Maria Sciortino | University of Messina (original) (raw)

Papers by Maria Sciortino

Journal of Virology, 2001

Following the lead of recent studies on the presence of RNA in virions of human cytomegalovirus, ... more Following the lead of recent studies on the presence of RNA in virions of human cytomegalovirus, we investigated the presence and identity of RNAs from purified virions of herpes simple virus 1. To facilitate these studies, we designed primers for all known open reading frames (ORFs) and also constructed cDNA arrays containing probes designed to detect all known transcripts. In the first series of experiments, labeled DNA made by reverse transcription of poly(A) ؉ RNA extracted from infected HEp-2 or rabbit skin cells hybridized to all but two of the probes in the cDNA array. A similar analysis of the RNA extracted from purified extracellular virions derived from infected HEp-2 cells hybridized to probes representing 24 of the ORFs. In the second series of analyses, we reverse transcribed and amplified by PCR RNAs from purified intracellular or extracellular virions derived from infected HEp-2 or Vero cell lines. The positive RNAs were retested by PCR with and without prior reverse transcription to ensure that the samples tested were free of contaminating DNA. The results were as follows. (i) Only a fraction of viral ORF transcripts were represented in virion RNA, and only nine RNAs ) were positive in all RT PCR assays. Of these, seven were positive by hybridization to cDNA arrays. (ii) RNA extracted from cells infected with a mutant virus lacking the U S 8 to U S 12 genes yielded results similar to those described above, indicating that U S 11, a known RNA binding protein, does not play a role in packaging RNA in virions.

Annals of The New York Academy of Sciences, 2007

Abstract: We have previously demonstrated that wild-type herpes simplex virus type 1 (HSV-1), as ... more Abstract: We have previously demonstrated that wild-type herpes simplex virus type 1 (HSV-1), as well as nonreplicating UV-inactivated HSV-1, promptly activates the nuclear factor-κB (NF-κB) in U937 monocytoid cells and that glycoprotein D (gD) of HSV-1 is sufficient by itself to exert a similar effect.We then investigated the signaling pathway used by HSV-1 to initiate NF-κB activation and, particularly, whether our observation could be related to the capability of HSV-1-gD to directly stimulate NF-κB through its interaction with the herpes virus entry receptor A (HveA). Here we report that: (a) co-cultivation of U937 cells with an adherent cell line expressing wild-type gD on its surface led to increased NF-κB activation, while co-cultivation with the same adherent cell line expressing a mutated form of gD, lacking the capability to bind HveA, did not cause the same effect; (b) exposure to UV-inactivated HSV-1 induced the activation of NF-κB in HveA-expressing U937 and THP-1 cells, but not in non-HveA-expressing HEp-2 cells; and (c) activation of NF-κB in U937 and THP-1 cells exposed to soluble gD was inhibited by an antibody able to interfere with gD–HveA interaction. These results suggest that HSV-1-gD–HveA interaction initiates a signal transduction pathway leading to NF-κB activation.

Journal of Virology, 2007

To generate a null U L 49 gene mutant of herpes simplex virus 1 (HSV-1), we deleted from the vira... more To generate a null U L 49 gene mutant of herpes simplex virus 1 (HSV-1), we deleted from the viral DNA, encoded as a bacterial artificial chromosome (BAC), the U L 49 open reading frame and, in a second step, restored it. Upon transfection into Vero cells, the BAC-⌬U L 49 DNA yielded foci of degenerated cells that could not be expanded and a few replication-competent clones. The replication-competent viral clones derived from independent transfections yielded viruses that expressed genes with some delay, produced smaller plaques, and gave lower yields than wild-type virus. A key finding is that the independently derived replication-competent viruses lacked the virion host shutoff (vhs) activity expressed by the RNase encoded by the U L 41 gene. One mutant virus expressed no vhs protein, whereas two others, derived from independent transfections, produced truncated vhs proteins consistent with the spontaneous in-frame deletion. In contrast, cells infected with the virus recovered upon transfection of the BAC-U L 49R DNA (R-U L 49) accumulated a full-length vhs protein, indicating that in the parental BAC-⌬U L 49 DNA, the U L 41 gene was intact. We conclude that expression of the vhs protein in the absence of U L 49 protein is lethal, a conclusion bolstered by the evidence reported elsewhere that in transfected cells vhs requires both VP16 and VP22, the product of U L 49, to be neutralized.

Antiviral Research, 2005

, is an acyclic nucleoside phosphonate known to inhibit HIV replication in vitro and to reduce vi... more , is an acyclic nucleoside phosphonate known to inhibit HIV replication in vitro and to reduce viremia in HIV-infected patients. Here we have investigated whether tenofovir is able to protect peripheral blood mononuclear cells (PBMCs) from healthy donors against human T-cell leukemia/lymphotropic virus type 1 (HTLV-1) infection in vitro. PBMCs were pre-treated with tenofovir and infected by exposure to an irradiated cell line chronically harbouring HTLV-1. Measurements of viral DNA, as well as viral gene and protein expression, at 4 weeks after infection, revealed that tenofovir at concentrations of 1 M and higher completely protected PBMCs against HTLV-1; lower concentrations did not fully prevent HTLV-1 infection of the cultures. Nevertheless, in the long term, cell growth of infected PBMCs was inhibited in vitro even by 0.1 M tenofovir. In addition, tenofovir directly inhibited HTLV-1 reverse transcriptase activity, in a cell-free assay that utilizes a crude preparation from HTLV-1 viral particles as a source of the enzyme. The selectivity index of tenofovir for HTLV-1, was about four times higher than that of azidothymidine. Taken together our results strongly encourage further studies to investigate the real impact of tenofovir towards HTLV-1 infection.

