Lucia Palmieri

ReSearch Pharmaceutical Services, Fort Washington, Pennsylvania, United States

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Publications (9)20.2 Total impact

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    ABSTRACT: The aim of this work was to evaluate the efficiency and duration of gene expression mediated by a VSV-G pseudotyped last generation lentiviral (LV) vector. We studied LV efficiency in ex-vivo models of respiratory epithelial cells, obtained from bronchial biopsies and nasal polyps, by GFP epifluorescence and cytofluorimetry. In vivo efficiency and persistence of gene expression was investigated by GFP immunohistochemistry and luciferase activity in lung cryosections and homogenates, respectively, upon intranasal and intratracheal administration protocols in C57Bl/6 mice. Both primary bronchial and nasal epithelial cells were transduced up to 70-80% 72 hr after the LV infection. In vivo nasal luciferase expression was increased by lysophosphatidylcholine pre-treatment of the nose. Conversely, the bronchial epithelium was transduced in the absence of any pre-conditioning treatment and luciferase expression lasted for at least 6 months without any decline. We conclude that a last generation LV vector is a promising gene transfer agent in the target organ of genetic and acquired lung diseases, as in the case of cystic fibrosis.
    Viruses 08/2010; 2(8):1577-88. · 2.51 Impact Factor
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    ABSTRACT: Lentiviruses (LVs) are considered one of the most promising tools for gene transfer, however, their potential to induce pro-inflammatory cytokines on delivery into the respiratory tissue remains to be established. Here we tested a third-generation vesicular stomatitis virus (VSV)-G pseudotyped LV vector in the two respiratory epithelial cell lines A549 and CFT1-C2. We observed that the VSV-G LV vector does not induce (a) activation of the nuclear factor (NF)-kappaB, which intervenes in transcription of pro-inflammatory genes; (b) expression of ICAM-1; and (c) transcription of a panel of cytokines, with the exception of a mild and transient (24h) increase of IFN-gamma mRNA. In contrast, an adenovirus-derived vector strongly activated NF-kappaB and different transcripts such as those of ICAM-1, IL-8, RANTES, IP-10, TNF-alpha, IL-6, IL-1 beta. In conclusion, this third-generation VSV-G pseudotyped LV vector does not elicit major pro-inflammatory signals in human airway epithelial cells and appears to be better suited for gene delivery strategies.
    Virus Research 05/2009; 144(1-2):8-17. · 2.75 Impact Factor
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    ABSTRACT: The involvement of surface molecules in HIV-1-derived lentivirus (LV)-mediated transduction of airway epithelial cells has not been studied so far. The present study aimed to evaluate the role of glycosaminoglycans (GAGs) in gene transfer mediated by a third generation vesicular stomatitis virus G glycoprotein (VSV-G) pseudotyped LV vector in an in vitro model of polarized airway epithelial cells. Human bronchial (16HBE-S1) and tracheal (CFT1-C2) epithelial cells were grown either on plastic or on filters and transduced with the LV vector polypurine tract (PPT)-green fluoresecent protein (GFP). Zonula Occludens (ZO)-1, a marker of tight junction, and GAG localization were assessed by cytofluorimetry and confocal microscopy. Soluble GAGs and removal of cell surface GAGs were used to affect LV-mediated transduction. Extensive optimization of experimental parameters (presence of polybrene during the infection, the incubation time in the presence of LV particles, period of time intercurring between infection and gene expression analysis) was carried out in plastic-adherent cells. Polybrene resulted to be cytotoxic and was not further used. In CFT1-C2 polarized cells, EGTA treatment determined a 20% decrease in transepithelial resistance, a diminished ZO-1 localization at the tight junction location and a 31% increase in GFP positive cells. Heparane sulfate was distributed evenly on the cell surface. Heparin and soluble chondroitin sulfate A and B inhibited LV-mediated transduction in a dose-dependent fashion. These results were confirmed upon enzymatic removal of GAGs from the cell surface. Taken together, these results show that GAGs are involved in VSV-G LV transduction of airway epithelial cells.
    The Journal of Gene Medicine 10/2008; 10(12):1294-302. · 2.16 Impact Factor
  • Journal of Cystic Fibrosis - J CYST FIBROS. 01/2007; 6.
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    Journal of Cystic Fibrosis 01/2007; 6:S10. · 2.87 Impact Factor
  • Journal of Cystic Fibrosis 01/2007; 6:S10. · 2.87 Impact Factor
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    ABSTRACT: Molecular Therapy (2006) 13, S268|[ndash]|S269; doi: 10.1016/j.ymthe.2006.08.772 694. Involvement of Glycosaminoglycans in VSV-G Pseudotyped Lentiviral Vector Mediated Gene Transfer into Airway Epithelial Cells|[ast]| Elena Copreni1, Lucia Palmieri1, Salvatore Carrabino1, Stefano Castellani1, Luigi Naldini2 and Massimo Conese11Institute for Experimental Treatment of Cystic Fibrosis, H.S. Raffaele, Milano, Italy2TIGET, H.S. Raffaele, Milano, Italy|[ast]|This work has been supported by a grant from the Italian Cystic Fibrosis Research Foundation and by the Associazione Lombarda Fibrosi Cistica |[ndash]| ONLUS.
    Molecular Therapy 04/2006; · 7.04 Impact Factor
