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cytometry part b

Diana Golden, Antonina Kolmakova, Sunitha Sura, Anthony T Vella, Ani Manichaikul, Xin-Qun Wang, Suzette J Bielinski, Kent D Taylor, Yii-Der Ida Chen, Stephen S Rich, Annabelle Rodriguez
BACKGROUND: The lipoprotein scavenger receptor BI (SCARB1) rs10846744 noncoding variant is significantly associated with atherosclerotic disease independently of traditional cardiovascular risk factors. We identified a potentially novel connection between rs10846744, the immune checkpoint inhibitor lymphocyte activation gene 3 (LAG3), and atherosclerosis. METHODS: In vitro approaches included flow cytometry, lipid raft isolation, phosphosignaling, cytokine measurements, and overexpressing and silencing LAG3 protein...
October 20, 2016: JCI Insight
Timothy B Lannin, Fredrik I Thege, Brian J Kirby
Advances in rare cell capture technology have made possible the interrogation of circulating tumor cells (CTCs) captured from whole patient blood. However, locating captured cells in the device by manual counting bottlenecks data processing by being tedious (hours per sample) and compromises the results by being inconsistent and prone to user bias. Some recent work has been done to automate the cell location and classification process to address these problems, employing image processing and machine learning (ML) algorithms to locate and classify cells in fluorescent microscope images...
October 2016: Cytometry. Part A: the Journal of the International Society for Analytical Cytology
Morten N Andersen, Sinan N H Al-Karradi, Tue W Kragstrup, Marianne Hokland
Nonspecific binding of monoclonal antibodies (mAbs) to Fc-receptors on leukocytes is an important cause of background fluorescence in flow cytometry, and failing to block such nonspecific binding can lead to erroneous results. A major part of previous studies on blocking reagents for flow cytometry have been done in mice, and published results are not completely in agreement. In humans, Fc-receptors are found on most leukocytes, with highest abundance on monocytes/macrophages. Therefore, in the present study our aim was to thoroughly investigate the efficiency of different commonly used blocking reagents regarding inhibition of nonspecific binding of mouse mAbs to human peripheral blood mononuclear cells (MNCs) and monocyte-derived macrophages (MDMs)...
October 12, 2016: Cytometry. Part A: the Journal of the International Society for Analytical Cytology
David B Guiliano, Helen North, Eleni Panayoitou, Elaine C Campbell, Kirsty McHugh, Fiona G M Cooke, Marine Silvestre, Paul Bowness, Simon J Powis, Antony N Antoniou
Objective - HLA-B27 is associated with the inflammatory spondyloarthropathies (SpAs). Of significance, subtypes HLA-B*27:06 and HLA-B*27:09 are not associated with the SpAs. These subtypes primarily differ from the HLA-B*27:05 disease associated allele at residues 114 and 116 of the heavy chain, part of the F pocket of the antigen-binding groove. Dimerisation of HLA-B27 during assembly has been implicated in disease onset. This study investigated the factors influencing differences in dimerisation between disease associated and non-associated HLA-B27 alleles...
October 9, 2016: Arthritis & Rheumatology
Sanjay B Rathod, Anuradha S Tripathy
BACKGROUND AND AIM: Literature on the role of Regulatory T cells (Tregs) in acute viral infections is limited. Having established that the Tregs in self-limiting hepatitis E infection are elevated and functional, this study has focused on characterizing the specificity, phenotypes and identifying the molecules or factors responsible for enhancement of Treg cells and abrogation of Treg-mediated suppression in hepatitis E. METHODS: HEV rORF2p specific (a) Treg frequency, subset analysis and expression of surface and intracellular markers on Tregs and CFSE based functional analysis by flow cytometry (b) key cytokines quantification by multiplex (c) suppressive functional assay in the presence of anti-TGF-β1 or anti-IL-10 or both antibodies or Transwell insert or in combination were performed on samples from 58 acute patients (AVH-E), 45 recovered individuals from hepatitis E and 55 controls...
