Elena Mattia

Agglutination of blastospores of Candida albicans by concanavalin A and its relationship with the distribution of mannan polymers and the ultrastructure of the cell wall.

Blastospores of Candida albicans were readily agglutinated by Concanavalin A (Con A) owing to the specific binding of this lectin to the mannan receptors of the cell surface. When mannan was extracted from the cell wall by neutral buffers, alkali and acid, the agglutination was decreased or lost depending on the degree of extraction. A relatively mild alkali treatment was sufficient to derange the multilayered wall organization and transform it into a uniform, medium-density structure having about the same thickness as the untreated wall. After a more drastic extraction, all the electron-dense components of the wall were lost, the residual, alkali-insoluble wall fabric being completely electron-transparent and of about the same thickness as the inner wall region of untreated cells. Thiol-reducing agents like mercaptoethanol or dithiothreitol also extracted wall materials, an effect which was enhanced by pronase. After dithiothreitol-pronase treatment, the outer wall layers were removed but the inner wall region was not apparently damaged and some electron-dense components remained. None of these treatments significantly affected blastospore agglutination by Con A--this was reduced (but not abolished) only by the sequential action of pronase and helicase, which led to sphaeroplast formation. These sphaeroplasts showed a varied amount of residual wall consisting of evenly distributed, fibrogranular components. Two main conclusions were drawn from these results: (i) mannan polymers extend throughout the wall of the blastospore of C. albicans; (ii) the layering of the wall, as seen by ordinary fixation and staining for electron microscopy, essentially reflects the distribution of the various alkali-soluble complexes, at different levels, both over and in the rigid, glucan-chitin matrix.

Resveratrol inhibits Epstein Barr Virus lytic cycle in Burkitt's lymphoma cells by affecting multiple molecular targets

Resveratrol (RV), a polyphenolic natural product present in many plants and fruits, exhibits anti-inflammatory, cardio-protective and anti-proliferative properties. Moreover, RV affects a wide variety of viruses including members of the Herpesviridae family, retroviruses, influenza A virus and polyomavirus by altering cellular pathways that affect viral replication itself. Epstein Barr Virus (EBV), the causative agent of infectious mononucleosis, is associated with different proliferative diseases in which it establishes a latent and/or a lytic infection. In this study, we examined the antiviral activity of RV against the EBV replicative cycle and investigated the molecular targets possibly involved. In a cellular context that allows in vitro EBV activation and lytic cycle progression through mechanisms closely resembling those that in vivo initiate and enable productive infection, we found that RV inhibited EBV lytic genes expression and the production of viral particles in a dose-dependent manner. We demonstrated that RV inhibited protein synthesis, decreased reactive oxygen species (ROS) levels, and suppressed the EBV-induced activation of the redox-sensitive transcription factors NF-kB and AP-1. Further insights into the signaling pathways and molecular targets modulated by RV may provide the basis for exploiting the antiviral activity of this natural product on EBV replication.

Resveratrol Inhibits Proliferation and Survival of Epstein Barr Virus–Infected Burkitt's Lymphoma Cells Depending on Viral Latency Program

Resveratrol (3,4′,5-trihydroxy-trans-stilbene), a polyphenolic natural product, shows chemopreventive properties against several cancers, heart diseases, inflammation, and viral infections. Epstein Barr virus (EBV), a γ-herpesvirus, contributes to the development of several human cancers including Burkitts lymphoma (BL). In this study, we asked whether treatment with resveratrol would affect the viability of EBV-positive BL cells displaying different forms of latency. We report here that resveratrol, regardless of EBV status, induces caspase-dependent apoptosis by arresting cell-cycle progression in G1 phase. However, resveratrol strongly induced apoptosis in EBV(−) and latency I EBV(+) cells, whereas latency II and latency III EBV(+) BL cells showed a survival advantage that increased with the extent of the pattern of viral gene expression. Resveratrol-induced cell-cycle arrest and apoptosis occurred in association with induction of p38 MAPK phosphorylation and suppression of ERK1/2 signaling pathway. Moreover, NF-κB DNA-binding activity was inhibited in all BL lines except EBV(+) latency III cells. LMP1 oncogene, which is expressed in latency III phenotype, is involved with the higher resistance to the antiproliferative effect of resveratrol because siRNA-mediated inhibition of LMP1 greatly increased the sensitivity of latency III BL cells as well as that of lymphoblastoid cell lines to the polyphenol. We propose that a combined resveratrol/siRNA strategy may be a novel approach for the treatment of EBV-associated B-cell malignancies in which the viral pattern of gene expression has been defined. Mol Cancer Res; 9(10); 1346–55. ©2011 AACR.

