Modulation of macrophage activation by prostaglandins

The effect of prostaglandtn E2, iloprost and cAMP on both nitric oxide and tumour necrosis factor-α release in J774 macrophages has been studied. Both prostaglandin E2 and iloprost inhibited, in a concentration-dependent fashion, the lipopolysaccharide-induced generation of nitric oxide and tumour necrosis factor-α. The inhibitory effect of these prostanoids seems to be mediated by an increase of the second messenger cAMP since it was mimicked by dibutyryl cAMP and potentiated by the selective type IV phosphodiesterase inhibitor RO-20-1724. Our results suggest that the inhibition of nitric oxide release by prostaglandin E2 and iloprost in lipopolysaccharide-activated J774 macrophages may be secondary to the inhibition of tumour necrosis factor-α generation, which in turn is likely to be mediated by cAMP

The inhibition by hydrocortisone of prostaglandin biosynthesis in rat peritoneal leucocytes is correlated with intracellular macrocortin levels

Hydrocortisone inhibits prostaglandin generation by rat peritoneal leucocytes by releasing the polypeptide phospholipase inhibitor, macrocortin. The susceptibility of these cells to hydrocortisone is directly correlated with their intracellular macrocortin content. Cells depleted of the peptide by prior incubation with steroid cannot respond to the steroid, until a fresh intracellular store has been synthesized. In vitro, this process requires 4-5 h. Cells remain sensitive to the inhibitory action of the peptide at all times

Enhanced antioxidant effect of trans-resveratrol: potential of binary systems with polyethylene glycol and cyclodextrin.

Abstract Trans-resveratrol, a polyphenol extracted from Vitis vinifera, has different beneficial effects following its administration on the skin. Here the potential use of binary systems to enhance in vitro and in vivo activity of trans-resveratrol was investigated. Thus the aqueous solubility of trans-resveratrol was investigated in the presence of growing concentrations of polyethylene glycol (PEG) or β-cyclodextrin (βCD) as solubilizing excipients. Then, the solid dispersion of trans-resveratrol with PEG or inclusion complexes trans-resveratrol/βCD were prepared and characterised by different methods. Cytotoxicity and inhibition of reactive oxygen species (ROS) following H2O2 challenge in the presence of trans-resveratrol, alone or associated to the excipients, was evaluated on human keratinocyte HaCaT cell line. Both the trans-resveratrol-containing binary systems induced significant reduction of H2O2-induced ROS production, especially in the case of βCD that was selected for the following phase of the study. Thus, the effect of a cream containing trans-resveratrol, alone or associated to βCD, on different skin parameters such as corneometry, colorimetry and elastometry, was evaluated on human volunteers. All patients showed a visible improvement of clinical conditions with a remarkable decrease of aging signs, but this effect was higher of the hemi face treated with the βCD-containing formulation versus formulation containing trans-resveratrol alone

Nanocarriers for topical delivery of resveratrol.

Purpose: The aim of the present work was to develop a nanocarrier-based formulation for topical delivery of resveratrol. Methods: Trans-resveratrol (t-res) was encapsulated in liposomes, ethosomes or transferosomes, by a modified hand-shaking method followed by extrusion. All the formulations were characterised in terms of mean diameter, size distribution (I.P.), t-res loading, t-res stability upon encapsulation during storage. The nanocarriers containing t-res were then introduced in cellulose-based gel to allow their final administration on the skin and the viscoelastic properties of the resulting formulation were investigated. Finally, we studied the inhibition of reactive oxygen species (ROS) in human keratinocyte (HaCaT) cell line stimulated with H2O2 for 24 h and then incubated with the t-res containing nanocarriers. Results: All the t-res containing carriers were characterised by a very high (close to 100%) encapsulation efficiency, a negligible t-res release at 4°C and stability of resveratrol in its trans form. The carriers only slightly influenced the viscoelastic characteristics of cellulose-based gels. Nanocarriers encapsulating t-res reduced, in a concentration-dependent manner, ROS production induced by H2O2 and this effect was higher when using t-res-encapsulating nanocarrier, with the higher effect observed in the case of ethosomes. Conclusions: In this work nanocarriers with high encapsulation efficiency, high physical stability and negligible t-res release during storage at 4°C were prepared. To allow their final administration on the skin, the nanocarrier can be easilisy loaded in cellulose-based gels without altering its rheological properties. Moreover, the use of t-res-encapsulating ethosomes led to an efficient antioxidant activity. Further ex vivo and in vivo studies will clarify the role of the different carrier when administered on the skin

Palmitoylethanolamide inhibits rMCP-5 expression by regulating MITF activation in rat chronic granulomatous inflammation

