A microRNA profile of human CD8(+) regulatory T cells and characterization of the effects of microRNAs on Treg cell-associated genes.

BACKGROUND: Recently, regulatory T (Treg) cells have gained interest in the fields of immunopathology, transplantation and oncoimmunology. Here, we investigated the microRNA expression profile of human natural CD8(+)CD25(+) Treg cells and the impact of microRNAs on molecules associated with immune regulation. METHODS: We purified human natural CD8(+) Treg cells and assessed the expression of FOXP3 and CTLA-4 by flow cytometry. We have also tested the ex vivo suppressive capacity of these cells in mixed leukocyte reactions. Using TaqMan low-density arrays and microRNA qPCR for validation, we could identify a microRNA 'signature' for CD8(+)CD25(+)FOXP3(+)CTLA-4(+) natural Treg cells. We used the 'TargetScan' and 'miRBase' bioinformatics programs to identify potential target sites for these microRNAs in the 3'-UTR of important Treg cell-associated genes. RESULTS: The human CD8(+)CD25(+) natural Treg cell microRNA signature includes 10 differentially expressed microRNAs. We demonstrated an impact of this signature on Treg cell biology by showing specific regulation of FOXP3, CTLA-4 and GARP gene expression by microRNA using site-directed mutagenesis and a dual-luciferase reporter assay. Furthermore, we used microRNA transduction experiments to demonstrate that these microRNAs impacted their target genes in human primary Treg cells ex vivo. CONCLUSIONS: We are examining the biological relevance of this 'signature' by studying its impact on other important Treg cell-associated genes. These efforts could result in a better understanding of the regulation of Treg cell function and might reveal new targets for immunotherapy in immune disorders and cancer.

Intraoperative ketorolac in high-risk breast cancer patients. A prospective, randomized, placebo-controlled clinical trial.

BACKGROUND: Ketorolac has been associated with a lower risk of recurrence in retrospective studies, especially in patients with positive inflammatory markers. It is still unknown whether a single dose of pre-incisional ketorolac can prolong recurrence-free survival. METHODS: The KBC trial is a multicenter, placebo-controlled, randomized phase III trial in high-risk breast cancer patients powered for 33% reduction in recurrence rate (from 60 to 40%). Patients received one dose of ketorolac tromethamine or a placebo before surgery. Eligible patients were breast cancer patients, planned for curative surgery, and with a Neutrophil-to-Lymphocyte Ratio≥4, node-positive disease or a triple-negative phenotype. The primary endpoint was Disease-Free Survival (DFS) at two years. Secondary endpoints included safety, pain assessment and overall survival. FINDINGS: Between February 2013 and July 2015, 203 patients were assigned to ketorolac (n = 96) or placebo (n = 107). Baseline characteristics were similar between arms. Patients had a mean age of 55.7 (SD14) years. At two years, 83.1% of the patients were alive and disease free in the ketorolac vs. 89.7% in the placebo arm (HR: 1.23; 95%CI: 0.65-2.31) and, respectively, 96.8% vs. 98.1% were alive (HR: 1.09; 95%CI: 0.34-3.51). CONCLUSIONS: A single administration of 30 mg of ketorolac tromethamine before surgery does not increase disease-free survival in high risk breast cancer patients. Overall survival difference between ketorolac tromethamine group and placebo group was not statistically significant. The study was however underpowered because of lower recurrence rates than initially anticipated. No safety concerns were observed. TRIAL REGISTRATION: ClinicalTrials.gov NCT01806259.

Focal expression and final activity of matrix metalloproteinases may explain irregular dysfunctional endometrial bleeding.

Irregular dysfunctional bleeding of the endometrium (ie, metrorrhagia without organic lesion) is common in women, whether treated or not with ovarian hormones. Several matrix metalloproteinases (MMPs) become normally expressed and/or activated at menstruation and cause extracellular matrix breakdown. We therefore explored whether episodes of irregular dysfunctional bleeding could be associated with untimely MMP activity. By histology, foci of stromal breakdown were exclusively found in the endometrium of metrorrhagic women at bleeding. In these foci, 1) expression of estrogen receptor-alpha and progesterone receptor was altered; 2) collagenase-1 (MMP-1), stromelysin-1 (MMP-3), and gelatinase B (MMP-9) became detected in stromal cells, together with MMP-9 in neutrophils; and 3) gelatinase A (MMP-2) was more expressed and immunolocalized at the membrane of stromal cells. By biochemistry, endometrial lysates from nonbleeding metrorrhagic patients contained more latent and active MMP-2 and -9 than age-matched controls; at bleeding, collagenase activity, MMP-9, and active MMP-2 were strikingly increased whereas tissue inhibitor of metalloproteinases-1 (TIMP-1) was considerably decreased. As a functional assay, in situ gelatin zymography revealed large areas of gelatinolytic activity only in endometrium of bleeding patients. Altogether, these results strongly suggest that inappropriate focal expression and activation of several MMPs, combined with decreased inhibition, trigger irregular dysfunctional endometrial bleeding.