Improving the immunomodulatory function of mesenchymal stem cells by defined chemical approach.

Mesenchymal stem cell (MSC) is a potential therapeutic material that has self-renewal, multilineage differentiation, and immunomodulation properties. However, the biological function of MSCs may decline due to the influence of donor differences and the in vitro expansion environment, which hinders the advancement of MSC-based clinical therapy. Here, we investigated a method for improving the immunomodulatory function of MSCs with the help of small-molecule compounds, A-83-01, CHIR99021, and Y27632 (ACY). The results showed that small-molecule induced MSCs (SM-MSCs) could enhance their immunosuppressive effects on T cells and macrophages. In vivo studies showed that, in contrast to control MSCs (Ctrl-MSCs), SM-MSCs could inhibit the inflammatory response in mouse models of delayed hypersensitivity and acute peritonitis more effectively. In addition, SM-MSCs showed the stronger ability to inhibit the infiltration of pro-inflammatory T cells and macrophages. Thus, small-molecule compounds ACY could better promote the immunomodulatory effect of MSCs, indicating it could be a potential improving method in MSC culture.

Correlation between miR-126 expression and DNA hypomethylation of CD4+ T cells in rheumatoid arthritis patients.

It has been known that the occurrence of rheumatoid arthritis (RA) was closely correlated with DNA hypomethylation in CD4+ T cells, in which DNA methyltransferase plays a certain role. This study therefore investigated the effect of miR-126 on CD4+ T cell subgroup in RA patients and the alternation of DNA hypomethylation, in an attempt to provide new sights into the pathogenesis and treatment of RA. CD4+ T cells separated from RA patients were transfected with miRNA (miR)-126 expression vector or miR-126 inhibitor expression vector. The expression levels of CD11a, CD70 and DNMT1 mRNA were examined by real-time PCR. Protein levels of CD11a and CD70 were tested by flow cytometry while DNMT1 protein level was quantified by Western blotting. DNA was modified by sodium bisulfite and was sequenced for the methylation status of promoters of CD11a and CD70 genes. Both mRNA and protein expressions of CD11a and CD70 genes in CD4+ T cells were elevated by miR-126 transfection, along with decreased DNMT1 protein level but not mRNA level. The methylation degree of promoters of both CD11a and CD70 genes were significantly depressed after miR-126 transfection. The transfection by miR-126 inhibitor effectively reversed such effects. In RA patients, elevated miR-126 may promote the expression of CD11a and CD70 via the induction of hypomethylation of gene promoters by depressing DNMTI1 protein levels.

Study of the metabolism on tobacco-specific N-nitrosamines in the rabbit by solid-phase extraction and liquid chromatography-tandem mass spectrometry.

Metabolism of four tobacco-specific N-nitrosamines (TSNAs), N'-nitrosonornicotine (NNN), N'-nitrosoanatabine (NAT), N'-nitrosoanabasine (NAB), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) has been studied by solid-phase extraction (SPE) and liquid chromatography-tandem mass spectrometry (LC-MS-MS). 4-(Methylnitrosamino)-4-(3-pyridyl)-1-butanol (iso-NNAL) was used as internal standard. SPE and LC-MS-MS was found to be a rapid, simple, sensitive, and selective method for analysis of TSNAs in rabbit serum. The relative standard deviation (R.S.D., n = 6) for analysis of 5 ng mL(-1) and 0.5 ng mL(-1) standards and of serum sample spiked with 5 ng mL(-1) standards of five TSNAs was 2.1-11% and recovery of 5 ng mL(-1) standards from serum was 100.2-112.9%. A good linear relationship was obtained between peak area ratio and concentration in the range of 0.2-100 ng mL(-1) for NNAL and 0.5-100 ng mL(-1) for other four TSNAs, with correlation coefficients (R2) >0.99 (both linear and log-log regression). Detection limits for standards in solvent were between 0.04 and 0.10 ng mL(-1). Doses of TSNAs administered to rabbits via the auricular vein were 4.67 microg kg(-1) and 11.67 microg kg(-1), in accordance with the different levels in cigarettes. Metabolic curves were obtained for the four TSNAs and for 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), a metabolite of NNK; on the basis of these curves we modeled metabolic kinetic equations for these TSNAs by nonlinear curve fitting.