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Query: UNIPROT:P08758 (annexin V)
 9,383 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Annexin V binding to phosphatidylserine was evaluated by flow cytometry to examine apoptosis in different lymphocyte subsets of peripheral blood mononuclear cells after a 24 h in vitro culture period. We also applied a 2 Gy dose gamma-irradiation prior to incubation to evaluate the additional apoptogenic effect of radiation on the lymphocyte subsets. Overall, B lymphocytes showed the highest number of apoptotic cells, followed by T lymphocytes. Within the T lymphocytes, CD4-positive and CD45RA-negative cells were more prone to apoptosis than the CD8-positive and CD45RA-positive cells. Natural killer cells turned out to be most apoptosis-resistant. In the irradiated samples about twice as many apoptotic cells were found and the differences between lymphocyte subpopulations remained. Backgating of the annexin V-positive cells showed that these cells had a clearly decreased forward scatter signal. The antibody binding capacity (ABC) of lymphocyte membrane antigens was determined with CD3-fluorescein isothiocyanate (FITC), CD45RA-FITC, CD4-phycoerythrin (PE), CD8-PE, CD56-PE, and CD20-PE in viable and apoptotic cells. In the apoptotic cells a decrease of ABC was found for all antigens, except for CD20. There was no significant cell loss in the cultures. We conclude that the change in scatter and in ABC must be considered in immunophenotyping experiments on cells kept in culture for 24 h. If these changes are taken into account, percentages of subpopulations or the numbers of cells that stain positive for the studied markers do not significantly change.
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PMID:Quantification of apoptosis in lymphocyte subsets and effect of apoptosis on apparent expression of membrane antigens. 938 41

The present article compares the reliability of four previously described cytofluorometric methods of apoptosis quantification for phenotyping apoptotic human lymphocytes. Each of these assays detects distinct cellular alterations of the apoptotic process. Alteration in plasma membrane integrity can be evaluated following 7-AAD incorporation and the translocation of phosphatidylserine from the inner to the outer layer of the plasma membrane can be detected through the FITC annexin V staining. DNA strand breaks in apoptotic nuclei can be evidenced by the ISNT assay and finally morphological modifications can be followed with FSC/SSC criteria. Comparative analysis of apoptosis in cultured PBMC from HIV-infected patients considering the FSC/SSC parameters, 7-AAD stainability and annexin V fixation revealed that the latter identifies early apoptotic cells, also characterized as 7-AAD(low) with a reduced FSC. Moreover these three methods proved to be reliable and gave statistically similar results when combined with cell surface detection of antigens such as CD4, CD8 and CD19 by specific mAbs. Importantly, the 7-AAD assay easily allowed the identification of debris/apoptotic bodies, which were still stained by anti-cell surface mAbs and might therefore significantly distort the apoptosis percentage in a given lymphocyte subset. In the present report we also point out that the ISNT assay is not appropriate for phenotyping apoptotic lymphocytes in PBMC. Indeed it can particularly underestimate the rate of apoptosis in the B-cell subset. This was found to be related to the apoptosis-associated decrease in cell surface antigen expression, which is dramatically exacerbated in the ISNT assay because of the stripper effect of ethanol used for cell permeabilization. Finally, we propose a three step analytical strategy to accurately phenotype apoptotic peripheral human lymphocytes. It includes two gating steps performed on FSC/SSC criteria and 7-AAD/FSC parameters to eliminate monocytes, granulocytes and debris-apoptotic bodies, the third step being the phenotyping step itself, performed in dual or triple staining experiments. Altogether these observations emphasize that it is essential to assess critically the ability of a cytofluorometric method to phenotype apoptotic cells in complex lymphoid populations and that inaccurate identification of cell subsets undergoing apoptosis can be readily overcome by gating properly the lymphoid population, and using assays which preserve cell surface structure.
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PMID:Strategies for phenotyping apoptotic peripheral human lymphocytes comparing ISNT, annexin-V and 7-AAD cytofluorometric staining methods. 946 28

