-
[show abstract]
[hide abstract]
ABSTRACT: Pseudomonas aeruginosa is an opportunistic pathogen that causes chronic endobronchial infections in cystic fibrosis (CF) patients. The role of bacterial internalization in the clearance of P. aeruginosa from the airways is controversial.
A xenograft model was used to study P. aeruginosa strain PAO-1 clearance and internalization by the human airways in vivo. Human lung and tracheal rudiments, obtained from therapeutic abortions (20±2 weeks of gestation), were subcutaneously implanted in the flanks of SCID mice and infected after 14-22 weeks of engraftment. Lungs were surgically exposed and P. aeruginosa was injected in the pulmonary parenchima. Opercula closing the tracheal openings were excised, mucus removed, and bacterial inoculum was injected into the lumen. Internalization was studied at 4 hours post-infection on single-cell suspensions, while clearance was evaluated after 24-72 hours from the infection on homogenized tissues.
Tracheae and lungs were morphologically identical to the adult human tissues, as evaluated by standard histology. Both types of xenografts showed a very low level of bacterial internalization (0.004-0.25% of total recovered bacteria), although tracheal xenografts presented more than 100 times greater internalization than did lung xenografts. Both lung and tracheal xenografts did not clear the injected bacteria for each inocolum, even at very low doses (100 colony forming units).
P. aeruginosa internalization by epithelial cells occurs, albeit at very low levels, and is not sufficient to clear bacteria in the airway xenograft model. This model could be used for studying chronic respiratory infections in CF patients.
Medical science monitor: international medical journal of experimental and clinical research 11/2010; 16(12):BR361-6. · 1.70 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Lentiviral (LV) vectors are able to only slowly and inefficiently transduce nondividing cells such as those of the airway epithelium. To address this issue, we have exploited the magnetofection technique in in vitro models of airway epithelium.
Magnetofectins were formed by noncovalent interaction between LV particles and polycation-coated iron oxide nanoparticles. Efficiency of LV-mediated transduction (as evaluated through green fluorescent protein (GFP) expression by cytofluorimetric analysis) was measured in bronchial epithelial cells in the presence or absence of a magnetic field. Cytotoxicity was evaluated by lactate dehydrogenase (LDH) release; cell monolayer integrity by measurement of transepithelial resistance (TER) and evaluation of correct zonula occludens-1 (ZO-1) localization at tight junctions (TJs) by immunofluorescence and confocal microscopy.
In nonpolarized cells, magnetofectins enhanced LV-mediated transduction at multiplicity of infection (MOI) of 50 up to 3.9-fold upon a 24-h incubation, to levels that approached those achieved at MOI of 200 for LV alone, in the presence or absence of the magnetic field. Magnetofection significantly increased the percentage of transduced cells up to 186-fold already after 15 min of incubation. In polarized cells, magnetofection increased GFP+ cells up to 24-fold compared to LV alone. Magnetofection did not enhance LDH release and slightly altered TER but not ZO-1 localization at the TJs.
We conclude that magnetofection can facilitate in vitro LV-mediated transduction of airway epithelial cells, in the absence of overt cytotoxicity and maintaining epithelial integrity, by lowering the necessary vector dose and reducing the incubation time required to achieve efficient transduction.
