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K Kuritz, D Stöhr, N Pollak, F Allgöwer
Cyclic processes, in particular the cell cycle, are of great importance in cell biology. Continued improvement in cell population analysis methods like fluorescence microscopy, flow cytometry, CyTOF or single-cell omics made mathematical methods based on ergodic principles a powerful tool in studying these processes. In this paper, we establish the relationship between cell cycle analysis with ergodic principles and age structured population models. To this end, we describe the progression of a single cell through the cell cycle by a stochastic differential equation on a one dimensional manifold in the high dimensional dataspace of cell cycle markers...
November 28, 2016: Journal of Theoretical Biology
Sheri M Krams, Steven Schaffert, Audrey H Lau, Olivia M Martinez
Single-cell flow cytometric techniques have been indispensable to improving our understanding of the phenotype and function of immune cell subsets that are important in both rejection and tolerance post-transplant. Mass cytometry, or Cytometry by Time-of-Flight (CyTOF), is a single cell-based platform that utilizes antibodies conjugated to rare heavy metal ions for analysis of cellular proteins by a time-of-flight mass spectrometer. This new technology allows for the evaluation of over 40 simultaneous cellular parameters in a single sample because the limitation of spectral overlap, seen in conventional flow cytometry, is eliminated...
November 26, 2016: American Journal of Transplantation
Yi Yao, Tobias Welp, Qing Liu, Naiqian Niu, Xiaomei Wang, Clemente J Britto, Smita Krishnaswamy, Geoff L Chupp, Ruth R Montgomery
Airway diseases affect over 7% of the U.S. population and millions of patients worldwide. Asthmatic patients have wide variation in clinical severity with different clinical and physiologic manifestations of disease that may be driven by distinct biologic mechanisms. Further, the immunologic underpinnings of this complex trait disease are heterogeneous and treatment success depends on defining subgroups of asthmatics. Due to the limited availability and number of cells from the lung, the active site, in-depth investigation has been challenging...
November 3, 2016: Cytometry. Part B, Clinical Cytometry
Bing Z Carter, Po Yee Mak, Hong Mu, Hongsheng Zhou, Duncan H Mak, Wendy Schober, Joel D Leverson, Bin Zhang, Ravi Bhatia, Xuelin Huang, Jorge Cortes, Hagop Kantarjian, Marina Konopleva, Michael Andreeff
BCR-ABL tyrosine kinase inhibitors (TKIs) are effective against chronic myeloid leukemia (CML), but they rarely eliminate CML stem cells. Disease relapse is common upon therapy cessation, even in patients with complete molecular responses. Furthermore, once CML progresses to blast crisis (BC), treatment outcomes are dismal. We hypothesized that concomitant targeting of BCL-2 and BCR-ABL tyrosine kinase could overcome these limitations. We demonstrate increased BCL-2 expression at the protein level in bone marrow cells, particularly in Lin(-)Sca-1(+)cKit(+) cells of inducible CML in mice, as determined by CyTOF mass cytometry...
September 7, 2016: Science Translational Medicine
Katja Kleinsteuber, Björn Corleis, Narges Rashidi, Nzuekoh Nchinda, Antonella Lisanti, Josalyn L Cho, Benjamin D Medoff, Douglas Kwon, Bruce D Walker
Mass cytometry (CyTOF), a mass spectrometry-based single cell phenotyping technology, allows utilization of over 35 antibodies in a single sample and is a promising tool for translational human immunology studies. Although several analysis tools are available to interpret the complex data sets generated, a robust method for standardization and quality control within and across studies is needed. Here we report an efficient and easily adaptable method to monitor quality of individual samples in human immunology studies and to facilitate reproducible data analysis...
October 2016: Cytometry. Part A: the Journal of the International Society for Analytical Cytology
Richard M Ransohoff
Microglial research has entered a fertile, dynamic phase characterized by novel technologies including two-photon imaging, whole-genome transcriptomic and epigenomic analysis with complementary bioinformatics, unbiased proteomics, cytometry by time of flight (CyTOF; Fluidigm) cytometry, and complex high-content experimental models including slice culture and zebrafish. Against this vivid background of newly emerging data, investigators will encounter in the microglial research literature a body of published work using the terminology of macrophage polarization, most commonly into the M1 and M2 phenotypes...