Biomaterials, 2009

The aim of this study was to develop nanoparticles made of the amphiphilic cyclodextrin heptakis ... more The aim of this study was to develop nanoparticles made of the amphiphilic cyclodextrin heptakis (2-Ooligo(ethyleneoxide)-6-hexadecylthio-)-b-CD (SC16OH) entrapping docetaxel (Doc) and establish their in vivo potential. Doc-loaded SC16OH nanoparticles were prepared by the emulsion-solvent evaporation technique and fully characterized for size, zeta potential, amount of entrapped drug, release rate and degradation rate. Spherical vesicular nanoparticles displaying a hydrodynamic radius of about 95 nm which did not change upon storage as an aqueous dispersion, a negative zeta potential and entrapment efficiency of Doc very close to 100% were produced. DSC study highlighted the crystalline nature of SC16OH, unloaded and Doc-loaded SC16OH nanoparticles which resulted in their very slow dissolution during release stage and well-modulated release of entrapped Doc for about 8 weeks. Doc-loaded SC16OH nanoparticles were not hemolytic toward red blood cells as compared to a commercial Doc formulation (Taxotere Ò ) which shows a dose-dependent toxicity. After exposure of HEp-2 cells to equivalent doses of free Doc and Doc-loaded SC16OH nanoparticles, superior cell killing and cell damage were observed for nanoparticles. Finally, cell damage was attributed to aberrant mitosis which was found to be significantly higher for HEp-2 cells treated with Doc-loaded SC16OH nanoparticles as compared to free Doc likely due to the ability of nanoparticles to slowly release the drug allowing prolonged cell arrest in mitosis. Taken together, these results highlights a great potential of nanoparticles based on SC16OH in solid tumors therapy.

Biomaterials, 2006

The photodynamic activity of a carrier-sensitizer system consisting of heterotopic colloidal nano... more The photodynamic activity of a carrier-sensitizer system consisting of heterotopic colloidal nanoparticles (diameter 100-1000 nm) of a cationic amphiphilic cyclodextrin, heptakis(2-omega-amino-O-oligo(ethylene oxide)-6-hexylthio)-beta-CD (SC6CDNH2) encapsulating the anionic 5,10,15,20-tetrakis(4-sulfonatophenyl)-21H,23H-porphyrin (TPPS) is investigated by an interdisciplinary approach involving the combination of time-resolved absorption and emission techniques with in vitro studies on cultured tumor cells. In a range of TPPS:SC6CDNH2 molar ratios between 1:10 and 1:50 these nanoparticles preserve the photodynamic properties of the entrapped photoactive agent. In fact, the triplet state of TPPS is efficiently populated, very long-lived and, as a consequence, able to produce singlet oxygen (the essential species for the photodynamic action) with quantum yield comparable to the free TPPS. Photodynamic efficacy of the carrier/sensitizer system is proven by in vitro studies on tumor Hela cells treated with TPPS:SC6CDNH2 at different molar ratio, showing significant cells death upon illumination with visible light.

Chemistry-a European Journal, 2003

Proceedings of The National Academy of Sciences, 2007

The virion host shutoff (vhs) protein encoded by the UL41 gene of herpes simplex virus 1 is an en... more The virion host shutoff (vhs) protein encoded by the UL41 gene of herpes simplex virus 1 is an endoribonuclease. The enzyme is introduced into the cell during unpackaging of the virion upon entry and selectively degrades mRNA for several hours. The RNase activity ceases after the onset of synthesis of late (␥) viral proteins. Here we report that vhs protein does not accumulate in cells transiently transfected with only a plasmid encoding the UL41 gene. However, vhs does accumulate in cells cotransfected with plasmids expressing two other tegument proteins, VP16 and VP22. vhs does not directly interact with VP22 but, instead, binds VP22 only in the presence of VP16. In contrast to these findings, the amounts of vhs mRNA accumulating in the cells transfected solely with vhs are not significantly different from those detected in cells coexpressing vhs, VP16, and VP22. We conclude from these studies that the steady state of vhs mRNA, reflecting synthesis and turnover of mRNA, is not affected by the interaction of vhs protein with VP16 with VP22. A model is proposed in which the vhs protein may function to sequester mRNAs in compartments inaccessible to the cellular translational machinery and that VP16 and VP22 rescue the mRNAs by interacting with the vhs protein.

Journal of Medicinal Chemistry, 2005

Phosphonated carbocyclic 2′-oxa-3′-aza-nucleosides have been synthesized in good yields by 1,3-di... more Phosphonated carbocyclic 2′-oxa-3′-aza-nucleosides have been synthesized in good yields by 1,3-dipolar cycloaddition methodology. The cytotoxicity and the reverse transcriptase inhibitory activity of the obtained compounds have been investigated. Phosphonated carbocyclic 2′-oxa-3′-aza-nucleosides, while showing low levels of cytotoxicity, exert a specific inhibitor activity on two different reverse transcriptases, which is comparable with that of AZT, opening new perspectives on their possible use as therapeutic agents, in anti-retroviral and anti-HBV chemotherapy.