The Journal of Gene Medicine 09/2010; 12(9):747-54. · 2.48 Impact Factor
-
[show abstract]
[hide abstract]
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. · 1.50 Impact Factor
-
Elena Copreni,
Elena Nicolis,
Anna Tamanini,
Valentino Bezzerri,
Stefano Castellani,
Lucia Palmieri,
Maria Grazia Giri,
Antonio Vella,
Marco Colombatti,
Paolo Rizzotti,
Massimo Conese,
Giulio Cabrini
[show abstract]
[hide abstract]
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. · 2.94 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Cystic Fibrosis (CF) is a still life-treathening disease, although therapies have augmented the life span of CF individuals. Isolation of the CF gene, named CFTR (CF Transmembrane Conductance Regulator), led to the discovery that it encodes for a protein kinase A-regulated chloride channel, expressed by epithelial cells mainly in mucosal tissues. Mutated or absent CFTR brings about altered muco-ciliary clearance, by a yet disputed mechanism (likely involving overfunction of the epithelial sodium channel [ENaC]), and ultimately to colonization and infection of the airways by a few opportunistic bacteria species, including Pseudomonas aeruginosa (P.a.). An exuberant and persistent acute inflammatory reaction is commonly observed in the lung from CF patients, representing a key pathogenetic event of lung damage and respiratory insufficiency. Lung disease is the chief cause of morbidity and mortality in CF patients and current therapies are aimed at controlling the respiratory symptoms by antibiotic and anti-inflammatory treatments. Major improvements in the strategy to fight pulmonary P.a. infection are based on treatment of first colonization with P.a., of chronic infection and of multidrug resistant bacteria. Controlling inflammation is a challenging, however crucial, task in CF therapy. Since classical steroidal and non-steroidal anti-inflammatory drugs are endowed with serious side-effects, alternative antiinflammatory strategies are being developed, including drugs which modulate cytokine expression, nitric oxide production and the oxidation unbalance in the CF airways. Soon after gene identification, gene transfer vectors to replace the defective gene were developed and underwent a number of human trials which have not yet produced a viable clinical gene therapy strategy. Novel gene therapy vectors and strategies for delivering them more efficiently and safely to the lung are being studied. In the last few years various in vitro studies have demonstrated the possibility of pharmacological intervention to correct the primary defect in CF. This may be obtained by directly addressing the CFTR protein (by means of so-called correctors and potentiators) or by modulating the activity of other types of ion transport in epithelial cells (such as with Moli1901 or denufosol). Novel interventions aimed to improve the mucociliary clearance, including hypertonic saline, mannitol and ENaC inhibitors, will be finally discussed.
Current Pediatric Reviews 01/2009; 5(1):8-27.
-
[show abstract]
[hide abstract]
ABSTRACT: Cystic Fibrosis (CF) is a still life-treathening disease, although therapies have augmented the life span of CF individuals. Isolation of the CF gene, named CFTR (CF Transmembrane Conductance Regulator), led to the discovery that it encodes for a protein kinase A-regulated chloride channel, expressed by epithelial cells mainly in mucosal tissues. Mutated or absent CFTR brings about altered muco-ciliary clearance, by a yet disputed mechanism (likely involving over-function of the epithelial sodium channel [ENaC]), and ultimately to colonization and infection of the airways by a few opportunistic bacteria species, including Pseudomonas aeruginosa (P.a.). An exuberant and persistent acute inflammatory reaction is commonly observed in the lung from CF patients, representing a key pathogenetic event of lung damage and respiratory insufficiency. Lung disease is the chief cause of morbidity and mortality in CF patients and current therapies are aimed at controlling the respiratory symptoms by antibiotic and anti-inflammatory treatments. Major improvements in the strategy to fight pulmonary P.a. infection are based on treatment of first colonization with P.a., of chronic infection and of multidrug resistant bacteria. Controlling inflammation is a challenging, however crucial, task in CF therapy. Since classical steroidal and non-steroidal anti-inflammatory drugs are endowed with serious side-effects, alternative anti-inflammatory strategies are being developed, including drugs which modulate cytokine expression, nitric oxide production and the oxidation unbalance in the CF airways. Soon after gene identification, gene transfer vectors to replace the defective gene were developed and underwent a number of human trials which have not yet produced a viable clinical gene therapy strategy. Novel gene therapy vectors and strategies for delivering them more efficiently and safely to the lung are being studied. In the last few years various in vitro studies have demonstrated the possibility of pharmacological intervention to correct the primary defect in CF. This may be obtained by directly addressing the CFTR protein (by means of so-called correctors and potentiators) or by modulating the activity of other types of ion transport in epithelial cells (such as with Moli1901 or denufosol). Novel interventions aimed to improve the mucociliary clearance, including hypertonic saline, mannitol and ENaC inhibitors, will be finally discussed.