October 6, 2016: Human Immunology
M Kabuto, N Fujimoto, T Takahashi, T Tanaka
BACKGROUND: hile the frequency of IL-10-producing B cells (B10 cells) is reported to have an inverse correlation with disease activity in some human autoimmune diseases, the association between B10 cells and autoimmune blistering diseases (AIBD) has not been well-evaluated. Although several phenotypes of human regulatory B cells have been proposed, the most appropriate one was not established. OBJECTIVE: This study aimed to evaluate B10 cells in AIBD including their phenotypes...
October 7, 2016: British Journal of Dermatology
J Shi, B H Sun, L R Zhou, X S Wang
Mycobacterium tuberculosis (Mtb) is known to be responsible for tuberculosis (TB), but the pathogenesis of this disease and the host defense mechanisms involved are, for the most part, poorly understood. In this study, we divided 30 male C57BL/6 mice into control and infection groups, and following injection with physiological saline or Mtb, respectively, euthanized five mice from each group on days 1, 3, and 7. TNF-α and IL-10 levels were measured by enzyme-linked immunosorbent assay and flow cytometry, with the latter also being performed to assess apoptosis rates...
September 23, 2016: Genetics and Molecular Research: GMR
Isabel Corrales, Carlos Solano, Paula Amat, Estela Giménez, Rafael de la Cámara, José Nieto, Javier López, Ana García-Noblejas, José Luis Piñana, David Navarro
A single nucleotide polymorphism (SNP), 3 kbp upstream of the IL28B gene (rs12979860; C/T), has been shown to influence the dynamics of cytomegalovirus (CMV) replication in allogeneic stem cell transplant recipients (Allo-SCT). We investigated whether this SNP had any effect on the dynamics of CMV-specific T-cell immunity in these patients. CMV pp65/IE-1 IFN-γ CD8(+) and CD4(+) T cells were enumerated by flow cytometry in 85 patients with no prior CMV DNAemia (group A) and in 57 after the onset of CMV DNAemia (group B)...
September 3, 2016: Journal of Medical Virology
Ryuichi Hotta, Masahiro Ohira, Toshiharu Matsuura, Izumi Muraoka, Panagiotis Tryphonopoulos, Ji Fan, Akin Tekin, Gennaro Selvaggi, David Levi, Phillip Ruiz, Camillo Ricordi, Rodrigo Vianna, Hideki Ohdan, Herman Waldmann, Andreas G Tzakis, Seigo Nishida
BACKGROUND: T-cell depleting strategies have become an integral part of immunosuppressive regimens in organ transplantation. Alemtuzumab is a humanized monoclonal antibody against CD52, a cell-surface antigen on several immune cells. It has been suggested that lymphocyte depletion increases the risk of serious infections. However, this has not been observed with short-term alemtuzumab treatment in an organ transplant setting. For induction therapy using alemtuzumab following liver transplantation, we found that T- and B-cell numbers declined rapidly after alemtuzumab therapy; however, the natural killer (NK) cell number was sustained...
2016: PloS One
André Sulen, Stein-Erik Gullaksen, Lucius Bader, David W McClymont, Jørn Skavland, Sonia Gavasso, Bjørn T Gjertsen
Hydrogen sulfide (H2S) is an endogenous gasotransmitter in human physiology and inflammatory disease, however, with limited knowledge of how signal transduction pathways are involved in immune cells. To examine the effects of sulfide on relevant intracellular signaling in human peripheral blood mononuclear cells (PBMCs), we stimulated healthy donor PBMCs with sodium hydrosulfide (NaHS, 1-1000μM) to mimic H2S stimulation, and analyzed phosphorylation of p38 mitogen activated protein kinase (MAPK) (pT180/pY182), NF-κB p65 (pS529), Akt (pS473) and CREB/ATF1 (pS133/pS63) with flow and mass cytometry...
