Effect of the intake of resveratrol, resveratrol phosphate, and catechin-rich grape seed extract on markers of oxidative stress and gene expression in adult obese subjects.

BACKGROUND: The preventive effect of resveratrol (RES) on the development of human diseases has been verified by numerous epidemiological studies. Resveratrol triphosphate (RTP) is a stable derivative of RES in which phosphate groups protect the phenolic groups. AIMS: This study compared the effect of RTP on biochemical and molecular markers of oxidative stress to equimolar doses (0.66 mmol) of RES and catechin-rich grape seed extract (CGSE) in a model of oxidative and metabolic stress associated with obesity in humans. METHODS: Thirty-two obese subjects (BMI between 30 and 40) were enrolled. They all received 1 capsule of placebo/day for 28 days before being randomly devised into three arms receiving 1 capsule/day of RES, CGSE, or RTP during the following consecutive 28 days. Blood samples were collected at baseline, after the end of placebo intake, and after the end of the investigational product intake. Biochemical parameters of oxidative stress and blood expression of 200 redox-related genes were determined at each time point. RESULTS: RTP and CGSE showed better antioxidant activities compared to RES and induced important modulations of gene expression. CONCLUSION: The results suggest that RTP and CGSE could contribute to a significant reduction of oxidative stress in obese subjects.

Functionally active macrophage-derived myeloperoxidase in the skin of drug-induced toxic epidermal necrolysis.

BACKGROUND: Drug-induced toxic epidermal necrolysis (TEN) probably results from a complex and specific immune cell reaction involving lymphocytes and macrophages. OBJECTIVE: To assess the functional role of macrophages in TEN. METHODS: Immunohistochemistry was performed on biopsies from early blisters developed in 9 TEN patients. The amount of extracellular myeloperoxidase (MPO) was measured by ELISA in TEN blister fluid and serum. Controls were blister fluids taken from 9 second-degree burns. In addition, 3-chlorotyrosine (a specific marker of MPO activity) was searched for using liquid mass chromatography both in TEN and burn blister fluids. RESULTS: Immunohistochemistry revealed numerous CD68+ macrophages in 8/9 TEN patients; 5-20% of these cells and rare CD15+ neutrophils exhibited MPO immunoreactivity, while keratinocytes were negative. The amount of MPO was significantly higher in TEN blister fluid than in TEN serum, suggesting macrophage production of MPO in the skin. In addition, MPO was significantly more abundant in TEN blister fluid than in burn blister fluid. 3-Chlorotyrosine was detected in 7/9 TEN blister fluids, but in only 2/9 burn blister fluids. DISCUSSION: MPO produced by macrophages was functionally active in most TEN patients, leading to the production of hypochlorous acid, a potent oxidative compound that alters keratinocytes.

Effects of oral contraception with ethinylestradiol and drospirenone on oxidative stress in women 18-35 years old.

BACKGROUND: Oral contraceptives (OCs) with estrogens and progestins may affect oxidative stress (OS) status. STUDY DESIGN: A group of 32 women using oral contraceptives (OCU) containing 0.03 mg ethinylestradiol and 3 mg drospirenone have been compared to a matched control group of 30 noncontraception users (NCU). Blood levels of antioxidants, trace elements and markers of lipid peroxidation were assessed by biochemical methods. A microarray analysis of whole blood mRNA levels of 200 genes involved in OS-dependant pathway was also performed. RESULTS: Levels of zinc, vitamin E and antibodies to oxidized low-density lipoproteins (LDLs) were not significantly different between the two groups. On the other hand, significant increases in the mean levels of lipid peroxides (+176%, p<.001), oxidized LDLs (+145%, p<.002), copper (+103%, p<.001), Cu/Zn ratio (+100%, p<.001) and a significant decrease in the mean level of beta-carotene (-41%, p<.01) were observed in the OCU compared to NCU. There was a highly significant positive correlation between the lipid peroxide levels and the copper-to-zinc ratio. From the 200 genes tested by microarray, one coding for HSP70 was significantly up-regulated (log(2) fold change=+ 0.45, p<.02) and one coding for inducible nitric oxide synthase significantly down-regulated (log(2) fold change=-0.24, p<.05) in the OCU compared to the NCU. CONCLUSIONS: The recently introduced combination of ethinylestradiol and drospirenone induced the heightening of lipid peroxidation correlated with high levels of copper, a situation that could be associated with increased cardiovascular risk.

Monitoring of cellular responses after vaccination against tetanus toxoid: comparison of the measurement of IFN-gamma production by ELISA, ELISPOT, flow cytometry and real-time PCR.

One of the challenges for immunomonitoring in clinical trials is to detect an antigen specific T cell-mediated immune response. In an attempt to define the most suitable assay, tetanus toxoid was used to compare the capacity of 4 different methods to detect cytokine responses, before and after recall vaccination, in peripheral blood mononuclear cells (PBMC) of 14 healthy volunteers. ELISA, ELISPOT, intracytoplasmic detection and real-time RT-PCR were chosen to measure IFN-gamma production before and after vaccination. As far as the detection of memory T cell status (before vaccination) was concerned, we found that ELISPOT was the most sensitive method to discriminate TT-induced from spontaneous responses. On the other hand, intracytoplasmic cytokine detection was the most efficient method to detect the restimulating effect of TT vaccination.

Immune monitoring in whole blood using real-time PCR.

There is a need for simple and sensitive assays to assess innate and adaptive immune responses to microbial agents and vaccines. Herein, we describe a whole blood method allowing to measure the induction of cytokine synthesis at the mRNA level. The originality of this method consists in the combination of PAXgene tubes containing an mRNA stabilizer for blood collection, the MagNA Pure instrument as an automated system for mRNA extraction and RT-PCR reagent mix preparation, and the real-time PCR methodology on the Lightcycler for accurate and reproducible quantification of transcript levels. We first demonstrated that this method is adequate to measure the induction of interleukin (IL)-1beta and IL-1 receptor antagonist (IL-1 RA) mRNA upon the addition of bacterial lipopolysaccharide (LPS) to whole blood. We then showed that this approach is also suitable to detect the production of mRNA encoding T cell-derived cytokines in whole blood incubated with tetanus toxoid as a model of in vitro immune response to a recall antigen. Finally, we demonstrated that this methodology can be used successfully to assess inflammatory as well as T cell responses in vivo, as it allowed to detect the induction of IL-1beta and IL-1 RA after injection of LPS in healthy volunteers, and also the induction of IL-2 upon recall immunisation with tetanus vaccine.