Effects of Inhibition of IKK Kinase Phosphorylation On the Cellular Defence System and HSP90 Activity.

The present study was conceived to examine the effects of inhibition of BMS-345541 mediated IKK kinase phosphorylation on the cellular defence system as well as on anti-inflammatory response and HSP90 activity. The analysis was conducted in A549 cell line, since such cells carry a homozygous Keap1 mutation (G333C) that alters its interaction with Nrf2. Recent data have highlighted that Keap1, HSP90 protein and IKK kinase interact reciprocally and particularly Keap1 protein is involved in HSP90 and anti-oxidative pathway regulation. The activities of COX2 and HO1 were investigated by real time and immunoblot analysis along with the synthesis and activity of inducible forms of heat shock protein HSP90. Pre-treatment with IKK kinase inhibitor proved to be a protective means to lower the activity of inflammatory cascade, so preventing the formation of excessive amounts of pro-inflammatory molecules. The inhibitor of IKK kinase BMS-345541 was added to cultured A549 cells before the Escherichia coli lipopolysaccharide (LPS) addition. The viability of the cells was determined after 1-24 h incubation with BMS-345541 at concentrations ranging from 1,25-5 µM. It was found that 1 µM concentration does not significantly affected cell viability (data not shown). As a result, the treatment with 1 µM of BMS-345541 induces the inhibition of IKK phosphorylation. In the A549 cells treated with BMS-345541 and LPS, COX2 activity is not induced: mRNA and protein levels have not increased, while there is an increase in the level of HSP90, HO1 proteins and mRNA. The results suggest that the IKK inhibition is effective in the reduction of the inflammatory response thanks to mechanisms involving both the heat shock cellular defense system and the antioxidative pathway.

The Keap1 signaling in the regulation of HSP90 pathway.

The Keap1 protein is the master modulator of Nrf2 pathway; moreover, it is the hub of such important processes as cancer, cell stress, inflammation, and chemio- and radio-resistance. That is why Keap1 has become an intriguing pharmacological target. Many recent data show that Keap1 interacts with HSP90 protein. In this study, we use ferulic acid (FA) as antioxidant and anti-inflammatory agent, able to relieve inflammatory response. It is known that treatment with 100 µg of FA can significantly decrease the oxidative stress, so it turns to be useful to study the antioxidant regulation. The RAW 264.7 cells transfected with si-Keap1 and LPS treated are the in vitro model used to study the effects of Keap1 silencing on HSP90 activities and the FA antioxidant modulation. Immunoblot data and qPCR analysis show that Keap1 is involved in HSP90 modulation and on anti-oxidative response. Keap1 silencing affects negatively COX2 activation; in fact western blot and qPCR analysis conducted on RAW 264.7 cells Keap1silenced highlight that LPS treatment does not induce COX2 activation. In addition, the FA anti-oxidative and modulatory effect is abolished in COX2 pathway. The same results are point out using human A549 cell line with an allelic mutation on Keap1 gene, and the protein results are partially inactive. This preliminary study points out that Keap1protein is involved in HSP90 and anti-oxidative pathway regulation.

Toxic mineral elements in Mytilus galloprovincialis from Sicilian coasts (Southern Italy).

We assessed the relationship between V, Cr, Mn, Hg, As, Cd, Sn, Sb and Pb concentrations in Mytilus galloprovincialis samples from the coasts of Sicily and the expression of metallothioneins. Toxic mineral elements assessment was carried out by A.A. Spectrometry and ICP-MS. The metallothioneins expression was performed by q-PCR method. Low metals' levels were found in the mussel samples examined, in comparison with what was reported in literature. The highest mean values of toxic mineral elements were found in Gela (Cr 0.178 ± 0.03 mg/Kg, Mn 4.325 ± 0.012 mg/Kg, As 3.706 ± 0.009 mg/Kg, Sn 0.148 ± 0.014 mg/Kg, Sb 0.009 ± 0.004 mg/Kg e Pb 0.364 ± 0.01 mg/Kg). Significant levels of Hg were found in samples from Catania (0.014 ± 0.005 mg/Kg). Only vanadium and lead concentrations showed significant differences between sampling areas (p < 0.05). Molecular analysis verified a basal expression of Mt1 and the absence of over-expression of Mt2, confirming the low mineral's concentrations found in the samples examined.