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    ABSTRACT: HIV-1-derived lentiviral (LV) vectors are considered promising vehicles for Cystic Fibrosis (CF) gene therapy since they integrate in the host genome, transduce non-dividing cells and ensure long-term expression of the transgene. Although some knowledge on the infection of respiratory epithelial cells has been accumulated, the receptors involved in the transduction of these cells by VSV-G-pseudotyped LV vectors are not well known. Previous studies have shown that heparin inhibited infection of HT1080 cells by Moloney Murine Leukemia retrovirus pseudotyped with VSV-G (Guibinga, 2002), indicating that interaction with glycosaminoglycans (GAGs) of cellular membrane plays a role in infectivity of viral particles with VSV-G envelope. Our study investigated the involvement of heparan sulfate (HS) and chondroitin sulfate B in gene transfer mediated by a VSV-G pseudotyped LV vector in a respiratory epithelial cell line of tracheal origin (CFT1-C2). The HIV-1 derived LV vector PPT-GFP carries the Green Fluorescence Protein (GFP) reporter gene under the control of the CMV promoter (Follenzi, 2000). To evaluate the interaction between GAGs expressed on the apical surface of respiratory epithelial cells and lentiviral particles, the LV vector (3.2×10E8 TU, corresponding to MOI 2000) was pre-incubated with heparin or chondroitin sulfate B at different concentrations (10–250 micrograms/ml) for 60 minutes in binding buffer and then added to polarized CFT1-C2 cells (polarization was evaluated by the measure of transepithelial resistance and apical expression of ZO-1). The virus-containing solution was incubated with cells for 24 hours and GFP expression was analyzed by flow cytometry 72 hours after. Heparin and chondroitin sulfate B determined a dose-dependent inhibition of transduction efficiency up to 86% and 83% of untreated vector, respectively. HS expression in CFT1-C2 cells was analyzed by flow cytometry and confocal microscopy. HS was expressed by 85–88% of cells, the expression being much stronger on the basolateral side of polarized cells. Cells were incubated with 12 mM EGTA before LV administration to disrupt the epithelial tight junctions and allow the lentiviral particles to access the basolateral side of the epithelium. In the absence of EGTA, 60% of cells resulted GFP positive, whereas the pre-treatment with EGTA increased the percentage of GFP-expressing cells up to 80.5%. Our data strongly indicate that the little amount of GAGs restricted to the apical surface might be sufficient to mediate transduction of respiratory epithelial cells by a VSV-G pseudotyped LV vector. However, we can not exclude that other receptor(s) could be involved.
    Molecular Therapy - MOL THER. 01/2006; 13.
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    ABSTRACT: HIV-1 derived lentiviral (LV) vectors seem to be promising vehicles for gene transfer to the airway epithelium since they integrate in the host genome of non-dividing cells ensuring long-term expression of the transgene. Previous studies have shown that LV vectors could transduce the airway epithelium in vivo from the apical surface, only when tight junctions are disrupted or LV are pseudotyped with heterologous envelopes other than glycoprotein G of the vesicular stomatitis virus (VSV-G). Our study was aimed to investigate the efficiency of an improved VSV-G pseudotyped lentiviral vector, which contains the central polypurine tract (cPPT) and the Woodchuck post-translational regulatory elements, in human respiratory cells and in the murine lung. The vector PPT-GFP was injected intratracheally in C57BL/6 mice at the dose of 4.5×10E6 TU. After 48 hours, lungs were fixed in paraformaldehyde and cryosections stained with an antibody against GFP. Results show that PPT-GFP transduced the murine airway epithelium in the bronchial and alveolar compartments. Transgene expression was observed also after two weeks of infection with a LV dose of 1×10E8 TU. Hystological analysis showed no evidence of tissue damage in the lung, suggesting that this LV vector is not cytotoxic. Since heparan sulfate (HS) has been described to mediate infection by VSV-G-pseudotyped retroviral vectors and HIV-1, the expression of this glycosaminoglycan by human respiratory cells and its involvement in LV-mediated gene transfer were studied. Human bronchial epithelial cells (16HBE) and CF tracheal epithelial cells (CFT1) were analyzed for HS expression by flow cytometry and confocal microscopy. HS was expressed by 85-88% of cells, the expression being much stronger on the basolateral side in polarized epithelial cells. To correlate the efficiency of LV mediated transduction with HS expression, polarized CFT1 cells were infected with the lentivirus and some samples were pre-incubated with EGTA (Ethylene glycol-O,O-bis-[2-amino-ethyl]-N,N,N,N,-tetraacetic acid), which disrupts the epithelial tight junctions and allows the LV particles to access the basolateral side of the epithelium. Analysis of GFP expression by FACS indicates that the pre-conditioning increased the transduction efficiency, being the percentage of positive cells of 55%, when the infection was performed without pretreatment, and 71% when the cells were incubated with EGTA. To evaluate the interaction between glycosaminoglycans expressed on the apical surface and lentiviral particles, the LV vector was pre-incubated with heparin and then incubated with 16HBE cells. Heparin determined a dose-dependent inhibition of transduction efficiency up to 65% of untreated vector. Our study shows that 1) The PPT-GFP vector transduce murine airways and human polarized cells, without requirement of disruption of the epithelial barrier integrity and without cytotoxicy 2) HS might be a receptor involved in VSV-G-pseudotyped LV-mediated gene transfer.
    Molecular Therapy - MOL THER. 01/2005; 11.