Elisabetta Melloni

Cyclooxygenase-2 expression is induced during human megakaryopoiesis and characterizes newly formed platelets

Cyclooxygenase (COX)-1 or -2 and prostaglandin (PG) synthases catalyze the formation of various PGs and thromboxane (TX) A2. We have investigated the expression and activity of COX-1 and -2 during human megakaryocytopoiesis. We analyzed megakaryocytes from bone marrow biopsies and derived from thrombopoietin-treated CD34+ hemopoietic progenitor cells in culture. Platelets were obtained from healthy donors and patients with high platelet regeneration because of immune thrombocytopenia or peripheral blood stem cell transplantation. By immunocytochemistry, COX-1 was observed in CD34+ cells and in megakaryocytes at each stage of maturation, whereas COX-2 was induced after 6 days of culture, and remained detectable in mature megakaryocytes. CD34+ cells synthesized more PGE2 than TXB2 (214 ± 50 vs. 30 ± 10 pg/106 cells), whereas the reverse was true in mature megakaryocytes (TXB2 8,440 ± 2,500 vs. PGE2 906 ± 161 pg/106 cells). By immunostaining, COX-2 was observed in <10% of circulating platelets from healthy controls, whereas up to 60% of COX-2-positive platelets were found in patients. A selective COX-2 inhibitor reduced platelet production of both PGE2 and TXB2 to a significantly greater extent in patients than in healthy subjects. Finally, we found that COX-2 and the inducible PGE-synthase were coexpressed in mature megakaryocytes and in platelets. We conclude that both COX-isoforms contribute to prostanoid formation during human megakaryocytopoiesis and that COX-2-derived PGE2 and TXA2 may play an unrecognized role in inflammatory and hemostatic responses in clinical syndromes associated with high platelet turnover.

Supravital exposure to propidium iodide identifies apoptosis on adherent cells.

BACKGROUND Several studies indicate that plasma membrane changes during apoptosis are a general phenomenon. Among the flow cytometric methods to measure apoptosis, the Annexin V assay that detects the membrane exposure of phosphatidylserine (PS) is one of the most commonly used. However, the various treatments used for the detachment of adherent cells generally interfere with the binding of Annexin V to membrane PS, making apoptosis measurement a technical problem. Materials and Methods Apoptosis of different cell lines was investigated by fluorescence microscopy and multiple flow assays designed to assess loss of membrane integrity, translocation of PS, DNA fragmentation, and light scatter changes. Results and Conclusions We show that supravital propidium iodide (PI) assay stains adherent apoptotic cells, allowing flow cytometric quantification. Moreover, supravital exposure to PI without prior permeabilization identifies apoptotic cells as well as Annexin V and permits the simultaneous surface staining by FITC- and PE-conjugated monoclonal antibodies. As in the case of necrotic or permeabilized cells, fluorescence microscopy has revealed that PI staining of apoptotic cells is localized in the nucleus. This suggests that the binding of PI to the DNA/RNA structures is stable enough to withstand the trypsinization and/or washing procedures necessary to detach adherent cells.

Role of CREB transcription factor in c‐fos activation in natural killer cells

In natural killer (NK) cells, interleukin‐2 (IL‐2) differentially regulates the expression of several transcription factors, including JunB and c‐fos. The cAMP response element binding protein, CREB, is a key transcriptional regulator of a large number of genes containing the octanucleotide CRE consensus sequence in their upstream regulatory regions. We studied here thefunctional role of CREB in the IL‐2‐mediated transcriptional regulation of c‐fos in human NK cells. Our results show that IL‐2 activates CREB in human NK cells and that CREB activation hasa prominent regulatory role on the IL‐2‐induced expression of functional c‐fos and AP‐1 in NK cells. We identify two domains of the c‐fos promoter, containing three CRE sites, which are critical for the transcriptional activity induced by IL‐2. The first domain is located within the first 220 nucleotides of the c‐fos promoter, while the second encompasses the nucleotides − 440 and − 220. Our results show that CREB has a relevant role in the cytokine‐mediated activation of NK cells, and are particularly remarkable in the light of the several genes that are positively regulated by c‐fos and AP‐1, such as IFN‐γ, IL‐2 and GM‐CSF genes.