An in vitro inflammation model to study the Nrf2 and NF-κB crosstalk in presence of ferulic acid as modulator.

The aim of this study was to evaluate the crosstalk between Nrf2 and NF-κB signaling pathways and to explore the modulating activity actuated by ferulic acid. In the inflammation process, a key player is the nuclear factor-κB (NF-κB) transcription factor pathway. On the contrary, the activation of Nrf2 inhibits inflammation and impairs degenerative disease providing an interface between redox and anti-inflammatory responses. Recent studies have demonstrated that protein phosphorylation of IKK complex is a potential mechanism for the activation of both Nrf2 and NF-κB pathways. The IKK complex is as an integration point for signals emanating from these different pathways. In this study, we demonstrated that ferulic acid is able to regulate NF-κB and Nrf2 activities. Interestingly, we showed that ferulic acid mimics the potent IKK inhibitor such as BMS, down-regulating the NF-κB response, TAK 1 activation and turning off Nrf2 activities in LPS-stimulated RAW 264.7 cells. Immunoblot data showed that the release of Nrf2 from Keap1 is maintained at low levels also in the presence of LPS stimulus. Nrf2 controls the expression of many antioxidant and detoxification genes, by binding to antioxidant response elements (AREs) that are commonly found in the promoter region of antioxidant (and other) genes. We demonstrated that in the pARE-Luc transfected cells the pre-treatment with FA significantly reduced LPS-induced (p<0.01) and BMS-induced (p<0.01) transcriptional activities. Analysis of well-known Nrf2 transcriptional targets showed that mRNAs expression of Nrf2-dependent antioxidant and phase II enzymes such as dehydrogenase quinone1 (NQO1) and glutathione S-transferase A2 (GSTA2) were up-regulated by BMS and significantly increase more by association with LPS, but are down-regulated in the presence of FA. Interestingly, cells depleted of Keap1 showed increased response of the Nrf2 transcriptional activity also in the presence of FA, strongly suggesting its modulating role in Keap1-Nrf2 signaling pathway.

Substance P Induces HO-1 Expression in RAW 264.7 Cells Promoting Switch towards M2-Like Macrophages.

Substance P (SP) is a neuropeptide that mediates many physiological as well as inflammatory responses. Recently, SP has been implicated in the resolution of inflammation through induction of M2 macrophages phenotype. The shift between M1-like and M2-like, allowing the resolution of inflammatory processes, also takes place by means of hemeoxygenase-1 (HO-1). HO-1 is induced in response to oxidative stress and inflammatory stimuli and modulates the immune response through macrophages polarisation. SP induces HO-1 expression in human periodontal ligament (PDL), the latter potentially plays a role in cytoprotection. We demonstrated that SP promotes M2-like phenotype from resting as well as from M1 macrophages. Indeed, SP triggers the production of interleukine-10 (IL-10), interleukine-4 (IL-4) and arginase-1 (Arg1) without nitric oxide (NO) generation. In addition, SP increases HO-1 expression in a dose- and time-dependent manner. Here we report that SP, without affecting cell viability, significantly reduces the production of pro-inflammatory cytokines and enzymes, such as tumor necrosis factor-alpha (TNF-α), interleukine-6 (IL-6), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), and ameliorates migration and phagocytic properties in LPS-stimulated RAW 264.7 cells. M2-like conversion required retention of NF-κB p65 into the cytoplasm and HO-1 induced expression. Silencing of the HO-1 mRNA expression reversed the induction of pro-inflammatory cytokines in RAW 264.7 stimulated by LPS and down-regulated anti-inflammatory hallmarks of M2 phenotype. In conclusion, our data show that SP treatment might be associated with anti-inflammatory effects in LPS-stimulated RAW 264.7 cells by suppressing NF-κB activation and inducing HO-1 expression.

