Autocrine stimulation of P2Y1 receptors is part of the purinergic signaling mechanism that regulates T cell activation.

Previous studies have shown that T cell receptor (TCR) and CD28 coreceptor stimulation involves rapid ATP release, autocrine purinergic feedback via P2X receptors, and mitochondrial ATP synthesis that promote T cell activation. Here, we show that ADP formation and autocrine stimulation of P2Y1 receptors are also involved in these purinergic signaling mechanisms. Primary human CD4 T cells and the human Jurkat CD4 T cell line express P2Y1 receptors. The expression of this receptor increases following T cell stimulation. Inhibition of P2Y1 receptors impairs the activation of mitochondria, as assessed by mitochondrial Ca2+ uptake, and reduces cytosolic Ca2+ signaling in response to TCR/CD28 stimulation. We found that the addition of exogenous ADP or overexpression of P2Y1 receptors significantly increased IL-2 mRNA transcription in response to TCR/CD28 stimulation. Conversely, antagonists or silencing of P2Y1 receptors reduced IL-2 mRNA transcription and attenuated T cell functions. We conclude that P2Y1 and P2X receptors have non-redundant, synergistic functions in the regulation of T cell activation. P2Y1 receptors may represent potential therapeutic targets to modulate T cell function in inflammation and host defense.

Intronic miRNA-641 controls its host Gene's pathway PI3K/AKT and this relationship is dysfunctional in glioblastoma multiforme.

MicroRNAs have established their role as important regulators of the epigenome. A considerable number of human miRNA genes are found in intronic regions of protein-coding host genes, in many cases adopting their regulatory circuitry. However, emerging evidence foreshadows an unprecedented importance for this relationship: Intronic miRNAs may protect the cell from overactivation of the respective host pathway, a setting that may trigger tumor development. AKT2 is a well-known proto-oncogene central to the PI3K/AKT pathway. This pathway is known to promote tumor growth and survival, especially in glioblastoma. Its intronic miRNA, hsa-miR-641, is scarcely investigated, however. We hypothesized that miR-641 regulates its host AKT2 and that this regulation may become dysfunctional in glioblastoma. We found that indeed miR-641 expression differs significantly between GBM tissue and normal brain samples, and that transfection of glioma cells with miR-641 antagonizes the PI3K/AKT pathway. Combining clinical samples, cell cultures, and biomolecular methods, we could show that miR-641 doesn't affect AKT2's expression levels, but down-regulates kinases that are necessary for AKT2-activation, thereby affecting its functional state. We also identified NFAT5 as a miR-641 regulated central factor to trigger the expression of these kinases and subsequently activate AKT2. In summary, our study is the first that draws a connecting line between the proto-oncogene AKT2 and its intronic miRNA miR-641 with implication for glioblastoma development.

Down-regulation of MicroRNA-31 in CD4+ T Cells Contributes to Immunosuppression in Human Sepsis by Promoting TH2 Skewing.

BACKGROUND: Immunosuppression has been recognized as a major cause of sepsis-related mortality. Currently, there is much interest in identifying central hubs controlling septic immunoparalysis. In this context, in this study, the authors investigate the role of microRNA-31 (miR-31) as a regulator of T cell functions. METHODS: Primary human T cells were separated from healthy volunteers (n = 16) and from sepsis patients by magnetic beads (n = 23). Expression of mRNA/microRNA (miRNA) was determined by real-time polymerase chain reaction. Gene silencing was performed by small interfering RNA transfection, and miRNA-binding sites were validated by reporter gene assays. Effects of miR-31 or anti-miR-31 transfection were analyzed by real-time polymerase chain reaction, Western blotting, and flow cytometry. RESULTS: Overexpression of miR-31 in stimulated CD4 T cells promoted a proinflammatory phenotype with increased levels of interferon-γ (1.63 ± 0.43; P = 0.001; means ± SD) and reduced expression of interleukin (IL)-2 (0.66 ± 0.19; P = 0.005) and IL-4 (0.80 ± 0.2; P = 0.0001). In contrast, transfection of anti-miR-31 directed cells toward a TH2 phenotype. Effects on IL-2 and IL-4 were mediated by targeting of nuclear factor-kappa B-inducing kinase and factor-inhibiting hypoxia-inducible factor-1α. Interferon-γ, however, was influenced via control of signaling lymphocytic activation molecule (SLAM)-associated protein, an essential adaptor molecule of immunomodulatory SLAM receptor signaling, which was identified as a novel target gene of miR-31. In sepsis patients, an epigenetically driven down-regulation of miR-31 was found (0.44 ± 0.25; P = 0.0001), associated with increased nuclear factor-kappa B-inducing kinase, factor-inhibiting hypoxia-inducible factor-1α, SLAM-associated protein expression, and a cytokine shift toward TH2. CONCLUSIONS: In this study, the authors provide novel evidence of miR-31 as an emerging key posttranscriptional regulator of sepsis-associated immunosuppression. The study results contribute to a further understanding of septic immunoparalysis and provide new perspectives on miRNA-based diagnostic approaches.

