Objective To examine the role of lipoprotein(a) [Lp(a)] on the inflammatory response of cells in the nervous system by investigating its effect on lipopolysaccharide (LPS)-induced interleukin-6 (IL-6) secretion. Materials and methods Human astrocytoma U373 cells were treated with recombinant apolipoprotein(a) [r-apo(a)] A10K (175–11 nM), alone or in combination with LPS (100 and 10 ng/ml). IL-6 levels were evaluated by immunoblotting. Statistical analysis was performed by one-way ANOVA. Results r-apo(a) caused dose-dependent inhibition of LPS-induced IL-6 secretion (100 ng/ml LPS, p = 0.0205; 10 ng/ml LPS, p = 0.0005). Pre-treatment of cells with 88 nM r-apo(a), rinsing, and activation with 10 ng/ml LPS did not reverse the inhibition (p = 0.0048), which could be reversed by supplementation with excess serum (5–20%) (p = 0.0454) or recombinant CD14 (2.0–0.05 lg/ml) (p = 0.0230). Conclusions Our data indicate that apo(a) plays a natural anti-endotoxin role which relies on its interference with cell-associated and serum components of LPS signaling.

The demosponge Tethya citrina Sarà & Melone was subjected to long-term aquarium farming, considering that several spe- cies belonging to this genus are a rich source of useful bioactive compounds, and in particular interesting bioactive proteins. The rearing method in the present research was mainly based on applying parameters taken from the current literature. The biological activity of the sponge was assessed in terms of survival and growth over time, status of ltering apparatus, and protein banding pro les. Farmed sponges showed a high survival rate and marked reduction in size. In addition, they were frequently affected by massive production of asexual buds. Histological and ultrastructural observations showed a gradual disorganization of the sponge choanocyte chambers, with a drastic reduction in choanocytes and a progressive increase in spherulous cells. Comparative electro- phoretic analysis of the protein pro les of wild and reared specimens showed differences in protein composition and abundance between the tested groups. Such data are consistent with the increase in spherulous cells whose content was markedly electron- dense, a feature stressing the proteinaceous nature of these inclusions. Experimental tank-rearing of Tethya provided promising responses in terms of high survival rate of the species and easy reproduction, though the rearing protocol used led to profound morpho-functional changes in the sponge. This con rms the inadequate state of knowledge on farming techniques for this taxon, while also highlighting potential applications in biotechnology of ex situ breeding techniques, in order to modify the sponges’ biological responses.

Marine natural products extracted from sponges represent a new source for drug discovery. Here we describe a simple method for preparing aqueous extracts from 7 Mediterranean demosponges, which allowed the extraction of water-soluble compounds, such as proteins by homogenization of sponge tissue in phosphate buffered saline (PBS).The comparative analysis by SDS-PAGE showed differences in number of bands, bandwidth and intensity among the sponges analyzed. The PAS/silver staining revealed a substantial and different glycoprotein assortment among the demosponges studied.To further study the biological activities present in the sponge extracts, we determined the non-cytotoxic doses on four different mammalian cell types demonstrating that the optimal non-cytotoxic doses were cell type- and extract-dependent.In conclusion, the extraction method described in this paper represents a fast and efficient procedure for the extraction of water-soluble proteins from marine sponges. Furthermore, the cell viability data suggest the feasibility of this method for the direct in vitro cell-based assays.

FAD is a redox cofactor ensuring the activity of many flavoenzymes mainly located in mitochondria but also relevant for nuclear redox activities. The last enzyme in the metabolic pathway producing FAD is FAD synthase (EC 2.7.7.2), a protein known to be localized both in cytosol and in mitochondria. FAD degradation to riboflavin occurs via still poorly characterized enzymes, possibly belonging to the NUDIX hydrolase family. By confocal microscopy and immunoblotting experiments, we demonstrate here the existence of FAD synthase in the nucleus of different experimental rat models. HPLC experiments demonstrated that isolated rat liver nuclei contain ∼300 pmol of FAD·mg(-1) protein, which was mainly protein-bound FAD. A mean FAD synthesis rate of 18.1 pmol·min(-1)·mg(-1) protein was estimated by both HPLC and continuous coupled enzymatic spectrophotometric assays. Rat liver nuclei were also shown to be endowed with a FAD pyrophosphatase that hydrolyzes FAD with an optimum at alkaline pH and is significantly inhibited by adenylate-containing nucleotides. The coordinate activity of these FAD forming and degrading enzymes provides a potential mechanism by which a dynamic pool of flavin cofactor is created in the nucleus. These data, which significantly add to the biochemical comprehension of flavin metabolism and its subcellular compartmentation, may also provide the basis for a more detailed comprehension of the role of flavin homeostasis in biologically and clinically relevant epigenetic events.