The mechanisms accounting for T cell depletion in AIDS patients are not yet fully understood, nor are the roles of host factors in HIV pathogenesis. We show here that an ongoing humoral immune response to HIV gp120 can sensitize non-infected cells towards apoptosis. Thus, i.v. injection of 1 microg recombinant(r) gp120 into gp120-immunized human CD4-transgenic mice (huCD4 Tg), which express huCD4 on both T and B cells, results in T and B cell depletion in peripheral blood and lymphoid tissues. On day 6 after a bolus injection of gp120, the numbers of peripheral T cells and B cells in gp120-immunized huCD4 Tg decreased sevenfold and two- to threefold, respectively. Annexin V staining revealed a higher percentage of early apoptotic cells on day 1 of gp120 i.v. injection from gp120-primed huCD4 Tg spleens compared to gp120-primed controls. Boosting the primed huCD4 Tg mice with soluble gp120 and hen egg-white lysozyme led to lower secondary titers to both antigens than found in controls. Furthermore, splenocytes from gp120-pretreated immunized huCD4 Tg had a lower level of stimulation in response to anti-CD3 treatment. These in vivo results are consistent with in vitro data demonstrating that cross-linking CD4 on splenocytes of huCD4 Tg by rgp120SF2 and anti-gp120 not only sensitizes T cells for apoptosis, but also induces apoptosis per se, and suggest that anti-gp120 responsiveness can contribute to T cell depletion in AIDS.
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PMID:An ongoing immune response to HIV envelope gp120 in human CD4-transgenic mice contributes to T cell decline upon intravenous administration of gp120. 971 Feb 3

We have previously developed a human macrophage hybridoma model system to study the effect of HIV-1 infection on monocytic function. Upon coculture of one chronically (35 days postinfection) HIV-1-infected human macrophage hybridoma cell line, 43HIV, there was a dose-dependent decrease in the viability of cocultured Ag-stimulated T cells associated with an increase in DNA strand breaks. Enhanced apoptosis was determined by labeling with biotinylated dUTP and propidium iodide, increased staining with annexin V, increased side light scatter and expression of CD95, and decreased forward light scatter and expression of Bcl-2. There was also increased DNA strand breaks as determined by propidium iodide staining in unstimulated T cells cocultured with 43HIV and in T cells stimulated with anti-CD3 mAb and PHA. Pretreatment with 5145, a human polyclonal anti-gp120 Ab that recognizes the CD4 binding region, as well as with an anti-Fas ligand mAb blocked apoptosis in CD4+ T cells but not in CD8+ T cells. A soluble factor with a Mr below 10,000 Da was defined that induced apoptosis in CD4+ and CD8+ T cells and B cells. SDS-PAGE analysis of the active fractions revealed a band of 6000 Da that, after electroelution, had proapoptotic activity. The pI of the activity was estimated to be between 6.5 and 7.0. In conclusion, chronically HIV-1-infected monocytic cells induce apoptosis in bystander-, Ag-, anti-CD3-, and mitogen-stimulated T cells by multiple factors, which may contribute to the depletion of lymphocytes induced by HIV-1.
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PMID:Chronically HIV-1-infected monocytic cells induce apoptosis in cocultured T cells. 1705 88

The study of apoptosis in relation to various human disease states, particularly HIV infection, has seen a tremendous increase in activity. In this article, values obtained by seven different assays, designed to quantify apoptosis and applicable to the study of HIV infection, are compared in two cell systems: (1) stimulus-induced apoptosis in Jurkat cells treated with anti-Fas antibody and (2) spontaneous apoptosis in PBMCs isolated from HIV-infected children. The methods used included measurement of cells with subdiploid DNA content, labeling of DNA strand breaks by the TUNEL reaction, annexin V surface labeling for the detection of exposed phosphatidylserine, cytoplasmic antigen labeling with the apoptosis-specific antibody Apo 2.7, detection of changes in flow cytometric light-scattering properties, trypan blue dye exclusion by light microscopy, and detection of changes in cellular chromatin by fluorescence microscopy. These methods produced well-correlated values in the Jurkat system, whereas the same set of methods produced more discrepant values in the PBMC analyses, especially in those patients with low CD4 counts. Specifically, our results showed that the trypan blue test was unacceptable for quantification of apoptosis during HIV infection, whereas TUNEL, of all the methods tested, showed excellent overall correlation in both cell systems, was highly specific, and matched microscopic observation of the cells. Although many of the methods were suited to the study of a homogeneous cell line, caution must be exercised when examining cell death in a heterogeneous cell mixture from an HIV-infected individual.
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PMID:Comparison of seven quantitative assays to assess lymphocyte cell death during HIV infection: measurement of induced apoptosis in anti-Fas-treated Jurkat cells and spontaneous apoptosis in peripheral blood mononuclear cells from children infected with HIV. 982 19