The molecular events behind ferulic acid mediated modulation of IL-6 expression in LPS-activated Raw 264.7 cells.

Identification of new antioxidant and anti-inflammatory bioactive molecules is an important tool for selecting effective formulations for the treatment of inflammation. The mouse macrophage cell line RAW 264.7, lipopolysaccharide (LPS)-activated, is associated with an inflammation response. Activated macrophages produce reactive oxygen species (ROS), nitric oxide (NO) and inflammatory cytokines such as IL-6, TNF-α and IL-10. In the present study we have showed that pre-treatment with Ferulic Acid (FA) reduces NO accumulation in the culture medium of LPS-induced macrophage cells. Moreover, real-time experiments have revealed that FA has an inhibitory effect at the transcriptional level on the expression of some inflammatory mediators such as IL-6, TNF-α and iNOS and an activation effect on the expression of some antioxidant molecules such as Metallothioneins (MT-1, MT-2). Importantly, we have found that FA reduced the translocation of NF-E2-related factor 2 (Nrf2) and nuclear transcription factor-κB (NF-κB) into the nuclei through a reduction of the expression of phosphorylated IKK and consequently inhibited IL-6 and NF-κB promoter activity in a luciferase assay. Our data clearly suggest that FA anti-inflammatory effects are mainly mediated through IKK/NF-κB signalling pathway. Therefore, FA could represent a new natural drug extremely useful to improve anti-inflammatory treatment.

An allergen-polymeric nanoaggregate as a new tool for allergy vaccination.

A recombinant hybrid composed of the two major allergens of the Parietaria pollen Par j 1 and Par j 2 has been generated by DNA recombinant technology (PjED). This hybrid was produced in E. coli at high levels of purity. Then, the engineered derivative has been combined with a synthetic polyaminoacidic derivative having a poly(hydroxyethyl)aspartamide (PHEA) backbone and bearing both butyryl groups (C4) and succinyl (S) moieties in the side chain (PHEA-C4-S). The allergen-copolymer nanoaggregate was characterized by means of DLS, zeta potential, electrophoretic mobility and atom force microscopy analysis displaying the formation of a stable complex. Its safety has been proved in vitro on a murine cell line, human erythrocytes and basophils. Moreover, the formation of the complex did not alter the ability of the allergens to cross-link surface bound specific IgE demonstrating that the combination of an engineered hybrid with a copolymer did not interfere with its biological activity suggesting its employment as potential vaccine against Parietaria-induced allergies.

Lipid nanoparticles as delivery vehicles for the Parietaria judaica major allergen Par j 2.

Parietaria pollen is one of the major causes of allergic reaction in southern Europe, affecting about 30% of all allergic patients in this area. Specific immunotherapy is the only treatment able to modify the natural outcome of the disease by restoring a normal immunity against allergens. The preparation of allergen-solid lipid nanoparticles as delivery vehicles for therapeutic proteins, P. judaica major allergen Par j 2, was investigated. The Par j 2 allergen was expressed in a large amount in Escherichia coli and purified to homogeneity. Its immunological properties were studied by western blotting and enzyme-linked immunosorbent assay inhibition. Solid lipid nanoparticles were obtained by water-in-oil-in-water multiple emulsion method and characterized in terms of mean size and surface charge. These systems (approximately 250 nm diameter and negative surface charge) incorporated recombinant Par j 2 with 40% or greater efficiency. Moreover, the endotoxin level and anaphylactic activity of the empty solid lipid nanoparticles and recombinant Par j 2-loaded solid lipid nanoparticles were evaluated by looking at the overexpression of CD203c marker on human basophils. These results demonstrate that recombinant Par j 2-nanoparticles could be proposed as safe compositions for the development of new therapeutic dosage forms to cure allergic reactions.

Cloning and expression of a novel component of the CAP superfamily enhanced in the inflammatory response to LPS of the ascidian Ciona intestinalis.