Manganese (Mn) is an environmental contaminant and its overexposure contributes to the pathophysiological processes of numerous disorders of the central nervous system (CNS) in humans with mechanisms of action not completely understood. Activation of astrocytes and the subsequent release of neurotoxic factors has been implicated to contribute to neurodegeneration. Here we assessed the molecular basis of the effects of manganese on modulation of matrix metalloproteinases-2 (MMP-2) and -9 (MMP-9) in rat astrocyte cultures.Primary cultures of rat astrocytes were exposed to different doses of MnCl2. Culture supernatants and cell lysates were used for the detection of MMP-2 and MMP-9 levels and mRNA expression, respectively. The exposure of astrocytes to MnCl2 dose-dependently induced levels and expression of MMP-9. The addition of resveratrol (RSV) inhibited both levels and expression of MMP-9 in astrocytes, whereas N-acetylcysteine (NAC) and quercetin (QRC) were ineffective in inhibiting MMP-9. As a possible mechanism of Mn-induced MMP-9, we determined intracellular redox state in Mn-treated astrocytes by assessing superoxide dismutase (SOD) activity and intracellular reactive oxygen species (ROS) and found a significant increase of ROS and a decrease of SOD activity. RSV, NAC and QRC restored the redox state. The study of the mitogen activated protein kinases (MAPKs) signalling pathway demonstrated that MMP-9 transcription is mainly regulated by extracellular regulated kinase (ERK). Pre-treatment with RSV significantly reduced ERK activation suggesting that its ability to counteract MMP-9 overexpression is due not only to a general redox balance phenomenon, but also to the modulation of ERK signaling pathway.

BACKGROUND: Matrix metalloproteinases (MMPs) released by glial cells are important mediators of neuroinflammation and neurologic damage in HIV infection. The use of antiretroviral drugs able to combat the detrimental effect of chronic inflammation and target the exaggerated MMP activity might represent an attractive therapeutic challenge. Recent studies suggest that CCR5 antagonist maraviroc (MVC) exerts immunomodulant and anti-inflammatory activity beyond its anti-HIV properties. We investigated the in vitro effect of MVC on the activity of MMPs in astrocyte and microglia cultures. METHODOLOGY/PRINCIPAL FINDINGS: Primary cultures of rat astrocytes and microglia were activated by exposure to phorbol myristate acetate (PMA) or lypopolysaccharide (LPS) and treated in vitro with MVC. Culture supernatants were subjected to gelatin zymography and quantitative determination of MMP-9 and MMP-2 was done by computerized scanning densitometry. MMP-9 levels were significantly elevated in culture supernatants from both LPS- and PMA-activated astrocytes and microglia in comparison to controls. The treatment with MVC significantly inhibited in a dose-dependent manner the levels and expression of MMP-9 in PMA-activated astrocytes (p<0,05) and, to a lesser extent, in PMA-activated microglia. By contrast, levels of MMP-2 did not significantly change, although a tendency to decrease was seen in PMA-activated astrocytes after treatment with MVC. The inhibition of levels and expression of MMP-9 in PMA-activated glial cells did not depend on cytotoxic effects of MVC. No inhibition of MMP-9 and MMP-2 were found in both LPS-activated astrocytes and microglia. CONCLUSIONS: The present in vitro study suggests that CCR5 antagonist compounds, through their ability to inhibit MMP-9 expression and levels, might have a great potential for the treatment of HIV-associated neurologic damage.