How to improve prescription of inhaled salbutamol by providing standardised feedback on administration: a controlled intervention pilot study with follow-up.

BACKGROUND: The effectiveness of inhaled salbutamol in routine care depends particularly on prescribed dosage and applied inhalation technique. To achieve maximum effectiveness and to prevent drug-related problems, prescription and administration need to work in concert. METHODS: We performed a controlled intervention pilot study with 4 consecutive groups in a general paediatric unit and assessed problems in salbutamol prescribing and administration. Control group [i]: Routine care without additional support. First intervention group [ii]: We carried out a teaching session for nurses aimed at preventing problems in inhalation technique. Independently from this, a pharmacist counselled physicians on problems in salbutamol prescribing. Second intervention group [iii]: Additionally to the first intervention, physicians received standardised feedback on the inhalation technique. Follow-up group [iv]: Subsequently, without any delay after the second intervention group had been completed, sustainability of the measures was assessed. We performed the chi-square test to calculate the level of significance with p ≤ 0.05 to indicate a statistically significant difference for the primary outcome. As we performed multiple testing, an adjusted p ≤ 0.01 according to Bonferroni correction was considered as significant. RESULTS: We included a total of 225 patients. By counselling the physicians, we reduced the number of patients with problems from 55% to 43% (control [i] vs. first intervention [ii], n.s.). With additional feedback to physicians, this number was further reduced to 25% ([i] vs. [iii], p < 0.001). In the follow-up [iv], the number rose again to 48% (p < 0.01 compared to feedback group). CONCLUSIONS: Teaching nurses, counselling physicians, and providing feedback on the quality of inhalation technique effectively reduced problems in salbutamol treatment. However, for success to be sustained, continuous support needs to be provided. TRIAL REGISTRATION: German Clinical Trials register: DRKS00006792 .

Corticosteroid resistance in sepsis is influenced by microRNA-124--induced downregulation of glucocorticoid receptor-α.

OBJECTIVE: Acquired glucocorticoid resistance frequently complicates the therapy of sepsis. It leads to an exaggerated proinflammatory response and has been related to altered expression profiles of glucocorticoid receptor isoforms glucocorticoid receptor-α (mediating anti-inflammatory effects) and glucocorticoid receptor-ß (acting as a dominant negative inhibitor). We investigated the impact of glucocorticoid receptor isoforms on glucocorticoid effects in human T-cells. We hypothesized that 1) changes of the ratio of glucocorticoid receptor isoforms impact glucocorticoid resistance and 2) glucocorticoid receptor-α expression is controlled by microRNA-mediated gene silencing. DESIGN: Laboratory-based study. SETTING: University research laboratory. SUBJECTS AND PATIENTS: Healthy volunteers, sepsis patients. METHODS: First, T-cells from healthy volunteers (native and CD3/CD28-stimulated cells with or without addition of hydrocortisone) were analyzed for the expression of glucocorticoid receptor-isoforms by quantitative polymerase chain reaction. Additionally, effects of gene silencing of glucocorticoid receptor-ß by siRNA transfection were determined. Secondly, microRNA-mediated silencing was evaluated by cloning of a glucocorticoid receptor-α-specific 3'-untranslated-region reporter construct and subsequent transfection experiments in cell cultures. Effects of miRNA transfection on glucocorticoid receptor-α expression were analyzed in Jurkat T-cells and in T-cells from healthy volunteers (quantitative polymerase chain reaction and Western blotting). Finally, expression of glucocorticoid receptor-α, glucocorticoid receptor-ß, and miR-124 was tested in T-cells of sepsis patients (n=24). MEASUREMENTS AND MAIN RESULTS: Stimulation of T-cells induced a significant upregulation of glucocorticoid receptor-α (not glucocorticoid receptor-ß) thereby possibly rendering T-cells more sensitive to glucocorticoids; this T-cell response was hindered by hydrocortisone. Silencing of glucocorticoid receptor-ß doubled the inhibitory effects of glucocorticoids on interleukin-2 production. MicroRNA-124 was proved to specifically downregulate glucocorticoid receptor-α. Furthermore, a glucocorticoid-induced three-fold upregulation of microRNA-124 was found. T-cells of sepsis patients exhibited slightly decreased glucocorticoid receptor-α and slightly increased miR-124 expression levels, whereas glucocorticoid receptor-ß expression was two-fold upregulated (p<.01) and exhibited a remarkable interindividual variability. CONCLUSIONS: Glucocorticoid treatment induces expression of miR-124, which downregulates glucocorticoid receptor-α thereby limiting anti-inflammatory effects of glucocorticoids. Steroid treatment might aggravate glucocorticoid resistance in patients with high glucocorticoid receptor-ß levels.