Used Flow Cytometry (used + flow_cytometry)

Distribution by Scientific Domains

Selected Abstracts

Expression patterns and cell cycle profiles of PCNA, MCM6, cyclin D1, cyclin A2, cyclin B1, and phosphorylated histone H3 in the developing mouse retina

Kirston M. Barton
Abstract A challenge in studying organogenesis is the ability to identify progenitor cell populations. To address this problem, we characterized the expression patterns of cell cycle proteins during mouse retinal development and used flow cytometry to determine the expression profiles in the cell cycle. We found that MCM6 and PCNA are expressed in essentially all retinal progenitor cells throughout the proliferative period and these proteins are readily detectable in all cell cycle phases. Furthermore, their expression levels are downregulated as cells exit the cell cycle and differentiate. We also analyzed the expression of Cyclins D1, A2, and B1, and phosphorylated Histone H3 and found unexpected expression patterns and cell cycle profiles. The combined utilization of the markers tested and the use of flow cytometry should further facilitate the study of stem and progenitor cell behavior during development and in adult tissues. Developmental Dynamics 237:672,682, 2008. 2008 Wiley-Liss, Inc. [source]

Adenosine downregulates cytokine-induced expression of intercellular adhesion molecule-1 on rheumatoid synovial fibroblasts independently of adenosine receptor signaling

Takashi Nakazawa
Abstract Adhesion of fibroblast-like synoviocytes (FLSs) to T cells through the interaction of lymphocyte function-associated antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1) plays a pivotal role in the pathogenesis of rheumatoid arthritis (RA). We therefore used flow cytometry and quantitative polymerase chain reaction (PCR) to examine the effect of adenosine and its derivatives on expression of ICAM-1 induced by tumor necrosis factor-alpha and interferon-gamma in primary rheumatoid FLSs (RA-FLSs) and E11 cells, an RA-FLS line. Exposing cells to adenosine (5,500 M) for 24 h in the presence of coformycin, an adenosine deaminase inhibitor, concentration-dependently inhibited cytokine-induced transcription of ICAM-1 mRNA, as well as subsequent surface expression of the protein. Although transcription of all four adenosine receptor isoforms has been detected in FLSs, neither the A1 receptor agonist R-PIA, the A2A receptor agonist CGS21680 nor the A3 agonist Cl-IB-MECA had any effect on cytokine-induced ICAM-1 expression. Conversely, A1/A2 receptor antagonist xanthine amine congener and A2A antagonist ZM240385 both failed to suppress the effect of adenosine. Adenosine appears to inhibit cytokine-induced ICAM-1 expression in FLSs independently of adenosine receptor-mediated signaling. By contrast, the effect of adenosine was neutralized by nitrobenzylmercaptopurin, a nucleoside transporter inhibitor, or by ABT702, an adenosine kinase inhibitor. This suggests that adenosine taken up via the nucleoside transporter is phosphorylated by adenosine kinase, and the resultant phospho-adenosine interferes with the ICAM-1 transcription and cell surface expression. Downregulation of T cell,FLS interaction by adenosine may thus represent a novel approach to the treatment of RA. Drug Dev. Res. 58:368,376, 2003. 2003 Wiley-Liss, Inc. [source]

Effect of First Treatment with Aminobisphosphonates Pamidronate and Ibandronate on Circulating Lymphocyte Subpopulations

Martin Pecherstorfer
Abstract Up to 60% of patients receiving their first infusion of the bisphosphonate pamidronate experience an acute-phase reaction. In this study, we used flow cytometry to determine the effects of pamidronate treatment on circulating lymphocyte subpopulations, and we investigated whether pamidronate and ibandronate treatment affect lymphocyte subpopulations differently. Twenty patients received a pamidronate infusion, 20 patients received intravenously injected ibandronate, and 10 controls received a clodronate infusion. Pamidronate treatment was followed by a significant increase in median body temperature at the 10-hour measurement and a significant decrease in counts of circulating lymphocytes, natural killer cells, T cells, and CD4+ and CD8+ T-cell subsets. Ibandronate treatment did not affect median body temperature, and it was associated at the 10-hour measurement with maximum increases in total lymphocyte count, B cells, T cells, and CD4+ and CD8+ T-cell subsets. Thus, there is a substantial difference in the hematologic response to initial treatments with pamidronate and ibandronate. Clodronate treatment did not induce changes in body temperature or significantly affect the number of circulating T cells and NK cells. The reduction in lymphocyte subsets after initial pamidronate therapy might be mediated by the release of tumor necrosis factor ,, whose source in the acute-phase reaction could be T cells. [source]