July 26, 2016: Nature Neuroscience
Ling-Shiang Chuang, Nicole Villaverde, Ken Y Hui, Arthur Mortha, Adeeb Rahman, Adam P Levine, Talin Haritunians, Sok Meng Evelyn Ng, Wei Zhang, Nai-Yun Hsu, Jody-Ann Facey, Tramy Luong, Heriberto Fernandez-Hernandez, Dalin Li, Manuel Rivas, Elena R Schiff, Alexander Gusev, L Phillip Schumm, Beatrice M Bowen, Yashoda Sharma, Kaida Ning, Romain Remark, Sacha Gnjatic, Peter Legnani, James George, Bruce E Sands, Joanne M Stempak, Lisa W Datta, Seth Lipka, Seymour Katz, Adam S Cheifetz, Nir Barzilai, Nikolas Pontikos, Clara Abraham, Marla J Dubinsky, Stephan Targan, Kent Taylor, Jerome I Rotter, Ellen J Scherl, Robert J Desnick, Maria T Abreu, Hongyu Zhao, Gil Atzmon, Itsik Pe'er, Subra Kugathasan, Hakon Hakonarson, Jacob L McCauley, Todd Lencz, Ariel Darvasi, Vincent Plagnol, Mark S Silverberg, Aleixo M Muise, Steven R Brant, Mark J Daly, Anthony W Segal, Richard H Duerr, Miriam Merad, Dermot P B McGovern, Inga Peter, Judy H Cho
BACKGROUND & AIMS: Crohn's disease (CD) has the highest prevalence in Ashkenazi Jewish populations. We sought to identify rare, CD-associated frameshift variants of high functional and statistical effects. METHODS: We performed exome sequencing and array-based genotype analyses of 1477 Ashkenazi Jewish individuals with CD and 2614 Ashkenazi Jewish individuals without CD (controls). To validate our findings, we performed genotype analyses of an additional 1515 CD cases and 7052 controls for frameshift mutations in the colony-stimulating factor 2-receptor β common subunit gene (CSF2RB)...
October 2016: Gastroenterology
Tamar L Ben-Shaanan, Hilla Azulay-Debby, Tania Dubovik, Elina Starosvetsky, Ben Korin, Maya Schiller, Nathaniel L Green, Yasmin Admon, Fahed Hakim, Shai S Shen-Orr, Asya Rolls
Positive expectations contribute to the clinical benefits of the placebo effect. Such positive expectations are mediated by the brain's reward system; however, it remains unknown whether and how reward system activation affects the body's physiology and, specifically, immunity. Here we show that activation of the ventral tegmental area (VTA), a key component of the reward system, strengthens immunological host defense. We used 'designer receptors exclusively activated by designer drugs' (DREADDs) to directly activate dopaminergic neurons in the mouse VTA and characterized the subsequent immune response after exposure to bacteria (Escherichia coli), using time-of-flight mass cytometry (CyTOF) and functional assays...
August 2016: Nature Medicine
Shahram Kordasti, Benedetta Costantini, Thomas Seidl, Pilar Perez Abellan, Marc Martinez Llordella, Donal McLornan, Kirsten E Diggins, Austin Kulasekararaj, Cinzia Benfatto, Xingmin Feng, Alexander Smith, Syed A Mian, Rossella Melchiotti, Emanuele de Rinaldis, Richard Ellis, Nedyalko Petrov, Giovanni A M Povoleri, Sun Sook Chung, N Shaun B Thomas, Farzin Farzaneh, Jonathan M Irish, Susanne Heck, Neal S Young, Judith C W Marsh, Ghulam J Mufti
Idiopathic aplastic anemia (AA) is an immune-mediated and serious form of bone marrow failure. Akin to other autoimmune diseases, we have previously shown that in AA regulatory T cells (Tregs) are reduced in number and function. The aim of this study was to further characterize Treg subpopulations in AA and investigate the potential correlation between specific Treg subsets and response to immunosuppressive therapy (IST) as well as their in vitro expandability for potential clinical use. Using mass cytometry and an unbiased multidimensional analytical approach, we identified 2 specific human Treg subpopulations (Treg A and Treg B) with distinct phenotypes, gene expression, expandability, and function...