We previously reported that butyric acid, an extracellular metabolite from periodontopathic bacteria, induced apoptosis in murine thymocytes, splenic T cells, and human Jurkat T cells. In this study, we examined the ability of butyric acid to induce apoptosis in peripheral blood mononuclear cells (PBMC) and the effect of bacterial lipopolysaccharide (LPS) on this apoptosis. Butyric acid significantly inhibited the anti-CD3 monoclonal antibody- and concanavalin A-induced proliferative responses in a dose-dependent fashion. This inhibition of PBMC growth by butyric acid depended on apoptosis in vitro. It was characterized by internucleosomal DNA digestion and revealed by gel electrophoresis followed by a colorimetric DNA fragmentation assay to occur in a concentration-dependent fashion. Butyric acid-induced PBMC apoptosis was accompanied by caspase-3 protease activity but not by caspase-1 protease activity. LPS potentiated butyric acid-induced PBMC apoptosis in a dose-dependent manner. Flow-cytometric analysis revealed that LPS increased the proportion of sub-G1 cells and the number of late-stage apoptotic cells induced by butyric acid. Annexin V binding experiments with fractionated subpopulations of PBMC in flow cytometory revealed that LPS accelerated the butyric acid-induced CD3(+)-T-cell apoptosis followed by similar levels of both CD4(+)- and CD8(+)-T-cell apoptosis. The addition of LPS to PBMC cultures did not cause DNA fragmentation, suggesting that LPS was unable to induce PBMC apoptosis directly. These data suggest that LPS, in combination with butyric acid, potentiates CD3(+) PBMC T-cell apoptosis and plays a role in the apoptotic depletion of CD4(+) and CD8(+) cells.
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PMID:Lipopolysaccharide stimulates butyric acid-induced apoptosis in human peripheral blood mononuclear cells. 986 91

Examination of annexin V binding, an indicator of early apoptosis, on lymphocytes from HIV+ people immediately after isolation showed that both CD4(+) and CD8(+) T cells were apoptotic, whereas B cell apoptosis was induced mainly after incubation. CD8(+) T cell apoptosis correlated with fewer CD4(+) T cells, but not the level of viremia. To determine potential mechanisms for apoptosis, we examined FasL expression, which was dramatically elevated on CD14(+) monocytes; however, antibody to FasL did not reproducibly inhibit apoptosis. Rather, CD8(+) T cell apoptosis was caused by antigen-presenting cells because removal of monocytes or addition of antibodies to CD80 and CD86 reduced apoptosis. B cell apoptosis also involved costimulatory signals delivered by T cells but not monocytes. A unique CD8(bright)CD28(dim) T cell population died after costimulation by monocytes. Because this population was increased in patients with undetectable viremia, abnormal antigen-presenting cells may contribute to continued CD8(+) T cell exhaustion by inducing apoptosis.
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PMID:Costimulatory pathways mediate monocyte-dependent lymphocyte apoptosis in HIV. 1007 59