Journal of Medicinal Chemistry, 2007

Phosphonated isoxazolinyl nucleosides have been prepared via 1,3-dipolar cycloaddition reaction o... more Phosphonated isoxazolinyl nucleosides have been prepared via 1,3-dipolar cycloaddition reaction of nitrile oxides with corresponding vinyl or allyl nucleobases for antiviral studies. The cytotoxicity, the anti-HSV activity and the RT-inhibitory activity of the obtained compounds were evaluated and compared with those of AZT and diethyl{(1 0 SR,4 0 RS)-1 0 -[[(5-methyl-2,4-dioxo-3,4dihydropyrimidin-1(2H)-yl)]-3 0 -methyl-2 0 -oxa-3 0 -azacyclopent-4 0 -yl]}methylphosphonate, a saturated phosphonated dihydroisoxazole nucleoside analogue.

Vasopressin (AVP) is a hormone with antidiuretic properties that is also involved in cellular pro... more Vasopressin (AVP) is a hormone with antidiuretic properties that is also involved in cellular proliferation of breast, pulmonary, and pancreatic neoplasias, attributable to the interaction with specific receptors, among which is the V2-R. Using a culture model of CAKI-2 and A498 cancer cells, our study aimed to verify if renal carcinoma cells also express V2-R and whether receptor activation modulates their proliferation. Immunofluorescence and RT-PCR showed that both CAKI-2 and A498 cells effectively synthesize and express the V2-R. Administration of the vasopressin analogue DDAVP induced an evident growth in both CAKI-2 and A498 cell lines. However, this proliferative effect was completely avoided by the preventive addition of the V2-R antagonist SR121463B (satavaptan). Our study shows for the first time that renal cancer may effectively synthesize and express the V2-R. Furthermore, AVP exerts in vitro a proliferative effect by acting on this receptor, as the selective V2-R blockage is able to completely prevent the cellular growth. A validation of these findings with in vivo models is required to ascertain if the eventual presence of V2-R could influence the aggressiveness of human renal neoplasias. From this point of view, a new, interesting therapeutical application of V2-R antagonists in the treatment of renal cancer could also be proposed, similar to that successfully described in the treatment of autosomal polycystic kidney disease (ADPKD).

Journal of Biotechnology, 2010

Cellular Microbiology, 2008

The UV-inactivated herpes simplex virus 1 (HSV-1) and glycoprotein D (gD) of HSV-1 have been show... more The UV-inactivated herpes simplex virus 1 (HSV-1) and glycoprotein D (gD) of HSV-1 have been shown to activate nuclear factor κB (NF-κB) in U937 cells, but mechanisms involved in this activation have not been elucidated. Here we report that: (i) UV-inactivated HSV-1 induced an increased NF-κB activation in cells expressing human HVEM (for herpesvirus entry mediator) at surface level, naturally or following stable transfection, but not in cells in which this receptor was not detected by flow cytometry analysis, (ii) treatment with soluble gD induced a dose-dependent NF-κB activation in THP-1 cells naturally expressing HVEM, and a monoclonal antibody that prevents binding of gD to HVEM significantly reduced NF-κB activation by soluble gD in the same cells, (iii) coculture with transfectants expressing wild-type gD on their surface induced an approximately twofold increase in NF-κB activation in cells naturally expressing HVEM, while coculture with transfectants expressing a mutated form of gD, lacking its capability to bind HVEM, did not induce a similar effect and (iv) treatment with soluble gD induced a dose-dependent NF-κB activation in CHO transfectants expressing HVEM, but not in control CHO transfectants lacking any functional gD receptor. Overall, these results establish that HVEM is involved in NF-κB activation by HSV-1 gD.

Journal of Materials Chemistry, 2008

In this contribution we report the design, fabrication and properties of hydrosoluble platinum na... more In this contribution we report the design, fabrication and properties of hydrosoluble platinum nanoparticles decorated with a nitric oxide (NO) caging compound. Direct monitoring of NO through an ultrasensitive NO electrode demonstrate that the nanoparticles are stable in the dark ...

Virology, 2000

Increasing evidence suggests that regulation of apoptosis in infected cells is associated with se... more Increasing evidence suggests that regulation of apoptosis in infected cells is associated with several viral infections. The gammaherpesvirus bovine herpesvirus 4 (BHV-4) has been shown to harbor genes with antiapoptotic potentialities. However, here we have demonstrated that productive infection of adherent, permissive cell lines by BHV-4 resulted in a cytopathic effect characterized by induction of apoptosis. This phenomenon was confirmed using different techniques to detect apoptosis and using different virus strains and cell targets. Apoptosis induced by BHV-4 was inhibited by (1) treatment with doses of heparin, which completely inhibited virus attachment and infectivity; (2) UV treatment, which completely abrogated infectivity; and (3) treatment with a dose of phosphonoacetic acid, which blocked virus replication. Virus-induced apoptosis was associated with a down-regulation of Bcl-2 expression and was reduced by Z-VAD-FMK, but not by Z-DEVD-FMK (caspase-3-specific) caspase inhibitors. Inhibition of apoptosis by Z-VAD-FMK treatment during infection did not modify virus yield. Therefore, despite the presence of antiapoptotic genes in its genoma, BHV-4 could complete its cycle of productive infection while inducing apoptosis of infected cells. This finding might have implications for the pathobiology of BHV-4 and other gammaherpesviruses in vivo.