Immune Reconstitution Following Rabbit Antithymocyte Globulin

S. Gurkan
Depletional induction therapies are routinely used to prevent acute rejection and improve transplant outcome. The effects of depleting agents on T-cell subsets and subsequent T-cell reconstitution are incompletely defined. We used flow cytometry to examine the effects of rabbit antithymocyte globulin (rATG) on the peripheral T-cell repertoire of pediatric and adult renal transplant recipients. We found that while rATG effectively depleted CD45RA+CD27+ nave and CD45RO+CD27+ central memory CD4+ T cells, it had little effect on CD45RO+CD27, CD4+ effector memory or CD45RA+CD31,, CD45RO+CD27+ and CD45RO+CD27, CD8+ T cell subsets. When we performed a kinetic analysis of CD31+ recent thymic emigrants and CD45RA+/RO+ T cells, we found evidence for both thymopoiesis and homeostatic proliferation contributing to immune reconstitution. We additionally examined the impact of rATG on peripheral CD4+Foxp3+ T cells. We found that in adults, administration of rATG-induced peripheral expansion and new thymic emigration of T cells with a Treg phenotype, while CD4+Foxp3+ T cells of thymic origin predominated in children, providing the first evidence that rATG induces Treg in vivo. Collectively our data indicate that rATG alters the balance of regulatory to memory effector T cells posttransplant, providing an explanation for how it positively impacts transplant outcome. [source]

Increased cellular hypoxia and reduced proliferation of both normal and leukaemic cells during progression of acute myeloid leukaemia in rats

P.. Jensen
The microenvironmental changes in the bone marrow, spleen and liver during progression of the transplantable promyelocytic leukaemia in the Brown Norwegian rat (BNML) have been studied. We used flow cytometry to estimate cellular hypoxia and proliferation based on in vivo pulse-labelling with a mixture of 2-nitroimidazole linked to theophylline (NITP) and bromodeoxyuridine (BrdUrd). The leukaemic cells were identified with the RM124 antibody. In rats inoculated with leukaemic cells the fraction of RM124+ cells was significantly increased from day 20 onwards in the spleen and from day 27 in the bone marrow and liver, reaching a level of 65,87% in these organs at day 32. At day 32, the NITP+ fraction of RM124+ cells had increased significantly in the bone marrow and spleen to 88% and 90%, respectively. The corresponding fractions of NITP+ normal cells reached 63% and 65%, respectively. From day 13 to day 32, the DNA-synthesizing (BrdUrd+) fraction of RM124+ cells in the bone marrow decreased significantly from 52% to 25%, and of normal cells from about 20% to 6%. In the bone marrow and spleen at day 27 and 32, the S-phase and G2/M-phase fractions according to DNA content were higher for the NITP+ than for the NITP, cells. This could partly be explained by an impaired cell cycle progression due to hypoxia. Nevertheless, we found indications of leukaemic cells that were simultaneously labelled with NITP and BrdUrd, in the bone marrow and spleen. These latter findings suggest that in contrast to normal cells some of the leukaemic cells can proliferate even during hypoxia, and this subpopulation may consequently renew and expand the leukaemic cell load. [source]

Intracellular replication of Salmonella typhimurium strains in specific subsets of splenic macrophages in vivo

Suzana P. Salcedo
We used flow cytometry and confocal immunofluorescence microscopy to study the localization of Salmonella typhimurium in spleens of infected mice. Animals were inoculated intragastrically or intraperitoneally with S. typhimurium strains, constitutively expressing green fluorescent protein. Independently of the route of inoculation, most bacteria were found in intracellular locations 3 days after inoculation. Using a panel of antibodies that bound to cells of different lineages, including mononuclear phagocyte subsets, we have shown that the vast majority of S. typhimurium bacteria reside within macrophages. Bacteria were located in red pulp and marginal zone macrophages, but very few were found in the marginal metallophilic macrophage population. We have demonstrated that the Salmonella SPI-2 type III secretion system is required for replication within splenic macrophages, and that sifA, mutant bacteria are found within the cytosol of these cells. These results confirm that SifA and SPI-2 are involved in maintenance of the vacuolar membrane and intracellular replication in vivo. [source]

The use of small molecule high-throughput screening to identify inhibitors of the proteinase 3-NB1 interaction

M. Choi
Summary Anti-neutrophil cytoplasmic antibodies (ANCA) to proteinase 3 (PR3) are found in patients with small-vessel vasculitis. PR3-ANCA bind strongly to membrane PR3 (mPR3) that is presented by the NB1 receptor. We performed high-throughput screening using a small molecule library to identify compounds that inhibit PR3-NB1 binding. We established a human embryonic kidney (HEK293) cell-based system, where approximately 95 2% of the NB1-transfected cells expressed the NB1 receptor on the cell surface. Addition of 01 g/ml human PR3 to 104 NB1-expressing HEK293 cells resulted in PR3 binding that was detected by immunofluorescence using a fluorescence plate reader assay. We identified 13 of 20 000 molecules that inhibited PR3 binding by >70%. Seven of 13 substances showed reproducible inhibition in four additional validation experiments. Two selected compounds (27519 and 27549) demonstrated a dose-dependent inhibition over a range from 625 to 100 M as measured by the plate reader assay. We used flow cytometry as a second assay, and found that both compounds reproducibly inhibited PR3 binding to NB1-transfected HEK293 cells at 50 M (inhibition to 42 4% with compound 27519 and to 47 6% with compound 27549 compared to the dimethylsulphoxide control). Furthermore, compounds 27519 and 27549 also inhibited binding of exogenous PR3 to human neutrophils. In contrast, the compounds did not decrease mPR3 expression on resting neutrophils, but reduced the tumour necrosis factor-,-mediated mPR3 increase on NB1pos neutrophils when present continuously during the assay. The findings suggest that small inhibitory compounds provide a potential therapeutic tool to reduce mPR3 by preventing its binding to NB1. [source]