Extracellular Tat activates c-fos promoter in low serum-starved CD4+ T cells

The regulatory human immunodeficiency virus‐1 (HIV‐1) Tat protein shows pleiotropic effects on the survival and growth of both HIV‐1‐infected and uninfected CD4+ T lymphocytes. In this study, we have demonstrated that low concentrations (10 ng/ml) of extracellular Tat protein induce the expression of both c‐fos mRNA and protein in serum‐starved Jurkat CD4+ lymphoblastoid T cells. Using deletion mutants, we demonstrates that the SRE, CRE and, to a lesser extent, also the SIE domains (all placed in the first 356 bp of c‐fos promoter) play a key role in mediating the response to extracellular Tat. Moreover, the ability of Tat to activate the transcriptional activity of c‐fos promoter was consistently decreased by pretreatment with the ERK/MAPK kinase inhibitor PD98058. Activation of c‐fos is functional as demonstrated by induction of the AP‐1 transcription factor, which is involved in the regulation of critical genes for the activation of T lymphocytes, such as interleukin 2. The Tat‐mediated induction of c‐fos and AP‐1 in uninfected lymphoid T cells may contribute to explain the immune hyperactivation that characterizes the progression to autoimmuno deficiency syndrome and constitutes the optimal environment for HIV‐1 replication, occurring predominantly in activated/proliferating CD4+ T cells.

MIF Expression in Induced Peripheral Blood Mononuclear Cells by Vitamin D3 and its Potential Correlation with Resting Metabolic Rate in Obesity

Macrophage migration inhibitory factor (MIF) plays a pivotal role in systemic and local inflammatory and immune responses. The aim of this study is to assess MIF gene expression in PBMCs harvested from obese individuals and to compare it with that of lean subjects to analyze any potential relationship between resting metabolic rate as well as several different parameters and MIF expression in PBMC. We also aim to assess anti-inflammatory characteristics of vitamin D3 on MIF expression in vitro. Participants were 30 obese and 18 non-obese subjects who were assessed following an overnight fasting for RMR by means of indirect calorimetry. Body composition was measured using a Bodystat device. The PBMCs were separated from whole blood by the Ficoll-hypaque technique. The mRNA was extracted and the cDNA was synthesized. This process was followed by real-time PCR using primer pairs specific for MIF mRNA and beta actin as internal control. Our findings clearly demonstrate that there were significant differences in terms of BMI, BMR predict, fat proportion, fat mass, free fat mass, TBW, visceral fat, fasting serum glucose, TG, HDL, Hs-CRP and RMR between the two groups. Moreover, the level of MIF expression in the obese group was approximately 2.5 times higher compared to the lean group. An increased level of MIF expression in the obese group and a decreased expression of that non-obese was observed after inducing PBMCs with vitamin D3. One of the intriguing results of this study was the observed reverse correlation between MIF expression and fat mass as well as fat proportion after PBMCs were cultured in the presence of vitamin D3. Therefore, it could be concluded that MIF expression, which is in turn influenced by vitamin D3, has a role in the hyperactive immune profile and the pro-inflammatory state observed in obese individuals which is suggested to have a causal relationship with obesity.

Inverse Correlation Between Circulating Levels of TNF-Related Apoptosis-Inducing Ligand and 17β-Estradiol

CONTEXT The regulation of the circulating levels of TNF-related apoptosis-inducing ligand (TRAIL), a cytokine of the TNF family, playing a key role in the immune surveillance against cancer, is incompletely understood. OBJECTIVE The objective of the study was to investigate the potential link between TRAIL and 17β-estradiol. DESIGN, SETTING, AND PARTICIPANTS Circulating TRAIL levels were measured by an ELISA in plasma samples (n = 246) of healthy, age-matched (range 30-70 y) men and women and in the sera (n = 180) of females belonging to different physiopathological conditions (childhood, pregnancy, under gonadotropin treatment, menopause) characterized by different levels of circulating 17β-estradiol. RESULTS TRAIL plasma levels in women with aged younger than 50 years were significantly lower compared with age-matched men, whereas in woman 50 years old or older, TRAIL levels were significantly higher compared with the age-matched men and with the younger women. Moreover, an analysis of women with different conditions revealed a significant inverse correlation between the serum levels of TRAIL and 17β-estradiol, with the lowest levels of TRAIL being observed during pregnancy and the highest in childhood and in postmenopausal women. Moreover, gonadotropin treatment in women undergoing assisted reproduction was accompanied by an acute decrease of serum TRAIL levels. Finally, in vitro treatment with 17β-estradiol decreased the TRAIL expression levels in peripheral blood mononuclear cells. CONCLUSIONS Our data suggest that 17β-estradiol plays a role in regulating TRAIL circulating levels. The demonstration that postmenopausal women exhibit the highest TRAIL levels is of particular interest in light of a previous large study population showing that TRAIL is positively correlated to the overall survival.