Biochemical Pharmacology, 2008

A. Mastino). 1 This paper was presented in part in the form of a poster at the meeting ''Apoptosi... more A. Mastino). 1 This paper was presented in part in the form of a poster at the meeting ''Apoptosis World 2008: From mechanisms to applications'', Luxembourg, 23-26 January 2008. a v a i l a b l e a t w w w . s c i e n c e d i r e c t . c o m j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / b i o c h e m p h a r m 0006-2952/$ -see front matter #

Cell Death and Differentiation, 1997

Increasing evidence indicates that apoptosis can be associated with several viral infections. Her... more Increasing evidence indicates that apoptosis can be associated with several viral infections. Here we demonstrate, that infection of monocytoid cells by Herpes simplex virus 2 (HSV-2) resulted, in time-and dose-dependent induction of apoptosis as an exclusive cytopathic effect. The phenomenon was confirmed using four different techniques. Conversely, apoptosis was not observed in the Vero cell line. Virus yield in monocytoid cells was delayed and reduced, although well detectable, in comparison with that observed in Vero cells. Nevertheless, released virions exhibited full infecting capability. Apoptosis induced by HSV-2 was not inhibited by cycloheximide and only partially by an UV-treatment which completely abrogated infectivity. Virus-induced apoptosis was partly inhibited by indomethacin and was associated with a down-regulation of Bcl-2. A similar, but less pronounced, apoptosis-inducing effect in monocytoid cells was also observed with HSV-1 infection. Depending on the target cells, therefore, HSV could complete a cycle of infection which is characterized by apoptosis of infected cells.

Proceedings of The National Academy of Sciences, 2002

An earlier report has shown that herpes simplex virus 1 virions package RNA. Experiments designed... more An earlier report has shown that herpes simplex virus 1 virions package RNA. Experiments designed to reveal the identity of the virion proteins capable of binding the RNA and to show whether the mRNA carried in the newly infected cells was expressed showed the following: (i) 32P-labeled riboprobe generated by in vitro transcription of the US8.5 ORF bound three proteins identified as the products of US11, UL47, and UL49 (VP22) genes. (ii) Viral RNA was bound to UL47 or US11 proteins immune precipitated from cells transduced with baculoviruses expressing UL47 or US11 and then superinfected with HSV-1 under conditions that blocked DNA synthesis and assembly of virions. (iii) Virions were purified from cells transduced with a baculovirus encoding a US8.5 protein fused to green fluorescent protein and superinfected with an HSV-1 mutant lacking the US8-12 genes. HEp-2 cells infected with these virions expressed the chimeric protein in ≈1% of infected cells. (iv) In mixed cultures, untreated Vero cells acquired the mRNA encoding the green fluorescent-US8.5 chimeric protein from HEp-2 cells doubly transduced with the genes encoding VP22 and the chimeric protein. The transfer was RNase sensitive and VP22 dependent, indicating that the RNA encoded by the chimeric gene was transferred to Vero cells as mRNA. We conclude that (i) three virion proteins are capable of binding RNA; (ii) the packaged RNA can be expressed in newly infected cells; and (iii) the UL47 protein was earlier reported to shuttle from nucleus to the cytoplasm and may transport RNA. VP22 thus appears to be a member of a new class of viral proteins whose major function is to bind and transport infected cell mRNA to uninfected cells to create the environment for effective initiation of infection.

Proceedings of The National Academy of Sciences, 2002

An earlier report has shown that herpes simplex virus 1 virions package RNA. Experiments designed... more An earlier report has shown that herpes simplex virus 1 virions package RNA. Experiments designed to reveal the identity of the virion proteins capable of binding the RNA and to show whether the mRNA carried in the newly infected cells was expressed showed the following: (i) 32 P-labeled riboprobe generated by in vitro transcription of the U S 8.5 ORF bound three proteins identified as the products of US11, UL47, and UL49 (VP22) genes. (ii) Viral RNA was bound to U L47 or US11 proteins immune precipitated from cells transduced with baculoviruses expressing U L47 or US11 and then superinfected with HSV-1 under conditions that blocked DNA synthesis and assembly of virions. (iii) Virions were purified from cells transduced with a baculovirus encoding a U S8.5 protein fused to green fluorescent protein and superinfected with an HSV-1 mutant lacking the U S8 -12 genes. HEp-2 cells infected with these virions expressed the chimeric protein in Ϸ1% of infected cells. (iv) In mixed cultures, untreated Vero cells acquired the mRNA encoding the green fluorescent-U S8.5 chimeric protein from HEp-2 cells doubly transduced with the genes encoding VP22 and the chimeric protein. The transfer was RNase sensitive and VP22 dependent, indicating that the RNA encoded by the chimeric gene was transferred to Vero cells as mRNA. We conclude that (i) three virion proteins are capable of binding RNA; (ii) the packaged RNA can be expressed in newly infected cells; and (iii) the U L47 protein was earlier reported to shuttle from nucleus to the cytoplasm and may transport RNA. VP22 thus appears to be a member of a new class of viral proteins whose major function is to bind and transport infected cell mRNA to uninfected cells to create the environment for effective initiation of infection.

