Identification of potential anti-photoageing algal compounds using an in-vitro model of photoageing.

Stress-induced premature senescence (SIPS) has been proposed as an in-vitro model for testing the long-term effects of stressful events and to find molecules/natural extracts that protect against such stress. Premature senescence of human skin diploid fibroblasts (HDFs) can be induced by repeated subcytotoxic exposure to UVB, with the appearance of so-called biomarkers of senescence such as growth arrest, senescence-associated beta-galactosidase activity, senescence-associated gene over-expression and the common 4977-bp mitochondrial DNA deletion. This model of UVB-induced premature senescence has been acknowledged as a robust in-vitro model in photoageing research. In this study, the potential anti-photoageing effects of a series of algal extracts were tested. The appearance of the biomarkers of UVB-induced premature senescence of HDFs was studied with or without algal extracts. One algal extract was shown to be particularly protective against UVB-induced SIPS. The results obtained here reinforce the notion that UVB-induced premature senescence of HDFs can be used to screen potential anti-photoageing compounds.

Distinct pattern of IL-2 and IFN-gamma gene expression in CD4 and CD8 T cells: cytofluorometric analysis at a single cell level using non-radioactive probes.

IL-2 and IFN-gamma gene expression was analyzed using an original method for in situ hybridization (ISH) with non-isotopic probes and flow cytometric analysis (FC). This method permits rapid detection of mRNA at a single cell level among in vitro activated human peripheral blood mononuclear cells (PBMC) and purified CD4 and CD8 T cell subsets. After stimulation with PMA and ionomycin (PMA+Io), cells were fixed at different times and hybridized with digoxigenin (DIG)-labelled RNA antisense or sense probes specific for IL-2 and IFN-gamma. The level of cytokine gene expression in individual cells was visualized using FITC-conjugated anti-DIG antibodies and the fluorescent signal was analyzed by flow cytometry. Specific hybridization with IL-2 and IFN-gamma antisense probes was detected among activated PBMC within lymphoid cells identified by their light scattering properties. Kinetic analysis of the frequency of mRNA producing cells exhibited a biphasic pattern with an early peak at 6-8 hrs. when percentages of IL-2 and IFN-gamma expressing cells reached 35 +/- 7% and 18 +/- 4%, respectively. Similar data were obtained by enzymatic detection on cell smears using AP-conjugated anti-DIG. Combination of ISH with FC was applied to the comparison of the pattern of cytokine gene expression between CD4 and CD8 T cell subsets isolated by negative selection using immunobeads and magnetic separation. IL-2 was expressed by activated CD8 T cells (25-35%), but CD4 T cells were the major producers of IL-2 as assessed by the high frequency of mRNA expressing cells (60%) and the large amount of mRNA per cell relative to the mean fluorescence intensity. In contrast, IFN-gamma mRNA was preferentially expressed by CD8 T cells (27-37%) and a minority of CD4 T cells (17-23%). Despite quantitative differences, kinetic analysis of IL-2 gene expression in CD4 and CD8 T cells showed similar profiles with an early peak at 6-8 hrs. Upregulation of IL-2 gene expression in CD4 T cells by CD28 co-stimulation increases the amount of IL-2 mRNA per cell as visualized by mean fluorescence intensity. In addition the effect of CD28 co-stimulation on IL-2 mRNA stabilisation was demonstrated by the maintenance of a high frequency of IL-2 expressing CD4 T cells and an elevated level of mRNA per cell for prolonged period after PMA+Io stimulation. By contrast CD28 co-stimulation had no obvious effect on IFN-gamma expression.

In situ hybridization and cytofluorometric analysis of cytokine mRNA during in vitro activation of human T cells.

We present an original method for in situ hybridization (ISH) using non isotopic probes and flow cytometry analysis that permits rapid detection of lymphokine transcripts at single cell level in an in vitro activated human Jurkat T cell line and in peripheral blood T cell subsets. After PMA and either ionomycin or ConA stimulation, cells were fixed and hybridized with digoxigenin (DIG)-labelled RNA antisense or sense probes specific for IL-2 and IFN-gamma. The level of cytokine gene expression in individual cells was visualised using FITC-conjugated anti-DIG antibody, and the resultant signal was analysed by flow cytometry. IL-2 mRNA was first detected in activated Jurkat T cells. Addition of cycloheximide 4 hours after the beginning of stimulation increased both the frequency of labelled cells and the amount of mRNA per cell, as determined by the mean fluorescence intensity. The specificity and sensitivity of IL-2 mRNA detection were tested by comparison with Northern blot analysis and in situ hybridization (ISH) with immuno-cytochemical staining. IL-2 and IFN-gamma mRNA were detectable in PBMC as early as 3 hours after in vitro stimulation with PMA and ionomycin. The frequency of positive cells and the amount of mRNA per cell peaked at 6-8 hours, when the percentages of IL-2 and IFN-gamma mRNA-containing cells reached 30-40% and 15-20%, respectively. The two lymphokines were expressed in both CD4+ and CD8+ T cells, but the frequency of IL-2 expressing cells and the amount of IL-2 mRNA per cell were higher in CD4+ (60%) than in CD8+ T cells (25%), whereas IFN-gamma were preferentially transcribed by CD8+ T cells (40%). The results obtained by this method were in accordance with the data obtained by Northern blot analysis, with cellular protein content estimated by immuno-fluorescence staining, and with IL-2 titration by bioassay. We compared the performance of this method with ISH using radioactive probes.