Proteolytic enzymes have been implicated in the pathogenesis of Multiple Sclerosis (MS) for both their ability to degrade myelin proteins and for their presence in MS plaques.In this study we investigated whether interferon-beta (IFN-β) could differently modulate the activity and the expression of proteolytic activities against myelin basic protein (MBP) present in lipopolysaccharide (LPS)-activated astrocytes. Methodology/Principal Findings Rat astrocyte cultures were activated with LPS and simultanously treated with different doses of IFN-β. To assess the presence of MBP-cleaving proteolytic activity, culture supernatants and cellular extracts collected from astrocytes were incubated with exogenous MBP. A MBP-degrading activity was found in both lysates and supernatants from LPSactivated astrocytes and was dose-dependently inhibited by IFN-β. The use of protease inhibitors as well as the zymographic analysis indicated the presence of calpain II (CANP-2) in cell lysates and gelatinases A (MMP-2) and B (MMP-9) in cell supernatants. RT-PCR revealed that the expression of CANP-2 as well as of MMP-2 and MMP-9 was increased in LPS-activated astrocytes and was dose-dependently inhibited by IFN-β treatment. The expression of calpastatin, the natural inhibitor of CANPs, was not affected by IFN-β treatment. By contrast, decreased expression of TIMP-1 and TIMP-2, the natural inhibitors of MMP-9 and MMP-2, respectively, was observed in IFN-β-treated astrocytes compared to LPS-treated cells. The ratio enzyme/inhibitor indicated that the effect of IFN-β treatment is more relevant to CANP-2 than on MMPs. Conclusions: These results suggest that the neuroinflammatory damage during MS involves altered balance between multiple proteases and their inhibitors and indicate that IFN-β is effective in regulating different enzymatic systems involved in MS pathogenesis.

Multiple sclerosis is a complex and multifactorial neurological disease, and nutrition is one of the environmental factors possibly involved in its pathogenesis. At present, the role of nutrition is unclear, and MS therapy is not associated to a particular diet. MS clinical trials based on specific diets or dietary supplements are very few and in some cases controversial. To understand how diet can influence the course of MS and improve the wellness of MS patients, it is necessary to identify the dietary molecules, their targets and the molecular mechanisms involved in the control of the disease. The aim of this paper is to provide a molecular basis for the nutritional intervention in MS by evaluating at molecular level the effect of dietary molecules on the inflammatory and autoimmune processes involved in the disease.

The treatment of multiple sclerosis (MS) has seen important changes in the last two decades with the introduction of several drugs able to modify the evolution of this disease. Current MS therapies primarily target the peripheral immune response, although it has been suggested that their ef cacy could be in part the result of the bene cial effect on other non- speci c targets, such as matrix metalloproteinases (MMPs). Numerous experimental studies have suggested that MMPs may be involved in MS pathogenesis by contributing to blood–brain barrier disruption, migration of leukocytes into the central nervous system, demyelination and axonal damage. Therefore, MMPs have been considered important therapeutic targets in the course of MS. In this respect, different attempts have been made to develop synthetic, low-molecular-weight inhibitors of MMPs for the potential treatment of diseases in which MMPs play a major role. However, technical dif culties, side effects and reduced patient compliance because of parenteral administration have greatly limited the development in the clinical practice of speci c anti-MMP drugs. By contrast, interesting results have been obtained with compounds that are already used in the clinical practice, such as MS drugs and natural compounds with anti-in ammatory and antioxidant activity. Here, we discuss the evidence and potential mechanisms for altered MMP function in MS. Furthermore, we outline the possible medical implications for the use of compounds that target MMP activity and we propose that together with anti-MS drugs, other compounds with anti-in ammatory and antioxidant properties, such as natural ω3 fatty acids, polyphenols and tetracyclines, which inhibit MMP functions, might represent potential therapeutic approaches to mitigate MMP-related damage during MS.

We investigated whether polyphenols modulate the expression and activity of the enzymes gelatinases A (MMP-2) and B (MMP-9), involved in the pathogenesis of multiple sclerosis (MS). LPS-activated primary rat astrocytes were treated with the flavonoids quercetin (QRC) and cathechins [green tea extract (GTE)] and the non-flavonoids resveratrol (RSV) and tyrosol/hydroxytyrosol (Oliplus). As assessed by zymography and RT-PCR, RSV and Oliplus, but not QRC and GTE, dose-dependently inhibited the LPS-induced levels and mRNA expression of MMP-2 and MMP-9. By contrast, in cell-free systems direct inhibition of gelatinase activity in MS sera was determined by QRC and GTE, but not by RSV. Oliplus was only partially effective. Our results indicate that the flavonoids and non-flavonoids tested exert their inhibitory effect on MMPs, displaying different mechanisms of action, possibly related to their structure. Therefore, their combined use may represent a powerful tool for the down-regulation of MMPs in the course of MS.