August 16, 2016: Pharmacological Research: the Official Journal of the Italian Pharmacological Society
A Kubiak, P Matuszak, E Bembnista, M Kozlowska-Skrzypczak
BACKGROUND: Banking of hematopoietic stem cells (HSCs) is a rapidly growing part of the transplant field. The essence of the banking process is to maintain the optimal quality parameters throughout the storage period, allowing successful transplantation. METHODS: Our laboratory research was carried out on 126 HSC samples that were collected by means of leukapheresis from patients with lymphoproliferative diseases. The samples were frozen in a controlled rate and stored up to 76 months in containers in vapor phase of liquid nitrogen...
June 2016: Transplantation Proceedings
Isabelle Krämer, Melanie Engelhardt, Sabrina Fichtner, Brigitte Neuber, Sergej Medenhoff, Uta Bertsch, Jens Hillengass, Marc-Steffen Raab, Dirk Hose, Anthony D Ho, Hartmut Goldschmidt, Michael Hundemer
Immunomodulation is an important part of lenalidomide's mode of action. We analyzed the impact of lenalidomide on T cells from patients with multiple myeloma during lenalidomide therapy in vivo and in patients with lenalidomide-refractory disease in vitro Patients enrolled in the German Speaking Myeloma Multicenter Group (GMMG) MM5 trial received a consolidation therapy with two cycles of lenalidomide after autologous stem cell transplantation (ASCT). Half of the study population continued treatment with lenalidomide maintenance therapy for 2 y, while the other patients received lenalidomide maintenance therapy until complete remission...
May 2016: Oncoimmunology
Ayako Katagi, Li Sui, Kazuyo Kamitori, Toshisada Suzuki, Takeshi Katayama, Akram Hossain, Chisato Noguchi, Youyi Dong, Fuminori Yamaguchi, Masaaki Tokuda
Although various parts of J. curcas (Jatropha curcas L., Euphorbiaceae) have long been used as traditional folk medicines for their antiviral, analgesic, and/or antidotal efficacies, we are the first to investigate the role of anti-carcinogenicity of isoamericanol A (IAA) from the seed extract. Our results showed that IAA is capable of inhibiting cell proliferation in a dose-dependent manner on the human cancer cell lines of MCF-7, MDA-MB231, HuH-7, and HeLa. Flow cytometry analysis showed IAA significantly induces cell cycle arrest at G2/M on MCF-7 cells...
January 2016: Heliyon
Leo D Wang, Scott B Ficarro, John N Hutchinson, Roland Csepanyi-Komi, Phi T Nguyen, Eva Wisniewski, Jessica Sullivan, Oliver Hofmann, Erzsebet Ligeti, Jarrod A Marto, Amy J Wagers
Protein phosphorylation is a central mechanism of signal transduction that both positively and negatively regulates protein function. Large-scale studies of the dynamic phosphorylation states of cell signaling systems have been applied extensively in cell lines and whole tissues to reveal critical regulatory networks, and candidate-based evaluations of phosphorylation in rare cell populations have also been informative. However, application of comprehensive profiling technologies to adult stem cell and progenitor populations has been challenging, due in large part to the scarcity of such cells in adult tissues...
September 15, 2016: Blood
Anne Montfort, Oliver M T Pearce, Eleni Maniati, Benjamin Vincent, Lisa M Bixby, Steffen Böhm, Thomas Dowe, Edmund H Wilkes, Probir Chakravarty, Richard Thompson, Joanne Topping, Pedro R Cutillas, Michelle Lockley, Jonathan S Serody, Melania Capasso, Frances R Balkwill
PURPOSE: In high-grade serous ovarian cancer (HGSOC), higher densities of both B cells and the CD8+ T cell infiltrate were associated with a better prognosis. However, the precise role of B cells in the anti-tumor response remains unknown. As peritoneal metastases are often responsible for relapse, our aim was to characterize the role of B cells in the anti-tumor immune response in HGSOC metastases. EXPERIMENTAL DESIGN: Unmatched pre and post-chemotherapy HGSOC metastases were studied...