The CAP superfamily is a group of proteins that have been linked to several biological functions such as reproduction, cancer, and immune defense. A differential screening between lipopolysaccharide (LPS)-challenged and naive Ciona intestinalis has been performed to identify LPS-induced genes. This strategy has allowed the isolation of a full-length 1471-bp cDNA encoding for a 413-amino-acid protein (CiCAP). In silico analysis has shown that this polypeptide displays a modular structure with similarities to vertebrate CAP-superfamily proteins and to a collagen-binding adhesin of Streptococcus mutans. Domain organization analysis and alignment of CiCAP to other vertebrate CAP proteins have revealed a novel structure suggesting that this protein originated from a common ancestor gene that gave rise to many subfamilies of mosaic proteins with novel functions. Quantitative mRNA expression performed by real-time polymerase chain reaction analysis has demonstrated that this gene is rapidly activated in the pharynx of C. intestinalis a few hours after LPS injection. Moreover, in situ hybridization has shown that CiCAP mRNA is highly expressed by hemocytes with large granules contained inside the pharynx vessels. Thus, CiCAP represents a protein with novel structural domains involved in ascidian immune responses.

Ferulic acid inhibits oxidative stress and cell death induced by Ab oligomers: improved delivery by solid lipid nanoparticles.

Oxidative stress and dysfunctional mitochondria are among the earliest events in AD, triggering neurodegeneration. The use of natural antioxidants could be a neuroprotective strategy for blocking cell death. Here, the antioxidant action of ferulic acid (FA) on different paths leading to degeneration of recombinant beta-amyloid peptide (rAbeta42) treated cells was investigated. Further, to improve its delivery, a novel drug delivery system (DDS) was used. Solid lipid nanoparticles (SLNs), empty or containing ferulic acid (FA-SNL), were developed as DDS. The resulting particles had small colloidal size and highly negative surface charge in water. Using neuroblastoma cells and rAbeta42 oligomers, it was demonstrated that free and SLNs-loaded FA recover cell viability. FA treatment, in particular if loaded into SLNs, decreased ROS generation, restored mitochondrial membrane potential (Deltapsi(m)) and reduced cytochrome c release and intrinsic pathway apoptosis activation. Further, FA modulated the expression of Peroxiredoxin, an anti-oxidative protein, and attenuated phosphorylation of ERK1/2 activated by Abeta oligomers.

Abeta oligomers and fibrillar aggregates induce different apoptotic pathways in LAN5 neuroblastoma cell cultures.

Fibril deposit formation of amyloid beta-protein (Abeta) in the brain is a hallmark of Alzheimer's disease (AD). Increasing evidence suggests that toxicity is linked to diffusible Abeta oligomers, which have been found in soluble brain extracts of AD patients, rather than to insoluble fibers. Here we report a study of the toxicity of two distinct forms of recombinant Abeta small oligomers and fibrillar aggregates to simulate the action of diffusible Abeta oligomers and amyloid plaques on neuronal cells. Different techniques, including dynamic light scattering, fluorescence, and scanning electron microscopy, have been used to characterize the two forms of Abeta. Under similar conditions and comparable incubation times in neuroblastoma LAN5 cell cultures, oligomeric species obtained from Abeta peptide are more toxic than fibrillar aggregates. Both oligomers and aggregates are able to induce neurodegeneration by apoptosis activation, as demonstrated by TUNEL assay and Hoechst staining assays. Moreover, we show that aggregates induce apoptosis by caspase 8 activation (extrinsic pathway), whereas oligomers induce apoptosis principally by caspase 9 activation (intrinsic pathway). These results are confirmed by cytochrome c release, almost exclusively detected in the cytosolic fraction of LAN5 cells treated with oligomers. These findings indicate an active and direct interaction between oligomers and the cellular membrane, and are consistent with internalization of the oligomeric species into the cytosol.

EGFR signalling is required for Paracentrotus lividus endomesoderm specification.

