Identification of circulating regulatory T lymphocytes with membrane markers - a new multiparameter flow cytometry protocol.

INTRODUCTION: Regulatory T cells (Tregs) are a unique CD4+ T cell subset involved in the regulation of immune responses. The traditional immunophenotype used to define Tregs includes CD4+CD25high and the expression of the transcription factor Forkhead box protein 3 (FoxP3). A complex technique of intracellular staining, transient upregulation of FoxP3 in activated conventional T lymphocytes (Tcons), and the omission of naïve CD45RA+ Tregs with downregulated FoxP3 activity but a demethylated FOXP3 promoter region may lead to inaccurate quantification. In an attempt to meet the need for a reliable and simplified enumeration strategy, we investigated different membrane markers to capture the entire Treg compartment and to identify subpopulations of Tregs. MATERIAL AND METHODS: Analyses were performed on whole blood. Tested gating strategies were based on the expression of the following membrane antigens: CD45, CD3, CD4, CD25, CD127, CD26, CD6, CD39, CD71, HLA-DR, CD45RA and CD31. Double controls with FoxP3 were performed. RESULTS: The final enumeration panel consisted of the membrane markers CD45, CD3, CD4, CD25, CD127, CD26, CD39, CD45RA and CD31. A deep analysis of T cells with the CD4+CD25+CD127low/-CD26low/-CD45RAimmunophenotype revealed high expression of FoxP3 and/or CD39, while cells with the naïve immunophenotype, CD4+CD25+CD127low/-CD26low/-CD45RA+, presented lower expression of suppressor markers. Antigen CD31 is considered to be a valuable membrane marker of thymus-derived Tregs. CONCLUSIONS: The presented 9-color panel that can be easily applied in laboratories enables reliable enumeration of Tregs with additional information about the functionality, maturity and origin of T regulatory cells.

Impact of plant-based diet on lipid risk factors for atherosclerosis.

BACKGROUND: The aim of the study was to investigate the effect of a vegan diet on the serum lipid profile with particular regard to the parameters characterizing the high-density lipoprotein (HDL) fractions in subjects without subclinical atherosclerosis, measured by carotid Doppler ultrasonography. METHODS AND RESULTS: Forty-two 23 to 38 year old subjects (21 omnivores and 21 vegans) participated in the study. Compared to the omnivores, the vegan subjects were characterized by lower parameters of lipid profile: total cholesterol (p < 0.001), low-density lipoprotein (LDL)-cholesterol (p < 0.001), non-HDL-cholesterol (p < 0.001), apolipoprotein B (apoB) (p < 0.001) and phospholipids (p < 0.01). Concentration of HDL-cholesterol was apparently similar between groups. Furthermore, the parameters which characterize HDL particles (con-centration of apolipoproteins AI [apoAI] and AII, HDL-phospholipids, LpAI fraction and pre-b1-HDL fraction) were not significantly different between omnivore and vegan subjects. The apoB/apoAI ratio in vegans was lower than in omnivores (p < 0.01). There was no difference between serum concentration of triacylglycerols between omnivores and vegans. The activity of paraoxonase-1 and 8-iso-prostaglandin F2a concentration were also not different between the study groups. CONCLUSIONS: We suggest that a vegan diet may have a beneficial effect on serum lipid profile and cardiovascular protection, but it is not associated with changes in HDL composition.

PON-1 Activity and Plasma 8-Isoprostane Concentration in Patients with Angiographically Proven Coronary Artery Disease.

The aim of the study was to estimate association of the extent of angiographically proven coronary artery disease (CAD) with plasma 8-isoprostane F2 (8-iso-PGF2α) levels as a reliable marker of lipid peroxidation and serum activity of paraoxonase-1, which demonstrates the ability to protect against lipid oxidation. The study included 105 patients with angiographically documented CAD (CAD+) and 45 patients with negative results of coronary angiography (CAD-). Compared to the control group CAD+ patients were characterized by increased 8-iso-PGF2α levels (P = 0.007) and reduced activity of PON-1 towards paraoxon (PONase, P = 0.002) and phenyl acetate (AREase, P = 0.037). Univariate correlation analysis indicated that 8-iso-PGF2α concentrations were positively associated with the severity of CAD as evaluated by the Gensini score (R = 0.41, P < 0.001) while PONase activity (R = -0.26, P < 0.05) and AREase activity (R = -0.23, P < 0.05) were inversely correlated with CAD severity. PONase activity and 8-iso-PGF2α concentration remained independent determinant of atherosclerosis severity in multiple linear regression after adjusting for age, gender, smoking habits, hypertension, type 2 diabetes, statin therapy, and HDL-C and TAG concentration (ß coefficients -0.267; P < 0.05 and 0.368; P < 0.001, resp.). The results suggest that PON-1 activity and 8-iso-PGF2α concentration are associated with the presence and extent of coronary stenosis and may be considered additional markers of coronary artery disease.