin on clear cell renal cell carcinoma (ccRCC) cells and the possibility to combine the effects of chaetocin with the effects of cytokine-induced killer cells (CIK) assayed by MTT assay and FACS analysis. Chaetocin is a thiodioxopiperazine produced by fungi belonging to the chaetomiaceae family. In 2007, it was first reported that chaetocin shows potent and selective ex vivo anti-cancer activity by inducing reactive oxygen species. CIK cells are generated from CD3+/CD56- T lymphocytes with double negative CD4-/CD8- phenotype that are isolated from human blood...
2016: German Medical Science: GMS E-journal
https://www.readbyqxmd.com/read/27105585/utility-of-cd127-combined-with-foxp3-for-identification-of-operational-tolerance-after-liver-transplantation
#19
Hanaa Nafady-Hego, Ying Li, Hidenori Ohe, Hamed Elgendy, Xiangdong Zhao, Shimon Sakaguchi, G Alex Bishop, Takaaki Koshiba
Loss of cell surface expression of CD127 on CD4(+)CD25(++) regulatory T-cells (Tregs) may be a useful marker to efficiently isolate Tregs. As FOXP3 was specifically used to identify Tregs, combining these two markers could give better identification for patient with operational tolerance (OT) after liver transplantation. To testify this mixed lymphocyte reaction (MLR), the function of circulating CD4(+)CD25(++)CD127(dim) cells (CD127(dim) cells) was examined in immunosuppression (IS)-free pediatric recipients after liver transplantation (LTx) (group operational tolerance: OT) (Gr-tol n=25) compared to recipients who could not stop IS due to clinically overt rejection (group intolerance) (Gr-intol n=18), recipients who were weaning IS (Gr-weaning n=11) and age-matched healthy volunteers (Gr-vol n=11)...
May 2016: Transplant Immunology
https://www.readbyqxmd.com/read/27075616/human-endometrial-fibroblasts-derived-from-mesenchymal-progenitors-inherit-progesterone-resistance-and-acquire-an-inflammatory-phenotype-in-the-endometrial-niche-in-endometriosis
#20
Fatima Barragan, Juan C Irwin, Shaina Balayan, David W Erikson, Joseph C Chen, Sahar Houshdaran, Terhi T Piltonen, Trimble L B Spitzer, Ashley George, Joseph T Rabban, Camran Nezhat, Linda C Giudice
Human endometrium undergoes cyclic regeneration involving stem/progenitor cells, but the role of resident endometrial mesenchymal stem cells (eMSC) as progenitors of endometrial stromal fibroblasts (eSF) has not been definitively demonstrated. In endometriosis, eSF display progesterone (P4) resistance with impaired decidualization in vivo and in vitro. To investigate eMSC as precursors of eSF and whether endometriosis P4 resistance is inherited from eMSC, we analyzed transcriptomes of eutopic endometrium eMSC and eSF isolated by fluorescence-activated cell sorting (FACS) from endometriosis (eMSCendo, eSFendo) and controls (eMSCcontrol, eSFcontrol) and their derived primary cultures...
May 2016: Biology of Reproduction
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