Journal of Virology, 2001

Following the lead of recent studies on the presence of RNA in virions of human cytomegalovirus, ... more Following the lead of recent studies on the presence of RNA in virions of human cytomegalovirus, we investigated the presence and identity of RNAs from purified virions of herpes simple virus 1. To facilitate these studies, we designed primers for all known open reading frames (ORFs) and also constructed cDNA arrays containing probes designed to detect all known transcripts. In the first series of experiments, labeled DNA made by reverse transcription of poly(A) ؉ RNA extracted from infected HEp-2 or rabbit skin cells hybridized to all but two of the probes in the cDNA array. A similar analysis of the RNA extracted from purified extracellular virions derived from infected HEp-2 cells hybridized to probes representing 24 of the ORFs. In the second series of analyses, we reverse transcribed and amplified by PCR RNAs from purified intracellular or extracellular virions derived from infected HEp-2 or Vero cell lines. The positive RNAs were retested by PCR with and without prior reverse transcription to ensure that the samples tested were free of contaminating DNA. The results were as follows. (i) Only a fraction of viral ORF transcripts were represented in virion RNA, and only nine RNAs ) were positive in all RT PCR assays. Of these, seven were positive by hybridization to cDNA arrays. (ii) RNA extracted from cells infected with a mutant virus lacking the U S 8 to U S 12 genes yielded results similar to those described above, indicating that U S 11, a known RNA binding protein, does not play a role in packaging RNA in virions.

Annals of The New York Academy of Sciences, 2007

Abstract: We have previously demonstrated that wild-type herpes simplex virus type 1 (HSV-1), as ... more Abstract: We have previously demonstrated that wild-type herpes simplex virus type 1 (HSV-1), as well as nonreplicating UV-inactivated HSV-1, promptly activates the nuclear factor-κB (NF-κB) in U937 monocytoid cells and that glycoprotein D (gD) of HSV-1 is sufficient by itself to exert a similar effect.We then investigated the signaling pathway used by HSV-1 to initiate NF-κB activation and, particularly, whether our observation could be related to the capability of HSV-1-gD to directly stimulate NF-κB through its interaction with the herpes virus entry receptor A (HveA). Here we report that: (a) co-cultivation of U937 cells with an adherent cell line expressing wild-type gD on its surface led to increased NF-κB activation, while co-cultivation with the same adherent cell line expressing a mutated form of gD, lacking the capability to bind HveA, did not cause the same effect; (b) exposure to UV-inactivated HSV-1 induced the activation of NF-κB in HveA-expressing U937 and THP-1 cells, but not in non-HveA-expressing HEp-2 cells; and (c) activation of NF-κB in U937 and THP-1 cells exposed to soluble gD was inhibited by an antibody able to interfere with gD–HveA interaction. These results suggest that HSV-1-gD–HveA interaction initiates a signal transduction pathway leading to NF-κB activation.

Journal of Virology, 2007

To generate a null U L 49 gene mutant of herpes simplex virus 1 (HSV-1), we deleted from the vira... more To generate a null U L 49 gene mutant of herpes simplex virus 1 (HSV-1), we deleted from the viral DNA, encoded as a bacterial artificial chromosome (BAC), the U L 49 open reading frame and, in a second step, restored it. Upon transfection into Vero cells, the BAC-⌬U L 49 DNA yielded foci of degenerated cells that could not be expanded and a few replication-competent clones. The replication-competent viral clones derived from independent transfections yielded viruses that expressed genes with some delay, produced smaller plaques, and gave lower yields than wild-type virus. A key finding is that the independently derived replication-competent viruses lacked the virion host shutoff (vhs) activity expressed by the RNase encoded by the U L 41 gene. One mutant virus expressed no vhs protein, whereas two others, derived from independent transfections, produced truncated vhs proteins consistent with the spontaneous in-frame deletion. In contrast, cells infected with the virus recovered upon transfection of the BAC-U L 49R DNA (R-U L 49) accumulated a full-length vhs protein, indicating that in the parental BAC-⌬U L 49 DNA, the U L 41 gene was intact. We conclude that expression of the vhs protein in the absence of U L 49 protein is lethal, a conclusion bolstered by the evidence reported elsewhere that in transfected cells vhs requires both VP16 and VP22, the product of U L 49, to be neutralized.

Antiviral Research, 2005

, is an acyclic nucleoside phosphonate known to inhibit HIV replication in vitro and to reduce vi... more , is an acyclic nucleoside phosphonate known to inhibit HIV replication in vitro and to reduce viremia in HIV-infected patients. Here we have investigated whether tenofovir is able to protect peripheral blood mononuclear cells (PBMCs) from healthy donors against human T-cell leukemia/lymphotropic virus type 1 (HTLV-1) infection in vitro. PBMCs were pre-treated with tenofovir and infected by exposure to an irradiated cell line chronically harbouring HTLV-1. Measurements of viral DNA, as well as viral gene and protein expression, at 4 weeks after infection, revealed that tenofovir at concentrations of 1 M and higher completely protected PBMCs against HTLV-1; lower concentrations did not fully prevent HTLV-1 infection of the cultures. Nevertheless, in the long term, cell growth of infected PBMCs was inhibited in vitro even by 0.1 M tenofovir. In addition, tenofovir directly inhibited HTLV-1 reverse transcriptase activity, in a cell-free assay that utilizes a crude preparation from HTLV-1 viral particles as a source of the enzyme. The selectivity index of tenofovir for HTLV-1, was about four times higher than that of azidothymidine. Taken together our results strongly encourage further studies to investigate the real impact of tenofovir towards HTLV-1 infection.