Phosphatidylserine exposure in the exoplasmic leaflet of the plasma membrane is one of the early hallmarks of cells undergoing apoptosis. The shedding of membrane particles carrying Ags testifying to their tissue origin is another characteristic feature. Annexin V, a protein of as yet unknown specific physiologic function, presents a high Ca2+-dependent affinity for phosphatidylserine and forms two-dimensional arrays at the membrane surface. In this study, we report the delaying action of annexin V on apoptosis in the CEM human T cell line expressing CD4 and the normal cellular prion protein (PrPc), two Ags of particular relevance to cell degeneration and with different attachments to the membrane. The effect of annexin V was additive to that of z-Val-Ala-Asp-fluoromethyl ketone, a potent caspase inhibitor. Annexin V significantly reduced the degree of proteolytic activation of caspase-3, and totally blocked the release of CD4+ and PrPc+ membrane particles. z-Val-Ala-Asp-fluoromethyl ketone was a more powerful antagonist of caspase-3 processing, but prevented the shedding of CD4+ vesicles only partially and had no effect on that of PrPc+ ones. These results suggest that an external membrane constraint, such as that exerted by annexin V, has important consequences on the course of programmed cell death and on the dissemination of particular Ags. In vivo, annexin V had a significant protective effect against spleen weight loss in mice treated by an alkylating agent previously shown to induce lymphocyte apoptosis.
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PMID:Annexin V delays apoptosis while exerting an external constraint preventing the release of CD4+ and PrPc+ membrane particles in a human T lymphocyte model. 1022 3

Thymocytes and peripheral lymphocytes of BioBreeding (BB) diabetes-prone (BBDP) and diabetes-resistant (BBDR) rat were analysed by fluorescence-activated cell sorter (FACS). The number of CD4- CD8-, CD4+ CD8-, CD4- CD8+ and CD4+ CD8+ subsets was not different between BBDP and BBDR rat thymocytes, whereas spleen and lymph nodes in BBDP rats undergo severe T-cell lymphopenia. Notably, mature CD4- CD8+ [T-cell receptor (TCR)-alphabeta+ and CD5+] cells are certainly present in the BBDP rat thymus, unlike some previous reports, suggesting that the differentiation of CD4- CD8+ from CD4+ CD8+ cells occurs normally in the BBDP rat thymus. As a cause of peripheral T-cell lymphopenia we suspected apoptosis of recent thymic emigrants. By FACS analysis with fluorescein isothiocyanate-labelled annexin V, elevated apoptosis was evident in BBDP rat peripheral lymphocytes. Furthermore, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TUNEL) staining in BBDP rat splenic sections revealed that a number of TUNEL-positive cells were observed in the T-lymphocyte-rich area. From these results, we postulate that an abnormally elevated apoptosis of peripheral T lymphocytes, but not impaired thymocyte differentiation, is a cause of the peripheral T-cell lymphopenia in BBDP rats.
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PMID:Elevated apoptosis of peripheral T lymphocytes in diabetic BB rats. 1059 93

We previously reported that mice implanted with mammary tumors show a progressive thymic involution that parallels the growth of the tumor. The involution is associated with a severe depletion of CD4+8+ thymocytes. We have investigated three possible mechanisms leading to this thymic atrophy: 1) increased apoptosis, 2) decreased proliferation, and 3) disruption of normal thymic maturation. The levels of thymic apoptosis were determined by propidium iodide and annexin V staining. A statistically significant, but minor, increase in thymic apoptosis in tumor-bearing mice was detected with propidium iodide and annexin V staining. The levels of proliferation were assessed by in vivo labeling with 5'-bromo-2'-deoxyuridine (BrdU). The percentages of total thymocytes labeled 1 day following BrdU injection were similar in control and tumor-bearing mice. Moreover, the percentages of CD4-8- thymocytes that incorporated BrdU during a short term pulse (5 h) of BrdU were similar. Lastly, thymic maturation was evaluated by examining CD44 and CD25 expression among CD4-8- thymocytes. The percentage of CD44+ cells increased, while the percentage of CD25+ cells decreased among CD4-8- thymocytes from tumor-bearing vs control animals. Together, these findings suggest that the thymic hypocellularity seen in mammary tumor bearers is not due to a decreased level of proliferation, but, rather, to an arrest at an early stage of thymic differentiation along with a moderate increase in apoptosis.
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PMID:Early block in maturation is associated with thymic involution in mammary tumor-bearing mice. 1082 Feb 38


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