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Biomedical subjects

A C Pinder

Publications and source records attributed to A C Pinder.

At least 19 recordsLinked to original sources
PubMed2000

Apoptosis can be detected in attached colonic adenocarcinoma HT29 cells using annexin V binding, but not by TUNEL assay or sub-G0 DNA content.

BACKGROUND: Induction of apoptosis in adherent cell lines is associated with cell loss from the substratum. In this study the adenocarcinoma cell line, HT29, treated with indomethacin (400microM) has been employed as a model system to demonstrate how flow cytometric analysis can be used to quantify the changes that occur during this process. METHODS: Adherent and floating cell populations have been analyzed independently for effects on cell number, cell cycle characteristics and apoptosis using TUNEL assay and Annexin V binding. In addition apoptosis has been assessed using DNA laddering and morphology. RESULTS: Apoptosis was detected in adherent cells treated with indomethacin using Annexin V binding but not by other techniques employed in this study. In contrast, analysis of "floating" cells revealed the presence of apoptotic cells both in control and indomethacin treated cells using all the techniques employed. However quantification by flow cytometry showed that a significantly higher proportion of control "floaters" were late apoptotic/necrotic rather than apoptotic. DISCUSSION: The data here illustrate the need to interpret measures of apoptosis in adherent cell lines with care and the value of using flow cytometric techniques in the quantitative evaluation of the process.

Acridine Orange↗
PubMed2000

n-3 polyunsaturated fatty acids inhibit the antigen-presenting function of human monocytes.

Diets rich in n-3 polyunsaturated fatty acids (PUFAs) are associated with suppression of cell-mediated immune responses, but the mechanisms are unclear. We hypothesized that n-3 PUFAs can inhibit the function of human antigen-presenting cells. A prerequisite for this role of blood monocytes is the cell surface expression of major histocompatibility complex (MHC) class II molecules [human leukocyte antigen (HLA)-DR, -DP, and -DQ], aided by the presence of intercellular adhesion molecule-1 (ICAM-1) and leukocyte function associated antigens 1 and 3. We showed previously that the n-3 PUFA eicosapentaenoic acid (EPA) inhibits the expression of HLA-DR on unstimulated human monocytes in vitro, but that docosahexaenoic acid (DHA) enhances its expression. However, both n-3 PUFAs suppress the expression of HLA-DR, HLA-DP, and ICAM-1 on interferon-gamma-activated monocytes. We also established that dietary fish-oil supplementation can inhibit the expression of these surface molecules on circulating human monocytes. We subsequently showed that when EPA and DHA were combined in the same ratio as is commonly found in fish-oil-supplement capsules (3:2), there was no significant effect in vitro on the expression of HLA-DR on unstimulated monocytes, but the expression on activated monocytes remained significantly inhibited. In the same in vitro system, the ability of activated monocytes to present antigen to autologous lymphocytes was significantly reduced after culture with the combined n-3 PUFAs. These findings provide one potential mechanism for the beneficial effect of fish oil in the treatment of rheumatoid arthritis, a disorder associated with elevated expression of MHC class II and adhesion molecules on monocytes present within affected joints.

Adult↗
PubMed1999

Effect of eicosapentaenoic acid on the proliferation and incidence of apoptosis in the colorectal cell line HT29.

