Comparison of multiple assays for kinetic detection of apoptosis in thymocytes exposed to dexamethasone or diethylstilbesterol

Donner, K.J.; Becker, Karin; Hissong, Bruce D.; Ahmed, S. Ansar

doi:10.1002/(sici)1097-0320(19990101)35:1<80::aid-cyto11>3.3.co;2-#

Cited by 29 publications

(31 citation statements)

References 32 publications

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“…Apoptosis was evaluated by flow cytometric examination of cells stained with propidium iodide (PI), 7-aminoactinomycin D (7-AAD), or fluorescein isothiocyanate-annexin V; by forward/side scatter analysis, cell-size analyzer, and cytopathologic examination. In our extensive studies, apoptosis could only be detected in thymocyte cultures exposed to DEX but not in those exposed to DES (41). These studies imply that these two synthetic hormones induce atrophy of the thymus by dissimilar mechanisms in vivo.…”

Section: Animal Studesmentioning

confidence: 60%

Gender and risk of autoimmune diseases: possible role of estrogenic compounds.

Ahmed

Hissong

Verthelyi

et al. 1999

Environ Health Perspect

142

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A striking common feature of many autoimmune diseases in humans and experimental animals, despite differences in pathology, is that females are highly susceptible to autoimmune conditions compared to males. In several animal models, estrogens promote, whereas androgens abrogate, B-cell-mediated autoimmune diseases. To understand mechanisms by which estrogens regulate autoimmunity, it is first necessary to decipher estrogen effects on the normal immune system. Estrogen treatment of nonautoimmune mice diminished lymphocyte numbers in both developmental and mature lymphoid organs. Estrogen dysregulated T-and B-cell balance by inducing selective T-cell hypoactivity and B-cell hyperactivity. Even though estrogen did not alter the relative percentages of splenic T-cell subsets, splenic lymphocytes had a reduced proliferative response to T-cell stimulants and were refractory to rescue from activation-induced apoptosis compared to cells from placebo-treated mice. In contrast, estrogen induced B-cell hyperactivity (promoted autoantibodies to double-stranded DNA and phospholipids, increased numbers of plasma cells, and increased autoantibody yield per B cell). Note that treatment of normal mice with estrogen can alter T-and B-cell regulation and overcome B-cell tolerance to result in autoimmunity in normal individuals. Could environmental estrogens promote some human autoimmune disorders? Is there a link between environmental estrogens and autoimmune disorders, especially since these disorders are reported possibly more frequently? These provocative questions warrant investigation. Our findings on immunomodulatory effects may serve as a benchmark to examine whether endocrine-disrupting chemicals will have similar immunologic effects.

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Section: Animal Studesmentioning

confidence: 60%

Gender and risk of autoimmune diseases: possible role of estrogenic compounds.

Ahmed

Hissong

Verthelyi

et al. 1999

Environ Health Perspect

142

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“…Moreover, current apoptosis assays are often inadequately quantitative, leading to difficulties when quantitative information about disease pathways is desired. [20][21][22] …”

Section: Current Assay Methods For Apoptosismentioning

confidence: 99%

Wavelength-dependent backscattering measurements for quantitative real-time monitoring of apoptosis in living cells

2009

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Abstract. Apoptosis-programmed cell death-is a cellular process exhibiting distinct biochemical and morphological changes. An understanding of the early morphological changes that a cell undergoes during apoptosis can provide the opportunity to monitor apoptosis in tissue, yielding diagnostic and prognostic information. There is avid interest regarding the involvement of apoptosis in cancer. The initial response of a tumor to successful cancer treatment is often massive apoptosis. Current apoptosis detection methods require cell culture disruption. Our aim is to develop a nondisruptive optical method to monitor apoptosis in living cells and tissues. This would allow for real-time evaluation of apoptotic progression of the same cell culture over time without alteration. Elastic scattering spectroscopy ͑ESS͒ is used to monitor changes in light-scattering properties of cells in vitro due to apoptotic morphology changes. We develop a simple instrument capable of wavelength-resolved ESS measurements from cell cultures in the backward direction. Using Mie theory, we also develop an algorithm that extracts the size distribution of scatterers in the sample. The instrument and algorithm are validated with microsphere suspensions. For cell studies, Chinese hamster ovary ͑CHO͒ cells are cultured to confluence on plates and are rendered apoptotic with staurosporine. Backscattering measurements are performed on pairs of treated and control samples at a sequence of times up to 6-h posttreatment. Initial results indicate that ESS is capable of discriminating between treated and control samples as early as 10-to 15-min posttreatment, much earlier than is sensed by standard assays for apoptosis. Extracted size distributions from treated and control samples show a decrease in Rayleigh and 150-nm scatterers, relative to control samples, with a corresponding increase in 200-nm particles. Work continues to correlate these size distributions with underlying morphology. To our knowledge, this is the first report of the use of backscattering spectral measurements to quantitatively monitor apoptosis in viable cell cultures in vitro.