Biomaterials, 2009

The aim of this study was to develop nanoparticles made of the amphiphilic cyclodextrin heptakis ... more The aim of this study was to develop nanoparticles made of the amphiphilic cyclodextrin heptakis (2-Ooligo(ethyleneoxide)-6-hexadecylthio-)-b-CD (SC16OH) entrapping docetaxel (Doc) and establish their in vivo potential. Doc-loaded SC16OH nanoparticles were prepared by the emulsion-solvent evaporation technique and fully characterized for size, zeta potential, amount of entrapped drug, release rate and degradation rate. Spherical vesicular nanoparticles displaying a hydrodynamic radius of about 95 nm which did not change upon storage as an aqueous dispersion, a negative zeta potential and entrapment efficiency of Doc very close to 100% were produced. DSC study highlighted the crystalline nature of SC16OH, unloaded and Doc-loaded SC16OH nanoparticles which resulted in their very slow dissolution during release stage and well-modulated release of entrapped Doc for about 8 weeks. Doc-loaded SC16OH nanoparticles were not hemolytic toward red blood cells as compared to a commercial Doc formulation (Taxotere Ò ) which shows a dose-dependent toxicity. After exposure of HEp-2 cells to equivalent doses of free Doc and Doc-loaded SC16OH nanoparticles, superior cell killing and cell damage were observed for nanoparticles. Finally, cell damage was attributed to aberrant mitosis which was found to be significantly higher for HEp-2 cells treated with Doc-loaded SC16OH nanoparticles as compared to free Doc likely due to the ability of nanoparticles to slowly release the drug allowing prolonged cell arrest in mitosis. Taken together, these results highlights a great potential of nanoparticles based on SC16OH in solid tumors therapy.

Biomaterials, 2006

The photodynamic activity of a carrier-sensitizer system consisting of heterotopic colloidal nano... more The photodynamic activity of a carrier-sensitizer system consisting of heterotopic colloidal nanoparticles (diameter 100-1000 nm) of a cationic amphiphilic cyclodextrin, heptakis(2-omega-amino-O-oligo(ethylene oxide)-6-hexylthio)-beta-CD (SC6CDNH2) encapsulating the anionic 5,10,15,20-tetrakis(4-sulfonatophenyl)-21H,23H-porphyrin (TPPS) is investigated by an interdisciplinary approach involving the combination of time-resolved absorption and emission techniques with in vitro studies on cultured tumor cells. In a range of TPPS:SC6CDNH2 molar ratios between 1:10 and 1:50 these nanoparticles preserve the photodynamic properties of the entrapped photoactive agent. In fact, the triplet state of TPPS is efficiently populated, very long-lived and, as a consequence, able to produce singlet oxygen (the essential species for the photodynamic action) with quantum yield comparable to the free TPPS. Photodynamic efficacy of the carrier/sensitizer system is proven by in vitro studies on tumor Hela cells treated with TPPS:SC6CDNH2 at different molar ratio, showing significant cells death upon illumination with visible light.

Chemistry-a European Journal, 2003

Proceedings of The National Academy of Sciences, 2007

The virion host shutoff (vhs) protein encoded by the UL41 gene of herpes simplex virus 1 is an en... more The virion host shutoff (vhs) protein encoded by the UL41 gene of herpes simplex virus 1 is an endoribonuclease. The enzyme is introduced into the cell during unpackaging of the virion upon entry and selectively degrades mRNA for several hours. The RNase activity ceases after the onset of synthesis of late (␥) viral proteins. Here we report that vhs protein does not accumulate in cells transiently transfected with only a plasmid encoding the UL41 gene. However, vhs does accumulate in cells cotransfected with plasmids expressing two other tegument proteins, VP16 and VP22. vhs does not directly interact with VP22 but, instead, binds VP22 only in the presence of VP16. In contrast to these findings, the amounts of vhs mRNA accumulating in the cells transfected solely with vhs are not significantly different from those detected in cells coexpressing vhs, VP16, and VP22. We conclude from these studies that the steady state of vhs mRNA, reflecting synthesis and turnover of mRNA, is not affected by the interaction of vhs protein with VP16 with VP22. A model is proposed in which the vhs protein may function to sequester mRNAs in compartments inaccessible to the cellular translational machinery and that VP16 and VP22 rescue the mRNAs by interacting with the vhs protein.

Journal of Medicinal Chemistry, 2005

Phosphonated carbocyclic 2′-oxa-3′-aza-nucleosides have been synthesized in good yields by 1,3-di... more Phosphonated carbocyclic 2′-oxa-3′-aza-nucleosides have been synthesized in good yields by 1,3-dipolar cycloaddition methodology. The cytotoxicity and the reverse transcriptase inhibitory activity of the obtained compounds have been investigated. Phosphonated carbocyclic 2′-oxa-3′-aza-nucleosides, while showing low levels of cytotoxicity, exert a specific inhibitor activity on two different reverse transcriptases, which is comparable with that of AZT, opening new perspectives on their possible use as therapeutic agents, in anti-retroviral and anti-HBV chemotherapy.