Fish oil has been shown to reduce the induction of colorectal cancer in animal models by a mechanism which may involve suppression of mitosis, increased apoptosis, or both. We used the human colonic adenocarcinoma cell line HT29 to explore the effects of the long-chain n-3 polyunsaturated fatty acid eicosapentaenoic acid (EPA) on cell proliferation and death in vitro. Cells were cultured in media containing EPA at 5, 10, and 15 microg/mL. Cell number and thymidine incorporation were used to quantify proliferation, and cell cycle effects were studied using flow cytometry. Gel electrophoresis, annexin-V binding, and morphological criteria were used to characterize apoptosis. Adherent cells and freely floating detached cells were treated as two distinct populations. In the presence of EPA at 10 and 15 microg/mL there was a marked reduction in the growth rate of adherent HT29 colonies, owing to an increased detachment of adherent cells. After treatment with 10 or 15 microg/mL EPA the proportion of adherent cells in S-phase increased, indicating either a block in late S-phase or early G2. Floating cells showed evidence of extensive DNA cleavage, but the proportion of floating cells with sub GO DNA content declined on treatment with 10 or 15 microg/mL EPA even though the number of floating cells increased. We conclude that EPA does not inhibit mitosis of adherent cells, but increases the rate at which they become detached from the substrate, probably at an early stage in the initiation of apoptosis. This mechanism may be analogous to "anoikis," or induction of apoptosis in response to loss of cell contact, and may contribute to the anticarcinogenic effects of fish oil in vivo.

Acridine Orange↗
PubMed1998

Improved detection of bacteria by flow cytometry using a combination of antibody and viability markers.

A proprietary fluorogenic marker for cell viability (Chemchrome) was investigated for the detection of bacteria using flow cytometry. This marker was used in combination with fluorescently labelled monoclonal antibodies (against Salmonella typhimurium). Owing to the former's broad band emission spectrum, it was necessary to use the novel dye RED613 for the antibodies. This combined protocol, being sensitive only to the live Salm. typhimurium cells, reduced errors due to intrinsic fluorescence and non-specific binding. Detection of the order of 100 cells ml-1 was achieved in 30 min. This level was achieved even in the presence of large numbers of non-target or dead organisms.

Antibodies, Monoclonal↗
PubMed1997

The effect of beta-carotene supplementation on the immune function of blood monocytes from healthy male nonsmokers.

Although there is strong epidemiologic evidence that diets rich in carotenoids such as beta-carotene are associated with a reduced incidence of cancer, the cellular mechanisms underlying this phenomenon remain unknown. This article describes the effect of dietary beta-carotene supplementation on both the expression of functionally associated surface molecules on human monocytes and on the secretion of the cytokine tumor necrosis factor-alpha (TNF-alpha) by monocytes, all of which are involved in the initiation and regulation of immune responses involved in tumor surveillance. A double-blind, placebo-controlled, crossover study was undertaken in which 25 healthy, adult male nonsmokers were randomly assigned to receive beta-carotene (15 mg daily) or placebo for 26 days, followed by the alternative treatment for a further 26 days. The expression of functionally related monocyte surface molecules was quantified by flow cytometry, and ex vivo secretion of TNF-alpha was quantified by an enzyme-linked immunosorbent assay, before and after each treatment period. After dietary supplementation there were significant increases in plasma levels of beta-carotene and in the percentages of monocytes expressing the major histocompatibility complex class II molecule HLA-DR and the adhesion molecules intercellular adhesion molecule-1 and leukocyte function-associated antigen-3. In addition, the ex vivo TNF-alpha secretion by blood monocytes was significantly increased after supplementation. These findings suggest that moderate increases in the dietary intake of beta-carotene can enhance cell-mediated immune responses within a relatively short period of time, providing a potential mechanism for the anticarcinogenic properties attributed to beta-carotene.

Adult↗
PubMed1997

N-3 polyunsaturated fatty acids modulate the expression of functionally associated molecules on human monocytes and inhibit antigen-presentation in vitro.