September 1, 2016: Blood
David Pejoski, Nicolas Tchitchek, André Rodriguez Pozo, Jamila Elhmouzi-Younes, Rahima Yousfi-Bogniaho, Christine Rogez-Kreuz, Pascal Clayette, Nathalie Dereuddre-Bosquet, Yves Lévy, Antonio Cosma, Roger Le Grand, Anne-Sophie Beignon
Broadening our understanding of the abundance and phenotype of B cell subsets that are induced or perturbed by exogenous Ags will improve the vaccine evaluation process. Mass cytometry (CyTOF) is being used to increase the number of markers that can be investigated in single cells, and therefore characterize cell phenotype at an unprecedented level. We designed a panel of CyTOF Abs to compare the B cell response in cynomolgus macaques at baseline, and 8 and 28 d after the second homologous immunization with modified vaccinia virus Ankara...
June 1, 2016: Journal of Immunology: Official Journal of the American Association of Immunologists
Daniel E Lowther, Brittany A Goods, Liliana E Lucca, Benjamin A Lerner, Khadir Raddassi, David van Dijk, Amanda L Hernandez, Xiangguo Duan, Murat Gunel, Vlad Coric, Smita Krishnaswamy, J Christopher Love, David A Hafler
Immunotherapies targeting the immune checkpoint receptor programmed cell death protein 1 (PD-1) have shown remarkable efficacy in treating cancer. CD4(+)CD25(hi)FoxP3(+) Tregs are critical regulators of immune responses in autoimmunity and malignancies, but the functional status of human Tregs expressing PD-1 remains unclear. We examined functional and molecular features of PD-1(hi) Tregs in healthy subjects and patients with glioblastoma multiforme (GBM), combining functional assays, RNA sequencing, and cytometry by time of flight (CyTOF)...
April 21, 2016: JCI Insight
Alexander W Kay, Dara M Strauss-Albee, Catherine A Blish
Mass cytometry is a novel platform for high-dimensional phenotypic and functional analysis of single cells. This system uses elemental metal isotopes conjugated to monoclonal antibodies to evaluate up to 42 parameters simultaneously on individual cells with minimal overlap between channels. The platform can be customized for analysis of both phenotypic and functional markers. Here, we will describe methods to stain, collect, and analyze intracellular functional markers and surface phenotypic markers on natural killer cells...
2016: Methods in Molecular Biology
Carsten Marr, Joseph X Zhou, Sui Huang
Single-cell analyses of transcript and protein expression profiles-more precisely, single-cell resolution analysis of molecular profiles of cell populations-have now entered the center stage with widespread applications of single-cell qPCR, single-cell RNA-Seq and CyTOF. These high-dimensional population snapshot techniques are complemented by low-dimensional time-resolved, microscopy-based monitoring methods. Both fronts of advance have exposed a rich heterogeneity of cell states within uniform cell populations in many biological contexts, producing a new kind of data that has triggered computational analysis methods for data visualization, dimensionality reduction, and cluster (subpopulation) identification...
June 2016: Current Opinion in Biotechnology
Rosemary Fernandez, Holden Maecker
The ability to assess the function of a range of cytokine, antigen receptor, and Toll-like receptor (TLR) signaling pathways in a range of immune cells could provide a kind of fingerprint of the state of the human immune system. The mass cytometry or CyTOF, platform allows for the parallel application of about 40 labeled antibodies to a single sample, creating the possibility to read out many cell types and signaling pathways in a single small blood sample. We developed such a mass cytometry panel, consisting of 22 antibodies to cell surface lineage markers and 8 antibodies to phospho-specific epitopes of signaling proteins...