June 27, 2016: Clinical Cancer Research: An Official Journal of the American Association for Cancer Research
Elisa F D Canetti, J Keane, C P McLellan, A B Gray
PURPOSE: Compare capillary and venous blood in the analysis of concentration and function of leucocyte sub-populations. This study hypothesised that capillary samples may be used in a site-specific manner as an alternative source of blood samples for assays of leucocyte concentration and neutrophilic phagocytic function and reactive oxygen species (ROS) production, allowing acquisition of multiple samples to better monitor transient but significant post-exercise immune modulation. METHODS: Resting blood samples were simultaneously obtained from vein, finger and earlobe of healthy subjects (n = 10, age: 25...
August 2016: European Journal of Applied Physiology
Lisa Sutton, Nkechi Mba
Vitamin B12 deficiency is a known cause of megaloblastic anemia and bone marrow failure. Bone marrow biopsies are not frequently performed as part of the diagnostic workup and can demonstrate morphologic features that overlap with myelodysplastic syndrome (MDS) and acute leukemia. We describe a case of a dysplastic bone marrow with increased bone marrow hematogones detected by flow cytometry in a child with vitamin B12 deficiency. Hematogones are normal B cell precursors, and hyperplasia has been described in a variety of often reactive conditions and also disease...
June 10, 2016: Pediatric and Developmental Pathology
Xiantao Wang, Yuhan Sun, Huafeng Yang, Yuanxi Lu, Lang Li
This study was designed to investigate the apoptosis induced by oxidized low-density lipoprotein (ox-LDL) in cultured neonatal rat cardiomyocytes and explore the possible mechanisms. We evaluated whether ox-LDL-induced apoptosis depended in part on the activation of toll-like receptor-4(TLR4)/Nuclear factor κB (NF-κB) signaling pathway. Cells were cultivated with and without ox-LDL. Cell apoptosis was evaluated by flow cytometry. Immunofluorescence, western blot analysis and quantitative real-time polymerase chain reaction (qRT-PCR) were conducted to assess protein or mRNA expressions...
2016: Scientific Reports
Sumana R Chintalapudi, Levon Djenderedjian, Andrew B Stiemke, Jena J Steinle, Monica M Jablonski, Vanessa M Morales-Tirado
Loss of functional retinal ganglion cells (RGC) is an element of retinal degeneration that is poorly understood. This is in part due to the lack of a reliable and validated protocol for the isolation of primary RGCs. Here we optimize a feasible, reproducible, standardized flow cytometry-based protocol for the isolation and enrichment of homogeneous RGC with the Thy1.2(hi)CD48(neg)CD15(neg)CD57(neg) surface phenotype. A three-step validation process was performed by: (1) genomic profiling of 25-genes associated with retinal cells; (2) intracellular labeling of homogeneous sorted cells for the intracellular RGC-markers SNCG, brain-specific homeobox/POU domain protein 3A (BRN3A), TUJ1, and RNA-binding protein with multiple splicing (RBPMS); and (3) by applying the methodology on RGC from a mouse model with elevated intraocular pressure (IOP) and optic nerve damage...
2016: Frontiers in Aging Neuroscience
Sanchita Rauth, Sudipta Ray, Sayantan Bhattacharyya, Debapriya Ghosh Mehrotra, Neyaz Alam, Goutam Mondal, Partha Nath, Asoke Roy, Jaydip Biswas, Nabendu Murmu
Epidermal growth factor receptor (EGFR) pathway is overexpressed in head and neck cancer (HNC). Lupeol, a natural triterpene (phytosterol found in fruits, vegetables, etc.), has been reported to be effective against multiple cancer indications. Here we investigate the antitumor effects of Lupeol and underlying mechanism in oral cancer. Lupeol-induced antitumor response was evaluated in two oral squamous cell carcinoma (OSCC) cell lines (UPCI:SCC131 and UPCI:SCC084) by viability (MTT), proliferation, and colony formation assays...
June 2016: Molecular and Cellular Biochemistry
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