N-3 polyunsaturated fatty acid (PUFA)-rich diets are associated with suppression of cell-mediated immune responses, but the mechanisms are unclear. Specific immune responses are initiated by antigen-presenting cells (APC). We have previously shown in vitro that the n-3 PUFA, eicosapentaenoic acid (EPA), inhibits the expression of HLA-DR, an MHC class II molecule required for normal APC function on human blood monocytes. In contrast, docosahexaenoic acid (DHA) enhanced the expression of this molecule on unstimulated monocytes, but both n-3 PUFA suppressed its expression on interferon-gamma (IFN-gamma)-activated monocytes. In the present study we show that when EPA and DHA were combined at the same ratio as is commonly found in fish oil supplement capsules (3:2) there was no significant effect in vitro on the expression of HLA-DR on unstimulated monocytes, but the expression on IFN-gamma-activated monocytes remained significantly inhibited. In the same in vitro system a significant reduction in the ability of IFN-gamma-activated monocytes to present tetanus toxoid antigen to autologous lymphocytes was observed following culture with the combined n-3 PUFA. These findings support previous animal studies which suggest that n-3 PUFA can inhibit the antigen-presenting function of mononuclear phagocytes.

Adult↗
PubMed1996

Fish oil supplementation inhibits the expression of major histocompatibility complex class II molecules and adhesion molecules on human monocytes.

To test the hypothesis that fish oil supplementation can inhibit the expression of functionally associated molecules on the surface of human blood monocytes, we randomly assigned 12 healthy adults to receive either an n-3 polyunsaturated fatty acid-rich fish oil supplement for 21 d or to receive no supplement. The percentage of monocytes expressing major histocompatibility complex (MHC) class II molecules (HLA-DR, -DP, and -DQ), intercellular adhesion molecule-1, and leukocyte-function-associated antigen-1, and the intensity of expression of each molecule were quantified before and after the study period. Monocytes were examined immediately after blood sampling and again after incubation in serum-free culture medium for 24 h in the presence of interferon-gamma to up-regulate expression of MHC class II molecules by the monocytes. The intensity of expression of all the monocyte surface molecules examined was significantly reduced after fish oil supplementation (P < 0.025), although there was no change in the percentage of monocytes expressing each molecule. After incubation with interferon-gamma, there was a similar inhibition of surface molecule expression (with the exception of HLA-DQ) by monocytes from the fish oil-supplemented group, and there was a reduction in the percentage of monocytes expressing both HLA-DR and -DP molecules (P < 0.025). No significant changes were observed in the reference group. Dietary supplementation with fish oil can inhibit the expression of surface molecules involved in the function of human antigen-presenting cells, a potential mechanism by which n-3 fatty acids may suppress cell-mediated immune responses.

Adult↗
PubMed1996

(n-3) Polyunsaturated fatty acids modulate the expression of functionally associated molecules on human monocytes in vitro.

Diets rich in (n-3) polyunsaturated fatty acids (PUFA) are associated with suppression of the immune system, but the mechanisms are unclear. Specific immune responses are initiated by antigen-presenting cells. This study examines the in vitro effect of the (n-3) PUFA eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on the expression of cell surface molecules required for normal antigen-presenting cell function on human blood monocytes. Monocytes were incubated with or without EPA or DHA for 48 h at 37 degrees C. Following incubation, expression of major histocompatibility complex (MHC) class II molecules (HLA-DR, -DP and -DQ) and adhesion molecules [intercellular adhesion molecule-1 (ICAM-1) and leucocyte function associated antigen-1] was quantified by flow cytometry. In the presence of EPA alone there was a significantly lower median intensity of expression of HLA-DR and ICAM-1 relative to incubations without EPA. In contrast, significantly greater median intensities of expression of HLA-DR and -DP were observed following incubation with DHA. In parallel experiments, where monocytes were simultaneously activated by the addition of interferon-gamma to the cultures, median expression intensities of HLA-DR, -DP and ICAM-1 were significantly lower in the presence of either EPA or DHA compared with incubations without the (n-3) PUFA. These findings support previous animal studies that suggest that (n-3) PUFA can influence immune reactivity by modulating antigen-presenting cell function.

Adult↗
PubMed1994

Detection of low levels of specific Salmonella species by fluorescent antibodies and flow cytometry.