June 5, 2015: Bio-protocol
Julie M Yabu, Janet C Siebert, Holden T Maecker
BACKGROUND: Kidney transplantation is the most effective treatment for end-stage kidney disease. Sensitization, the formation of human leukocyte antigen (HLA) antibodies, remains a major barrier to successful kidney transplantation. Despite the implementation of desensitization strategies, many candidates fail to respond. Current progress is hindered by the lack of biomarkers to predict response and to guide therapy. Our objective was to determine whether differences in immune and gene profiles may help identify which candidates will respond to desensitization therapy...
2016: PloS One
Adeeb H Rahman, Leticia Tordesillas, M Cecilia Berin
The analysis of heterogeneous cell samples by mass cytometry (CyTOF) relies on the assumption that metal labeled antibodies accurately bind to their target antigens. We report a previously unappreciated experimental artifact of non-specific antibody binding by eosinophils during intracellular CyTOF analysis of human whole blood samples. We hypothesized that this non-specific binding results from a charge-based interaction between the metal-labeled antibodies and highly cationic proteins found in eosinophillic granules and found that this non-specific staining artifact could be reduced to background levels with a simple blocking protocol using heparin as a competing anionic protein...
June 2016: Cytometry. Part A: the Journal of the International Society for Analytical Cytology
Valentina Proserpio, Tapio Lönnberg
In the last lustrum single-cell techniques such as single-cell quantitative PCR, RNA and DNA sequencing, and the state-of-the-art cytometry by time of flight (CyTOF) mass cytometer have allowed a detailed analysis of the sub-composition of different organs from the bone marrow hematopoietic compartment to the brain. These fine-grained analyses have highlighted the great heterogeneity within each cell compartment revealing previously unknown subpopulations of cells. In this review, we analyze how this fast technological evolution has improved our understanding of the biological processes with a particular focus on rare cells of the immune system...
March 2016: Immunology and Cell Biology
Alan J Simmons, Amrita Banerjee, Eliot T McKinley, Cherie' R Scurrah, Charles A Herring, Leslie S Gewin, Ryota Masuzaki, Seth J Karp, Jeffrey L Franklin, Michael J Gerdes, Jonathan M Irish, Robert J Coffey, Ken S Lau
Understanding heterogeneous cellular behaviors in a complex tissue requires the evaluation of signaling networks at single-cell resolution. However, probing signaling in epithelial tissues using cytometry-based single-cell analysis has been confounded by the necessity of single-cell dissociation, where disrupting cell-to-cell connections inherently perturbs native cell signaling states. Here, we demonstrate a novel strategy (Disaggregation for Intracellular Signaling in Single Epithelial Cells from Tissue-DISSECT) that preserves native signaling for Cytometry Time-of-Flight (CyTOF) and fluorescent flow cytometry applications...
October 30, 2015: Molecular Systems Biology
Lorenz Wanke-Jellinek, Joshua W Keegan, James W Dolan, James A Lederer
T cell receptor γδ cells are known to be the primary effector T cells involved in the response to bacterial infections, yet their phenotypic characteristics are not as well established as other T cell subsets. In this study, we used cytometry by time-of-flight mass cytometry to better characterize the phenotypic response of T cell receptor γδ cells to Streptococcus pneumoniae lung infection. Mice were infected, and cells from lung washouts, spleen, and lymph nodes were stained to detect cell-surface, intracellular, and signaling markers...
March 2016: Journal of Leukocyte Biology
Jia-Ren Lin, Mohammad Fallahi-Sichani, Peter K Sorger
Single-cell analysis reveals aspects of cellular physiology not evident from population-based studies, particularly in the case of highly multiplexed methods such as mass cytometry (CyTOF) able to correlate the levels of multiple signalling, differentiation and cell fate markers. Immunofluorescence (IF) microscopy adds information on cell morphology and the microenvironment that are not obtained using flow-based techniques, but the multiplicity of conventional IF is limited. This has motivated development of imaging methods that require specialized instrumentation, exotic reagents or proprietary protocols that are difficult to reproduce in most laboratories...
September 24, 2015: Nature Communications
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