The present study investigates mRNA and protein levels of A 3 adenosine receptors in resting (R) and activated (A) human lymphocytes. The receptors were evaluated by the antagonist radioligand ϩ cell fraction. Real-time reverse transcription-polymerase chain reaction experiments confirmed the rapid increase of A 3 mRNA after T cell activation. Competition of radioligand binding by adenosine ligands displayed a rank order of potency typical of the A 3 subtype. Thermodynamic data indicated that the binding is enthalpy-and entropy-driven in both R and A cells, suggesting that the activation process does not involve, at a molecular level, receptor alterations leading to modifications in the A 3 -related binding mechanisms. Functionally, the up-regulation of A 3 adenosine receptors in A versus R cells corresponded to a potency increase of the A 3 agonist N 6 -(3-iodo-benzyl)-2-chloro-adenosine-5Ј-N-methyluronamide in inhibiting cAMP accumulation (IC 50 ϭ 1.5 Ϯ 0.4 and 2.7 Ϯ 0.3 nM, respectively); this effect was antagonized by MRE 3008F20 (IC 50 ϭ 5.0 Ϯ 0.3 nM). In conclusion, our results provide, for the first time, an in-depth investigation of A 3 receptors in human lymphocytes and demonstrate that, under activating conditions, they are up-regulated and may contribute to the effects triggered by adenosine.

Pulsed electromagnetic fields (PEMFs) play a regulatory role on osteoblast activity and are clinically beneficial during fracture healing. Human mesenchymal stem cells (MSCs) derived from different sources have been extensively used in bone tissue engineering. Compared with MSCs isolated from bone marrow (BMSCs), those derived from adipose tissue (ASCs) are easier to obtain and available in larger amounts, although they show a less osteogenic differentiation potential than BMSCs. The hypothesis tested in this study was to evaluate whether PEMFs favor osteogenic differentiation both in BMSCs and in ASCs and to compare the role of PEMFs alone and in combination with the biochemical osteogenic stimulus bone morphogenetic protein (BMP)-2. Early and later osteogenic markers, such as alkaline phosphatase (ALP) activity, osteocalcin levels, and matrix mineralization, were analyzed at different times during osteogenic differentiation. Results showed that PEMFs induced osteogenic differentiation by increasing ALP activity, osteocalcin, and matrix mineralization in both BMSCs and ASCs, suggesting that PEMF activity is maintained during the whole differentiation period. The addition of BMP-2 in PEMF exposed cultures further increased all the osteogenic markers in BMSCs, while in ASCs, the stimulatory role of PEMFs was independent of BMP-2. Our results indicate that PEMFs may stimulate an early osteogenic induction in both BMSCs and ASCs and they suggest PEMFs as a bioactive factor to enhance the osteogenesis of ASCs, which are an attractive cell source for clinical applications. In conclusion, PEMFs may be considered a possible tool to improve autologous cell-based regeneration of bone defects in orthopedics.

Novel biomaterials are of paramount importance for bone regrowth. In this study, we investigated human adipose stem cells (hASCs) for osteogenic, osteoconductivity, and osteoinductivity effects of an innovative collagen/hydroxylapatite hybrid scaffold. In hASCs that were grown on this scaffold, osteogenic genes were analyzed for their expression profiles, together with adhesion and extracellular matrix genes. In hASC integrins, basement membrane constituents and collagens were up-regulated, together with cell proliferation. In addition, expression of osteopontin and activated focal adhesion kinase was studied at the protein level. Our data indicate that hASCs, together with hybrid biomaterial, is an important model of study to investigate bone induction.-Mazzoni, E., D'Agostino, A., Manfrini, M., Maniero, S., Puozzo, A., Bassi, E., Marsico, S., Fortini, C., Trevisiol, L., Patergnani, S., Tognon, M. Human adipose stem cells induced to osteogenic differentiation by an innovative collagen/hydroxylapatite hybrid scaffold.

Tat, the trans activation protein of HIV, is produced early upon infection to promote and expand HIV replication and transmission. However, Tat appears to also have effects on target cells, which may affect Ag recognition both during infection and after vaccination. In particular, Tat targets dendritic cells and induces their maturation and Ag-presenting functions, increasing Th1 T cell responses. We show in this work that Tat modifies the catalytic subunit composition of immunoproteasomes in B and T cells either expressing Tat or treated with exogenous biological active Tat protein. In particular, Tat up-regulates latent membrane protein 7 and multicatalytic endopeptidase complex like-1 subunits and down-modulates the latent membrane protein 2 subunit. These changes correlate with the increase of all three major proteolytic activities of the proteasome and result in a more efficient generation and presentation of subdominant MHC-I-binding CTL epitopes of heterologous Ags. Thus, Tat modifies the Ag processing and modulates the generation of CTL epitopes. This may have an impact on both the control of virally infected cells during HIV-1 infection and the use of Tat for vaccination strategies.

Estrogens play a protective role in coronary artery disease. The mechanisms of action are still poorly understood, although a role for estrogens in stimulation of angiogenesis has been suggested. In several cell types, estrogens modulate the Notch pathway, which is involved in controlling angiogenesis downstream of vascular endothelial growth factor A (VEGF-A). The goal of our study was to establish whether estrogens modulate Notch activity in endothelial cells and the possible consequences on angiogenesis. Human umbilical vein endothelial cells (HUVECs) were treated with 17β-estradiol (E2) and the effects on Notch signalling were evaluated. E2 increased Notch1 processing as indicated by i) decreased levels of Notch1 transmembrane subunit ii) increased amount of Notch1 in nuclei iii) unaffected level of mRNA. Similarly, E2 increased the levels of the active form of Notch4 without altering Notch4 mRNA. Conversely, protein and mRNA levels of Notch2 were both reduced suggesting transcriptional repression of Notch2 by E2. Under conditions where Notch was activated by upregulation of Delta-like ligand 4 (Dll4) following VEGF-A treatment, E2 caused a further increase of the active form of Notch1, of the number of cells with nuclear Notch1 and of Hey2 mRNA. Estrogen receptor antagonist ICI 182.780 antagonized these effects suggesting that E2 modulation of Notch1 is mediated by estrogen receptors. E2 treatment abolished the increase in endothelial cells sprouting caused by Notch inhibition in a tube formation assay on 3D Matrigel and in mouse aortic ring explants. In conclusion, E2 affects several Notch pathway components in HUVECs, leading to an activation of the VEGF-A-Dll4-Notch1 axis and to a modulation of vascular branching when Notch signalling is inhibited. These results contribute to our understanding of the molecular mechanisms of cardiovascular protection exerted by estrogens by uncovering a novel role of E2 in the Notch signalling-mediated modulation of angiogenesis.