Axl, a prototypic member of the transmembrane tyrosine kinase receptor family, is known to regulate innate immunity. In this study, we show that Axl expression is induced by IFN-α during human dendritic cell (DC) differentiation from monocytes (IFN/DC) and that constitutively Axl-negative, IL-4-differentiated DC (IL-4/DC) can be induced to up-regulate Axl by IFN-α. This effect is inhibited by TLR-dependent maturation stimuli such as LPS, poly(I:C), TLR7/8 ligand, and CD40L. LPS-induced Axl down-regulation on the surface of human IFN-α-treated DC correlates with an increased proteolytic cleavage of Axl and with elevated levels of its soluble form. GM6001 and TAPI-1, general inhibitors of MMP and ADAM family proteases, restored Axl expression on the DC surface and diminished Axl shedding. Furthermore, stimulation of Axl by its ligand, Gas6, induced chemotaxis of human DC and rescued them from growth factor deprivation-induced apoptosis. Our study provides the first evidence that Gas6/Axl-mediated signaling regulates human DC activities, and identifies Gas6/Axl as a new DC chemotaxis pathway. This encourages one to explore whether dysregulation of this novel pathway in human DC biology is involved in autoimmunity characterized by high levels of IFN-α.
Saccharomyces cerevisiae pyruvate kinase 1 (Pyk1) was demonstrated to be associated to an immunoprecipitate of yeast protein kinase A holoenzyme (HATpk1⅐Bcy1) and to be phosphorylated in a cAMP-dependent process. Both glutathione S-transferase (GST)-Pyk1 and GST-Pyk2 were phosphorylated in vitro by the bovine heart protein kinase A (PKA) catalytic subunit and by immobilized yeast HA-Tpk1. The specificity constant for the phosphorylation of GSTPyk1 and GST-Pyk2 by bovine catalytic subunit was in the range of the value for Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide). Both fusion proteins were phosphorylated in vivo, in intact cells overexpressing the protein, or in vitro using crude extracts, as source of protein kinase A, when a wild type strain was used but were not phosphorylated when using a strain with only one TPK gene with an attenuated mutation (tpk1 w1 ). The effect of phosphorylation on Pyk activity was assayed in partially purified preparations from three strains, containing different endogenous protein kinase A activity levels. Pyk1 activity was measured at different phosphoenolpyruvate concentrations in the absence or in the presence of the activator fructose 1,6-bisphosphate at 1.5 mM. Preliminary kinetic results derived from the comparison of Pyk1 obtained from extracts with the highest versus those from the lowest protein kinase A activity indicate that the enzyme is more active upon phosphorylation conditions; in the absence of the activator it shows a shift in the titration curve for phosphoenolpyruvate to the left and an increase in the Hill coefficient, whereas in the presence of fructose 1,6-bisphosphate it shows an n H value of 1.4, as compared with an n H of 2 for the Pyk1 obtained from extracts with almost null protein kinase A activity.
BACKGROUND AND PURPOSEA crosstalk between the immune system and depression has been postulated, with monocytes/macrophages and cytokines having a key role in this interaction. In this study, we examined whether vortioxetine, a multimodal anti-depressive drug, was endowed with anti-inflammatory and antioxidative activity, leading to immunomodulatory effects on human monocytes and macrophages. EXPERIMENTAL APPROACHHuman monocytes were isolated from buffy coats and used as such or differentiated into M1 and M2 macrophages. Cells were treated with vortioxetine before or after differentiation, and their responsiveness was evaluated. This included oxy-radical and TNFα production, TNFα and PPARγ gene expression and NF-κB translocation. KEY RESULTSVortioxetine significantly reduced the PMA-induced oxidative burst in monocytes and in macrophages (M1 and M2), causing a concomitant shift of macrophages from the M1 to the M2 phenotype, demonstrated by a significant decrease in the expression of the surface marker CD86 and an increase in CD206. Moreover, treatment of monocytes with vortioxetine rendered macrophages derived from this population less sensitive to PMA, as it reduced the oxidative burst, NF-kB translocation, TNFα release and expression while inducing PPARγ gene expression. FACS analysis showed a significant decrease in the CD14 + /CD16 + /CD86 + M1 population. CONCLUSIONS AND IMPLICATIONSThese results demonstrate that in human monocytes/macrophages, vortioxetine has antioxidant activity and anti-inflammatory effects driving the polarization of macrophages towards their alternative phenotype. These findings suggest that vortioxetine, alongside its antidepressive effect, may have immunomodulatory properties. Abbreviations
The stabilization of the restriction enzyme EcoRI by its incorporation into aqueous glass-forming carbohydrate or polymer solutions, followed by vacuum-drying to low moisture, has been studied. Glass-forming solutes included trehalose, sucrose, lactose, maltose, raffinose, maltodextrin DE 10, and poly(vinylpyrrolidone) (molecular weight 40,000, PVP). Among the solutes examined, trehalose and sucrose protected the enzyme most effectively during storage at 37 and 45 degrees C. The restriction enzyme dried with trehalose or sucrose maintained its activity without detectable loss for at least 20 days at 37 degrees C and 12 days at 45 degrees C. In contrast, the activity of the enzyme dried with maltodextrin or PVP was reduced during vacuum desiccation and also it decreased remarkably during storage at the same temperatures. Stored (37/45 degrees C) vacuum-dried trehalose and sucrose systems were either a dense paste or a very viscous syrup, and this indicated that they were not glassy. Moreover, no relationship was found between the glass transition temperatures (Tg) of the pure added solute and enzyme protection during storage, since, e.g., sucrose which has significantly lower Tg values protected the enzyme much better than either maltose, lactose, maltodextrin, or PVP. The trisaccharide raffinose offered good protection of enzyme activity, and its role as a novel excipient matrix for labile enzyme stabilization deserves further investigation. The stability of enzyme EcoRI was rapidly lost when the vacuum-dried trehalose and sucrose systems were humidified to 58% relative humidity and stored at 45 degrees C, and this was attributed to disaccharide crystallization.
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