Inhibition of p38 MAP kinase pathway induces apoptosis and prevents Epstein Barr virus reactivation in Raji cells exposed to lytic cycle inducing compounds

BackgroundEBV lytic cycle activators, such as phorbol esters, anti-immunoglobulin, transforming growth factor β (TGFβ), sodium butyrate, induce apoptosis in EBV-negative but not in EBV-positive Burkitts lymphoma (BL) cells. To investigate the molecular mechanisms allowing EBV-infected cells to be protected, we examined the expression of viral and cellular antiapoptotic proteins as well as the activation of signal transduction pathways in BL-derived Raji cells exposed to lytic cycle inducing agents.ResultsOur data show that, following EBV activation, the latent membrane protein 1 (LMP1) and the cellular anti-apoptotic proteins MCL-1 and BCL-2 were quickly up-regulated and that Raji cells remained viable even when exposed simultaneously to P(BU)2, sodium butyrate and TGFβ. We report here that inhibition of p38 pathway, during EBV activation, led to a three fold increment of apoptosis and largely prevented lytic gene expression.ConclusionThese findings indicate that, during the switch from the latent to the lytic phase of EBV infection, p38 MAPK phosphorylation plays a key role both for protecting the host cells from apoptosis as well as for inducing viral reactivation. Because Raji cells are defective for late antigens expression, we hypothesize that the increment of LMP1 gene expression in the early phases of EBV lytic cycle might contribute to the survival of the EBV-positive cells.

Inhibition of Poly(ADP-ribose)polymerase impairs Epstein Barr Virus lytic cycle progression

BackgroundPoly(ADP-ribosylation) is a post-translational modification of nuclear proteins involved in several cellular events as well as in processes that characterize the infective cycle of some viruses. In the present study, we investigated the role of poly(ADP-ribosylation) on Epstein-Barr Virus (EBV) lytic cycle activation.ResultsInhibition of PARP-1 by 3-aminobenzamide (3-ABA) during EBV induction, diminished cell damage and apoptosis in the non-productive Raji cell line while markedly reducing the release of viral particles in the productive Jijoye cells. Furthermore, incubation with 3-ABA up-regulated the levels of LMP1 and EBNA2 latent viral proteins. At the same time, it slightly affected the expression of the immediate early BZLF1 gene, but largely down-regulated the levels of the early BFRF1 protein. The modulation of the expression of both latent and lytic EBV genes appeared to be post-transcriptionally regulated.ConclusionTaken together the data indicate that PARP-1 plays a role in the progression of EBV lytic cycle and therefore, PARP inhibitors might represent suitable pharmacological adjuncts to control viral spread in EBV productive infection.

Inhibition of in vitro proliferation of Epstein Barr virus infected B cells by an antisense oligodeoxynucleotide targeted against EBV latent membrane protein LMP1

Inhibition of in vitro proliferation of Epstein Barr Virus infected B cells by an antisense oligodeoxynucleotide targeted against EBV latent membrane protein LMP1

Antisense to Epstein Barr virus-encoded LMP1 does not affect the transcription of viral and cellular proliferation-related genes, but induces phenotypic effects on EBV-transformed B lymphocytes