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“…Dual-color immunophenotyping of cell samples was performed on a Coulter Epics XL/MXL flow cytometer (Hialeah, Fla.). A gated lymphocyte population was derived from a bivariate histogram display of forward and side scatter, and immunofluorescence data were analyzed with the Immuno-4 software program (7,27).…”

Section: Methodsmentioning

confidence: 99%

Induction of Specific Cytotoxic Lymphocytes in Mice Vaccinated withBrucella abortusRB51

Vemulapalli

Zeytun

et al. 2001

Infect Immun

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A safe, more sensitive, nonradioactive, neutral red uptake assay was adopted to replace the traditional 51 Cr release assay for detection of Brucella-specific cytotoxic T lymphocyte (CTL) activity. Our studies indicated that Brucella abortus strain RB51 vaccination of mice induced specific CTLs against both strain RB51-and strain 2308-infected J774.A1 macrophages but not against Listeria monocytogenes-infected J774.A1 cells. The antigenspecific cytotoxic activity was exerted by T lymphocytes but not by NK cells. CD3 ؉ CD4 ؉ T cells secreted the highest level of gamma interferon (IFN-␥) and were able to exert a low but significant level of specific lysis of Brucella-infected macrophages. They also exerted a low level of nonspecific lysis of noninfected macrophages. In contrast, CD3؉ CD8 ؉ T cells secreted low levels of IFN-␥ but demonstrated high levels of specific lysis of Brucella-infected macrophages with no nonspecific lysis. These findings indicate that B. abortus strain RB51 vaccination of mice induces specific CTLs and suggest that CD3 ؉ CD4 ؉ and CD3 ؉ CD8 ؉ T cells play a synergistic role in the anti-Brucella activity.Brucella abortus is a gram-negative, facultative intracellular bacterial pathogen that causes brucellosis in humans and cattle (4). In the infected host, B. abortus multiplies within the phagosomes of phagocytic cells (e.g., macrophages) by inhibiting the phagosome-lysosome fusion (4). Similar to most of the intracellular bacterial infections, cell-mediated immunity seems to play a critical role in protection against virulent Brucella infection, although antibodies specific for the O polysaccharide of the lipopolysaccharide can confer a certain level of protection in some host species (1, 2, 17, 31). Passive transfer assays with mice indicated that both CD4 ϩ and CD8 ϩ T-cell subsets are involved in the protective immunity to brucellosis (1, 2). One mechanism by which immune T cells confer protection from B. abortus infection is by secreting molecules such as gamma interferon (IFN-␥), which stimulates the antimicrobial activity of macrophages, allowing intracellular bacterial killing (16,35). The crucial role of IFN-␥ in resistance to Brucella infection has been demonstrated for mice by in vivo antibody neutralization experiments (35). Another mechanism of T-cellmediated immunity is the lysis of infected cells by the specific cytotoxic T lymphocytes (CTLs) (23). Present knowledge about the role of CTLs in the acquired resistance to brucellosis is limited. The development of Brucella-specific CTLs in vaccinated animals and the phenotypic and functional characterization of such CTL's have not been studied in detail. The classic CTL assay is based on determining the level of 51 Cr released from lysed target cells. Unfortunately, this assay is not very sensitive (23,24), and the use of radioactive 51 Cr in a biosafety level 3 environment restricts the usefulness of this assay in Brucella research even further. Therefore, we have developed a highly sensitive, nonradioactive assay for Brucella...

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Comparison of multiple assays for kinetic detection of apoptosis in thymocytes exposed to dexamethasone or diethylstilbesterol

Abstract: Two of the six assays, 7-AAD and annexin, were similar in detecting apoptosis at an early kinetic time point. Results of both assays were comparable at all time points studied. Our studies imply that DEX and DES induce thymic atrophy, in vivo, by different mechanisms.

Cited by 29 publications

References 32 publications

Gender and risk of autoimmune diseases: possible role of estrogenic compounds.

Gender and risk of autoimmune diseases: possible role of estrogenic compounds.

Wavelength-dependent backscattering measurements for quantitative real-time monitoring of apoptosis in living cells

Induction of Specific Cytotoxic Lymphocytes in Mice Vaccinated withBrucella abortusRB51

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