The use of fluorescently-labelled monoclonal antibodies, with detection by multi-parameter flow cytometry, was investigated for the rapid detection of salmonellas in pure cultures. Accurate detection of specific Salmonella serotypes was demonstrated down to levels of below 10(4) cells ml-1 (within 30 min) and 1 cell ml-1 (after 6 h non-selective pre-enrichment). This level of sensitivity was attained even in the presence of high levels of other bacterial species that would otherwise have interfered with the results. With combinations of different antibodies, each with a unique fluorescent label, simultaneous analysis for two species was possible.

Antibodies, Bacterial↗
PubMed1994

Rapid assay for pathogenic Salmonella organisms by immunofluorescence flow cytometry.

Multi-parameter flow cytometry was investigated for the rapid detection of specific serotypes of salmonellas (S. typhimurium and S. montevideo) labelled with fluorescent monoclonal antibodies, both in pure culture and in a typical food matrix (full-fat milk). In all cases, the method was accurate to levels of below 10(4) target cells per ml for a total assay time of about 30 min. After 6 h non-selective enrichment in the presence of a 10,000-fold excess of competing micro-organisms (Escherichia coli) the corresponding detection limit was about 20 cells ml-1. These results suggest that flow cytometry has significant potential for the detection of pathogenic micro-organisms in the food industry.

Animals↗
PubMed1994

Detection of Salmonella typhimurium in dairy products with flow cytometry and monoclonal antibodies.

Flow cytometry, combined with fluorescently labelled monoclonal antibodies, offers advantages of speed and sensitivity for the detection of specific pathogenic bacteria in foods. We investigated the detection of Salmonella typhimurium in eggs and milk. Using a sample clearing procedure, we determined that the detection limit was on the order of 10(3) cells per ml after a total analysis time of 40 min. After 6 h of nonselective enrichment, the detection limits were 10 cells per ml for milk and 1 cell per ml for eggs, even in the presence of a 10,000-fold excess of Escherichia coli cells.

Animals↗
PubMed1990

Validation of flow cytometry for rapid enumeration of bacterial concentrations in pure cultures.

Flow cytometry was investigated as a rapid detection and counting method for bacteria in pure cultures. A simple two-parameter detection scheme was employed: particle size was measured by forward angle light scatter and nucleic acid content by fluorescence of the DNA/RNA-binding dye ethidium bromide. The technique gave results that correlated exceptionally well with conventional plate counting for four species of bacteria, and concentrations in the range 10(2) to 10(7) cfu/ml. Cytometric counts were obtained in a few minutes, as compared with 2 d required for the plate counts. Under ideal conditions, each bacterial species examined exhibited a characteristic 'signature' on the cytometer, which could be explained by its known properties and morphology.

Aeromonas↗
PubMed1987

Flow cytometry and its applications in veterinary medicine.

Flow cytometry is a technique for analysing and separating populations of cells (and subcellular components). The cells are generally stained with fluorescent markers (eg, fluorescent antibodies or DNA-binding dyes). Each cell is analysed individually, at high speed. Thus, assays may be performed on small samples (less than 10,000 cells). A subpopulation of interest can be separated from the remainder of the cells to a high degree of purity (up to 99 per cent). The application of flow cytometry to veterinary science is increasing and is discussed here.

Animals↗
PubMed1986

The application of flow microfluorimetry to biomedical research and diagnosis: a review.

The application of flow cytometric or microfluorimetric techniques to the biomedical sciences is a major growth area. Flow microfluorimetry is the science of analyzing and separating populations of cells (and their sub-cellular components) using fluorescent markers (usually fluorescent antibodies or fluorescent DNA-binding dyes). Each cell is analysed individually allowing assays to be performed on small samples (less than 10,000 cells). Interesting cells can be sorted out to a high degree of purity (up to 99%). Flow microfluorimetric analysis enjoys multidisciplinary use, ranging from cell biology, microbiology and immunology to cell cycle analysis and "flow karyotyping" of cells. Examples taken from both clinical and research settings are discussed.

Animals↗