Journal of Medicinal Chemistry, 2007

Phosphonated isoxazolinyl nucleosides have been prepared via 1,3-dipolar cycloaddition reaction o... more Phosphonated isoxazolinyl nucleosides have been prepared via 1,3-dipolar cycloaddition reaction of nitrile oxides with corresponding vinyl or allyl nucleobases for antiviral studies. The cytotoxicity, the anti-HSV activity and the RT-inhibitory activity of the obtained compounds were evaluated and compared with those of AZT and diethyl{(1 0 SR,4 0 RS)-1 0 -[[(5-methyl-2,4-dioxo-3,4dihydropyrimidin-1(2H)-yl)]-3 0 -methyl-2 0 -oxa-3 0 -azacyclopent-4 0 -yl]}methylphosphonate, a saturated phosphonated dihydroisoxazole nucleoside analogue.

Vasopressin (AVP) is a hormone with antidiuretic properties that is also involved in cellular pro... more Vasopressin (AVP) is a hormone with antidiuretic properties that is also involved in cellular proliferation of breast, pulmonary, and pancreatic neoplasias, attributable to the interaction with specific receptors, among which is the V2-R. Using a culture model of CAKI-2 and A498 cancer cells, our study aimed to verify if renal carcinoma cells also express V2-R and whether receptor activation modulates their proliferation. Immunofluorescence and RT-PCR showed that both CAKI-2 and A498 cells effectively synthesize and express the V2-R. Administration of the vasopressin analogue DDAVP induced an evident growth in both CAKI-2 and A498 cell lines. However, this proliferative effect was completely avoided by the preventive addition of the V2-R antagonist SR121463B (satavaptan). Our study shows for the first time that renal cancer may effectively synthesize and express the V2-R. Furthermore, AVP exerts in vitro a proliferative effect by acting on this receptor, as the selective V2-R blockage is able to completely prevent the cellular growth. A validation of these findings with in vivo models is required to ascertain if the eventual presence of V2-R could influence the aggressiveness of human renal neoplasias. From this point of view, a new, interesting therapeutical application of V2-R antagonists in the treatment of renal cancer could also be proposed, similar to that successfully described in the treatment of autosomal polycystic kidney disease (ADPKD).

Journal of Biotechnology, 2010

Cellular Microbiology, 2008

The UV-inactivated herpes simplex virus 1 (HSV-1) and glycoprotein D (gD) of HSV-1 have been show... more The UV-inactivated herpes simplex virus 1 (HSV-1) and glycoprotein D (gD) of HSV-1 have been shown to activate nuclear factor κB (NF-κB) in U937 cells, but mechanisms involved in this activation have not been elucidated. Here we report that: (i) UV-inactivated HSV-1 induced an increased NF-κB activation in cells expressing human HVEM (for herpesvirus entry mediator) at surface level, naturally or following stable transfection, but not in cells in which this receptor was not detected by flow cytometry analysis, (ii) treatment with soluble gD induced a dose-dependent NF-κB activation in THP-1 cells naturally expressing HVEM, and a monoclonal antibody that prevents binding of gD to HVEM significantly reduced NF-κB activation by soluble gD in the same cells, (iii) coculture with transfectants expressing wild-type gD on their surface induced an approximately twofold increase in NF-κB activation in cells naturally expressing HVEM, while coculture with transfectants expressing a mutated form of gD, lacking its capability to bind HVEM, did not induce a similar effect and (iv) treatment with soluble gD induced a dose-dependent NF-κB activation in CHO transfectants expressing HVEM, but not in control CHO transfectants lacking any functional gD receptor. Overall, these results establish that HVEM is involved in NF-κB activation by HSV-1 gD.

Journal of Materials Chemistry, 2008

In this contribution we report the design, fabrication and properties of hydrosoluble platinum na... more In this contribution we report the design, fabrication and properties of hydrosoluble platinum nanoparticles decorated with a nitric oxide (NO) caging compound. Direct monitoring of NO through an ultrasensitive NO electrode demonstrate that the nanoparticles are stable in the dark ...

Virology, 2000

Increasing evidence suggests that regulation of apoptosis in infected cells is associated with se... more Increasing evidence suggests that regulation of apoptosis in infected cells is associated with several viral infections. The gammaherpesvirus bovine herpesvirus 4 (BHV-4) has been shown to harbor genes with antiapoptotic potentialities. However, here we have demonstrated that productive infection of adherent, permissive cell lines by BHV-4 resulted in a cytopathic effect characterized by induction of apoptosis. This phenomenon was confirmed using different techniques to detect apoptosis and using different virus strains and cell targets. Apoptosis induced by BHV-4 was inhibited by (1) treatment with doses of heparin, which completely inhibited virus attachment and infectivity; (2) UV treatment, which completely abrogated infectivity; and (3) treatment with a dose of phosphonoacetic acid, which blocked virus replication. Virus-induced apoptosis was associated with a down-regulation of Bcl-2 expression and was reduced by Z-VAD-FMK, but not by Z-DEVD-FMK (caspase-3-specific) caspase inhibitors. Inhibition of apoptosis by Z-VAD-FMK treatment during infection did not modify virus yield. Therefore, despite the presence of antiapoptotic genes in its genoma, BHV-4 could complete its cycle of productive infection while inducing apoptosis of infected cells. This finding might have implications for the pathobiology of BHV-4 and other gammaherpesviruses in vivo.