The EGFR pathway is critical for cell fate specification throughout the development of several organisms. Here we identified in sea urchin an EGFR-related antigen maternally expressed and showing a dynamic pattern of localization during development. To investigate the role played by the EGFR in Paracentrotus lividus development we blocked its activity by using the EGFR kinase inhibitor AG1478. This treatment produces decrease of EGFR phosphorylation, and embryos with various defects especially in the endomesoderm territory until to obtain an animalized phenotype. These effects are rescued by the addition of TGF-alpha, an EGFR ligand. The role played by EGFR-like along the animal/vegetal axis was also detected, after AG1478 treatment, by the extended distribution of HE and decreased nuclearization of beta-catenin in vegetal cells. Moreover, inhibition of EGFR-like reduced ERK phosphorylation, necessary for cell fate specification in the micromeres and their derivates. Taken together these results indicate that EGFR-like activity is required both for A/V axis formation and endomesoderm differentiation.

Employment of cationic solid-lipid nanoparticles as RNA carriers.

Gene transfer represents an important advance in the treatment of both genetic and acquired diseases. In this article, the suitability of cationically modified solid-lipid nanoparticles (SLN) as a nonviral vector for gene delivery was investigated, in order to obtain stable materials able to condense RNA. Cationic SLN were produced by microemulsion using Compritol ATO 888 as matrix lipid, Pluronic F68 as tenside, and dimethyldioctadecylammonium bromide (DDAB) as cationic lipid. The resulting particles were approximately 100 nm in size and showed a highly positive surface charge (+41 mV) in water. Size and shape were further characterized by scanning electron microscopy (SEM) measurements. Moreover, we utilized the sea urchin as a model system to test their applicability on a living organism. To evaluate cationic SLN ability to complex the in vitro transcribed Paracentrotus lividus bep3 RNA, we utilized both light scattering and gel mobility experiments, and protection by nuclease degradation was also investigated. By microinjection experiment, we demonstrated that the nanoparticles do not inference with the viability of the P. lividus embryo and the complex nanoparticles-bep3 permits movement of the RNA during its localization in the egg, suggesting that it could be a suitable system for gene delivery. Taken together, all these results indicate that the cationic SNL are a good RNA carrier for gene transfer system and the sea urchin a simple and versatile candidate to test biological properties of nanotechnology devices.

Maternal Paracentrotus lividus RNAs are differentially localized during the first cell division.

In Paracentrotus lividus eggs, there are RNAs localized at the animal and vegetal poles. During the first cell division, some of these RNAs are associated with the mitotic spindle, whereas others are free in the cytoplasm. Among the RNAs bound to mitotic apparatus (MA), we have found the mitochondrial 16S rRNA. By immunohistochemistry we have also detected hsp60, a mitochondrial membrane protein, localized around the MA, suggesting that the entire mitochondria are associated with it. Western blotting of proteins prepared by cellular fractionation after detergent treatment of P. lividus eggs revealed that both hsp60 and cytochrome c are not associated with cytoskeletal elements. All the above data have been confirmed by immunoblot analyses of preparations of microtubules and MA in which the presence of hsp60 and cytochrome c were detected only in the MA fraction. Moreover, mitochondrial succinate dehydrogenase activity was determined in MA and cytoplasm fractions during the first cell division, and the localization and vitality of the organelles were also confirmed by in vivo staining with Mito red. A possible role for mitochondria in the asymmetric distribution of RNAs and in cell division is discussed.

Paracentrotus lividus eggs contain different RNAs at the animal and vegetal poles.

Paracentrotus lividus eggs were divided by centrifugation into nucleated and anucleated halves. Fertilization and development of the two halves permitted us to establish that nucleated and anucleated fragments correspond, respectively, to the animal and vegetal parts. RNA was extracted from both egg halves and submitted to differential display. Northern blot analysis confirmed their maternal origin and showed that each transcript has a different expression pattern during development. By Northern blot and in situ hybridization experiments we ascertained that Bep2 and PlAn1 are localized in the animal part, whereas 16S rRNA, Plveg1, and L27 in the vegetal part, and that Plun1 is uniformly distributed. Moreover, by treating P. lividus eggs with detergent, in presence or not of drugs such as colchicine and cytochalasin B, we demonstrated the involvement of the cytoskeleton only in localization of Bep2, PlAn1, and Plun1, suggesting that different mechanisms are utilized for animal and vegetal distribution.