It is generally accepted that Epstein–Barr virus (EBV) latent genes EBNA-2, EBNA-3A, -3C, EBNA-LP and LMP1 are essential for growth transformation and immortalization of B lymphocytes. Among these genes, LMP1 plays a key role in the survival and dissemination of the infected B cells by inducing anti-apoptotic genes and surface expression of several activation antigens and adhesion molecules. We have previously shown that antisense oligodeoxynucleotides directed to LMP1 mRNA, effectively suppress LMP1 gene expression and substantially reduce B95.8 cell proliferation. In this study, we have used antisense LMP1 oligomers to investigate whether LMP1 suppression might influence the expression of latent EBV genes with oncogenic potential, anti-apoptotic genes, or affect the phenotype of EBV-infected B95.8 cells. Our data show that LMP1 suppression does not affect the transcription of EBNA-2, EBNA-3A, -3B and -3C genes, or that of bcl-2 and mcl-1 anti-apoptotic genes. In contrast, consistent modifications in the expression of CD39, CD54, CD23, CD11 and CD10 molecules were observed in B95.8 cells after treatment with antisense LMP1. Our findings support the possibility for using LMP1 antisense oligomers as therapeutics in EBV-associated tumors.

Interaction of EBV latent origin of replication with the nuclear matrix: identification of S/MAR sequences and protein components

During latency, Epstein Barr virus (EBV) genome, as an episome, is attached to the nuclear matrix (NM) via the latent origin of replication ori P. Within this element, we have found that a region, 580 bp long, encompassing the replicator DS element, shows the strongest affinity for the NM. In addition, by cross‐linking with cis‐diamminedichloroplatinum, we have identified two NM proteins with an apparent molecular weight of 85 and 60 kDa that, with high affinity and specificity, bind ori P. These proteins are not induced by EBV infection, but their interaction with ori P is lost upon induction of EBV lytic cycle. These data strongly suggest that the binding of ori P to specific components of the NM is required for EBV latent replication.

Resveratrol inhibits rhinovirus replication and expression of inflammatory mediators in nasal epithelia.

Human rhinoviruses (HRV), the cause of common colds, are the most frequent precipitants of acute exacerbation of asthma and chronic obstructive pulmonary disease, as well as causes of other serious respiratory diseases. No vaccine or antiviral agents are available for the prevention or treatment of HRV infection. Resveratrol exerts antiviral effect against different DNA and RNA viruses. The antiviral effect of a new resveratrol formulation containing carboxymethylated glucan was analyzed in H1HeLa cell monolayers and ex vivo nasal epithelia infected with HRV-16. Virus yield was evaluated by plaque assay and expression of viral capsid proteins by Western blot. IL-10, IFN-β, IL-6, IL-8 and RANTES levels were evaluated by ELISA assay. ICAM-1 was assessed by Western blot and immunofluorescence. Resveratrol exerted a high, dose-dependent, antiviral activity against HRV-16 replication and reduced virus-induced secretion of IL-6, IL-8 and RANTES to levels similar to that of uninfected nasal epithelia. Basal levels of IL-6 and RANTES were also significantly reduced in uninfected epithelia confirming an anti-inflammatory effect of the compound. HRV-induced expression of ICAM-1 was reversed by resveratrol. Resveratrol may be useful for a therapeutic approach to reduce HRV replication and virus-induced cytokine/chemokine production.

Neem oil nanoemulsions: characterisation and antioxidant activity

Abstract The aim of the present work is to develop nanoemulsions (NEs), nanosized emulsions, manufactured for improving the delivery of active pharmaceutical ingredients. In particular, nanoemulsions composed of Neem seed oil, contain rich bioactive components, and Tween 20 as nonionic surfactant were prepared. A mean droplet size ranging from 10 to 100 nm was obtained by modulating the oil/surfactant ratio. Physicochemical characterisation was carried out evaluating size, ζ-potential, microviscosity, polarity and turbidity of the external shell and morphology, along with stability in simulated cerebrospinal fluid (CSF), activity of Neem oil alone and in NEs, HEp-2 cell interaction and cytotoxicity studies. This study confirms the formation of NEs by Tween 20 and Neem oil at different weight ratios with small and homogenous dimensions. The antioxidant activity of Neem oil alone and in NEs was comparable, whereas its cytotoxicity was strongly reduced when loaded in NEs after interaction with HEp-2 cells. Graphical Abstract