Biochemical Pharmacology, 2008

A. Mastino). 1 This paper was presented in part in the form of a poster at the meeting ''Apoptosi... more A. Mastino). 1 This paper was presented in part in the form of a poster at the meeting ''Apoptosis World 2008: From mechanisms to applications'', Luxembourg, 23-26 January 2008. a v a i l a b l e a t w w w . s c i e n c e d i r e c t . c o m j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / b i o c h e m p h a r m 0006-2952/$ -see front matter #

Cell Death and Differentiation, 1997

Increasing evidence indicates that apoptosis can be associated with several viral infections. Her... more Increasing evidence indicates that apoptosis can be associated with several viral infections. Here we demonstrate, that infection of monocytoid cells by Herpes simplex virus 2 (HSV-2) resulted, in time-and dose-dependent induction of apoptosis as an exclusive cytopathic effect. The phenomenon was confirmed using four different techniques. Conversely, apoptosis was not observed in the Vero cell line. Virus yield in monocytoid cells was delayed and reduced, although well detectable, in comparison with that observed in Vero cells. Nevertheless, released virions exhibited full infecting capability. Apoptosis induced by HSV-2 was not inhibited by cycloheximide and only partially by an UV-treatment which completely abrogated infectivity. Virus-induced apoptosis was partly inhibited by indomethacin and was associated with a down-regulation of Bcl-2. A similar, but less pronounced, apoptosis-inducing effect in monocytoid cells was also observed with HSV-1 infection. Depending on the target cells, therefore, HSV could complete a cycle of infection which is characterized by apoptosis of infected cells.

Proceedings of The National Academy of Sciences, 2002

An earlier report has shown that herpes simplex virus 1 virions package RNA. Experiments designed... more An earlier report has shown that herpes simplex virus 1 virions package RNA. Experiments designed to reveal the identity of the virion proteins capable of binding the RNA and to show whether the mRNA carried in the newly infected cells was expressed showed the following: (i) 32P-labeled riboprobe generated by in vitro transcription of the US8.5 ORF bound three proteins identified as the products of US11, UL47, and UL49 (VP22) genes. (ii) Viral RNA was bound to UL47 or US11 proteins immune precipitated from cells transduced with baculoviruses expressing UL47 or US11 and then superinfected with HSV-1 under conditions that blocked DNA synthesis and assembly of virions. (iii) Virions were purified from cells transduced with a baculovirus encoding a US8.5 protein fused to green fluorescent protein and superinfected with an HSV-1 mutant lacking the US8-12 genes. HEp-2 cells infected with these virions expressed the chimeric protein in ≈1% of infected cells. (iv) In mixed cultures, untreated Vero cells acquired the mRNA encoding the green fluorescent-US8.5 chimeric protein from HEp-2 cells doubly transduced with the genes encoding VP22 and the chimeric protein. The transfer was RNase sensitive and VP22 dependent, indicating that the RNA encoded by the chimeric gene was transferred to Vero cells as mRNA. We conclude that (i) three virion proteins are capable of binding RNA; (ii) the packaged RNA can be expressed in newly infected cells; and (iii) the UL47 protein was earlier reported to shuttle from nucleus to the cytoplasm and may transport RNA. VP22 thus appears to be a member of a new class of viral proteins whose major function is to bind and transport infected cell mRNA to uninfected cells to create the environment for effective initiation of infection.

Proceedings of The National Academy of Sciences, 2002

An earlier report has shown that herpes simplex virus 1 virions package RNA. Experiments designed... more An earlier report has shown that herpes simplex virus 1 virions package RNA. Experiments designed to reveal the identity of the virion proteins capable of binding the RNA and to show whether the mRNA carried in the newly infected cells was expressed showed the following: (i) 32 P-labeled riboprobe generated by in vitro transcription of the U S 8.5 ORF bound three proteins identified as the products of US11, UL47, and UL49 (VP22) genes. (ii) Viral RNA was bound to U L47 or US11 proteins immune precipitated from cells transduced with baculoviruses expressing U L47 or US11 and then superinfected with HSV-1 under conditions that blocked DNA synthesis and assembly of virions. (iii) Virions were purified from cells transduced with a baculovirus encoding a U S8.5 protein fused to green fluorescent protein and superinfected with an HSV-1 mutant lacking the U S8 -12 genes. HEp-2 cells infected with these virions expressed the chimeric protein in Ϸ1% of infected cells. (iv) In mixed cultures, untreated Vero cells acquired the mRNA encoding the green fluorescent-U S8.5 chimeric protein from HEp-2 cells doubly transduced with the genes encoding VP22 and the chimeric protein. The transfer was RNase sensitive and VP22 dependent, indicating that the RNA encoded by the chimeric gene was transferred to Vero cells as mRNA. We conclude that (i) three virion proteins are capable of binding RNA; (ii) the packaged RNA can be expressed in newly infected cells; and (iii) the U L47 protein was earlier reported to shuttle from nucleus to the cytoplasm and may transport RNA. VP22 thus appears to be a member of a new class of viral proteins whose major function is to bind and transport infected cell mRNA to uninfected cells to create the environment for effective initiation of infection.