Current Pediatric Reviews 01/2009; 5:8-27.
-
[show abstract]
[hide abstract]
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. · 2.48 Impact Factor
-
Sante Di Gioia,
Joanna Rejman,
Salvatore Carrabino,
Ida De Fino,
Carsten Rudolph,
Ann Doherty,
Laura Hyndman,
Maurizio Di Cicco, Elena Copreni,
Alessandra Bragonzi,
Carla Colombo,
A Christopher Boyd,
Massimo Conese
[show abstract]
[hide abstract]
ABSTRACT: Efficient gene transfer to the airways by nonviral vectors is a function of different parameters, among which the size and the charge of the transfecting particles. The aim of this study was to determine the transfection efficiency of polyethylenimine (PEI)/albumin polyplexes in ex vivo and in vivo models of respiratory epithelium and to correlate it with biophysical characteristics of the particles. Complexes were obtained by adding different amounts of human serum albumin (HSA) to PEI polyplexes preformed in saline. The presence of HSA caused the formation of bigger and more negative polyplexes and increased PEI transfection efficiency in primary respiratory epithelial cells by 4-6-fold. For in vivo administration to the lung, PEI polyplexes were formed in water and optimized with respect to the N/ P ratio. PEI/pC-Luc complexes gave the highest luciferase expression at N/ P 15 when administered through the trachea. At this N/ P ratio, the size and the surface charge of albumin-containing polyplexes were not different as compared with plain PEI polyplexes. Formulation of PEI polyplexes in the presence of HSA or murine serum albumin (MSA) resulted in a 2-fold increase in luciferase expression. In mice treated with PEI or PEI/MSA polyplexes containing the nuclear beta-gal gene, X-gal staining revealed that transfected cells localized at the bronchiolar epithelium and that PEI/MSA transfected four times as many cells as PEI ( p < 0.05). Finally, double administration of PEI/MSA polyplexes resulted in a further enhancement of transfection of the lung. Our data show that serum albumin enhances PEI-mediated gene transfer to airway epithelial cells in vivo, likely facilitating the uptake of polyplexes, and indicate that this formulation would fulfill the requirement of repeated administration, as necessary in chronic lung diseases like cystic fibrosis.
Biomacromolecules 03/2008; 9(3):859-66. · 5.48 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: It is not clear whether cystic fibrosis (CF) airway inflammation is a consequence of bacterial infection or is intrinsically dysregulated. The aim of this study was to investigate IL-8 secretion and NF-kappaB activity in primary respiratory epithelial cells cultured from nasal polyps obtained from CF and non-CF subjects.
NF-kappaB activity was studied by electrophoretic mobility-shift and quantitative colorimetric assays in nuclear extracts. Immunoreactive IL-8 levels were assessed by ELISA in cell culture supernatants. Both parameters were studied at baseline and following challenge with Pseudomonas aeruginosa or stimulation with pro-inflammatory cytokines.
Under basal conditions, CF cells presented a significant higher activity of NF-kappaB than non-CF cells (P=0.0004). P. aeruginosa challenge and IL-1beta/H2O2 co-stimulation caused four and two fold induction of NF-kappaB activity in non-CF and CF cells, respectively. IL-8 levels in unstimulated CF cells were significantly higher than in non-CF cells (P=0.0025). Upon incubation with P. aeruginosa and IL-1beta/H2O2, non-CF cells produced 6.3 times more IL-8 than unstimulated cells, whereas IL-8 secretion increased only of 1.4 times in CF cells.
CF respiratory epithelial cells exhibit a basal dysregulated production of IL-8 that partially correlates to enhanced NF-kappaB activity. Our data corroborate the hypothesis of a basal exaggerated inflammatory response in the CF respiratory epithelium.