Chronic inflammation, a condition frequently associated with several pathologies, is characterized by angiogenic and fibrogenic responses that may account for the development of granulomatous tissue. We previously demonstrated that the chymase, rat mast cell protease-5 (rMCP-5), exhibits pro-inflammatory and pro-angiogenic properties in a model of chronic inflammation sustained by mast cells (MCs), granuloma induced by the subcutaneous carrageenan-soaked sponge implant in rat. In this study, we investigated the effects of palmitoylethanolamide (PEA), an anti-inflammatory and analgesic endogenous compound, on rMCP-5 mRNA expression and Microphtalmia-associated Transcription Factor (MITF) activation in the same model of chronic inflammation. The levels of rMCP-5 mRNA were detected using semi-quantitative RT-PCR; the protein expression of chymase and extracellular signal-regulated kinases (ERK) were analyzed by western blot; MITF/DNA binding activity and MITF phosphorylation were assessed by electrophoretic mobility shift assay (EMSA) and immunoprecipitation, respectively. The administration of PEA (200, 400 and 800 µg/ml) significantly decreased rMCP-5 mRNA and chymase protein expression induced by λ-carrageenan. These effects were associated with a significant decrease of MITF/DNA binding activity and phosphorylated MITF as well as phosphorylated ERK levels. In conclusion, our results, showing the ability of PEA to inhibit MITF activation and chymase expression in granulomatous tissue, may yield new insights into the understanding of the signaling pathways leading to MITF activation controlled by PEA

Novel particulate systems for the delivery of an oligonucleotide decoy to Nuclear Factor-kappaB: a potential strategy for treating cystic fibrosis

Objectives To develop safe and effective particulate systems for cystic fibrosis gene therapy by an oligonucleotide decoy to Nuclear Factor-kB. Background/Rationale Decoy oligonucleotide (decoy ODN) able to interfere with NF-kB pathway may be of great help in reducing NF-kB mediated pulmonary chronic inflammation, which is the primary cause of bronchioectasis, respiratory failure and death of CF patients. To overcome issues related to the use of oligonucleotide (ODN) in therapy, delivery through poly(lactide-co-glycolide) (PLGA) microparticles, able to protect ODN toward the biological environment and/or release the drug in a controlled way, has been suggested. Recent studies indicate that PLGA microparticles allow an increase of ODN biological stability, are able to maintain its hybridization capability and provide a sustained ODN pharmacological activity. Conceiving PLGA microspheres for CF treatment, another important challenge to be taken into account is their potential in ODN pulmonary delivery. On this matter, recent data indicate that adequately formulated PLGA particles may effectively transport high-molecular weight drugs to the lungs. Preliminary results In our previous studies, PLGA microspheres for ODN delivery have been developed. We investigated the potential of PLGA microspheres as delivery systems for a decoy phosphorotioate ODN against NF-kB in RAW 264.7 macrophages stimulated with lipopolysaccharide. In particular, in vitro incubation of macrophages with decoy ODN-releasing microspheres resulted in an inhibition of NF-kB activation eighty-times higher than that achieved with naked decoy ODN. We have also demonstrated that the use of polyethylenimine, a cationic polymer, can be of help in improving cell internalization and nuclear characterization of ODN. These findings suggest that due to their sustained release properties PLGA microspheres allow the full optimization of ODN pharmacokinetics. More recently, we have designed and developed PLGA-based dry powders intended for drug pulmonary delivery. In vitro/in vivo experimental results suggest that large porous particles (LPP) with flow properties and size suitable for aerosolization and deposition in deep regions of the lung following inhalation can be achieved. Project description (experimental plan, methods, timetable) The project will be carried out in two years. PLGA microspheres loaded with a decoy ODN against NFkB, eventually containing PEI, will be developed. Particular attention will be paid to particle for pulmonary delivery by evaluating in depth their aereodynamic properties which will affect their potential as pulmonary carriers. All the particles will be fully characterized for morphology, size, loading efficiency and in vitro release. The optimized systems will be tested on specific CF cell lines and animal models of CF. Anticipated output Research results such as patents, scientific publications and presentations at international symposia can be expected. We believe that this will occur in a reasonable short period (by the end of the 1st year) due to the advanced knowledge and expertise of the research teams involved in the project as well as the fact that the groups already collaborate on similar topics. Relevance to Italian CF Foundation The project could be of great interest for pharmaceutical and biotechnological industries involved in the development of novel therapeutic approaches for rare diseases. In fact, thanks to a specific EU legislation, industries which desire to sponsor an orphan drug can be the recipients of several economic incentives. Due to the high industrial applicability of the research, CF foundation could attract industrial attention and benefit of new opportunity of financial support

Characterization of vasocortin-like proteins induced by dexamethasone in endothelial cells.

In this study we have shown that dexamethasone induces vasocortin-like proteins in bovine endothelial cells as well as in the bovine aortic endothelial cell line, GM 7373. Vasocortin-like proteins have been characterized by their ability to mimic the glucocorticoid inhibition of rat dextran oedema and histamine release induced by concanavalin-A in rat mast cells. Following partial purification of these proteins by gel filtration, vasocortin activity was found to be associated to proteins with molecular weight between 20-35 kD. This study showed that dexamethasone induces vasocortin-like proteins in endothelial cells and suggests that endothelial cells are target cells of glucocorticoid activity in vascular tissue