Journal of Cystic Fibrosis 06/2006; 5(2):113-9. · 3.19 Impact Factor
-
[show abstract]
[hide abstract]
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; · 6.87 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The interaction of polyethylenimine (PEI) polyplexes with proteins in cystic fibrosis (CF) airway secretions poses a significant hurdle to this nonviral delivery system. The aim of this study was to evaluate whether albumin may increase the efficiency of PEI complexes in mediating gene transfer into respiratory epithelial cells in the presence of CF mucus.
PEI (25 kDa) was complexed to DNA in the presence of human serum albumin (HSA) and used to transfect confluent A549 and 9HTEo- cells. Alternatively, albumin was added to preformed PEI-DNA complexes. The cytotoxicity of complexes was analysed by the LDH (lactate dehydrogenase) assay. CF CFT1-C2 cells were allowed to polarise and were transfected either with luciferase- or CFTR-expressing plasmids. To evaluate the effect of CF respiratory secretions on transfection efficiency, confluent cells were transfected in the presence of sputum obtained from two CF patients.
The ternary PEI-HSA complexes increased luciferase expression in confluent cultures in a dose-dependent fashion up to 100 times as compared to PEI-DNA. The number of GFP-expressing cells, as evaluated by epifluorescence, was augmented several-fold. When HSA was added to preformed PEI-DNA complexes, a further 5-10-fold increase in gene expression was observed. No significant cytotoxicity was observed with either PEI or PEI-HSA polyplexes. The ternary complexes determined detectable CFTR gene transfer and expression at the apical membrane in polarised CFT1-C2 cells, as evaluated by confocal microscopy. CF sputum inhibited PEI-mediated gene transfer by 7-186-fold. Although luciferase expression mediated by PEI-HSA was still inhibited by CF sputum, these levels were 18-83.8-fold higher than with PEI.
Our results demonstrate that albumin increases PEI gene transfer efficiency in confluent and polarised respiratory epithelial cells and can allow CFTR gene expression in the appropriate cellular compartment. PEI-HSA complexes display a higher efficiency than PEI also in the presence of CF sputum, indicating that albumin-containing polyplexes may help overcome barriers imposed by CF airway secretions.
The Journal of Gene Medicine 01/2006; 7(12):1555-64. · 2.48 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: It is not known whether cytokine levels in sputum may be used as outcome measures after parenteral antibiotic therapy in cystic fibrosis (CF) patients. Here, we assessed the effects of antibiotic therapy on cytokine levels in sputum and serum obtained from young CF patients. Thirty-two CF patients (14 females; mean age, 18.6 years; range, 11.4-35.7 years), consecutively admitted at the CF Center of Milan for parenteral antibiotic therapy during pulmonary exacerbation, were enrolled in the study. Before and after 21 days (range, 5-41) of intravenous antibiotic treatment, all patients underwent routine laboratory determinations (including white blood cell (WBC) count and C-reactive protein (CRP)), a chest X-ray, pulmonary function tests (forced expiratory volume in 1 sec (FEV1) and forced vital capacity (FVC) as % predicted), and sputum cultures. Interleukin (IL)-6, IL-8, IL-10, and tumor necrosis factor (TNF)-alpha levels in serum and sputum samples were determined by means of immunometric assays. After therapy, FEV1 and FVC significantly improved (median increase of 7.5% and 8.5% predicted, respectively), while CRP and WBC count were significantly decreased (median values from 14 to 5.5 mg/dl and from 8,350 to 7,400 n/mm3, respectively). While levels of IL-6 and IL-10 in sputum were generally undetectable, IL-8 and TNF-alpha were always measurable, and IL-8 levels significantly decreased after antibiotic treatment (median values from 7,165 to 5,415 pg/ml). Following antibiotic therapy, IL-8 and TNF-alpha levels in sputum were inversely related with both FEV(1) and FVC. In conclusion, TNF-alpha and IL-8 levels in sputum of young CF patients with pulmonary exacerbation were always detectable and may be useful, noninvasive outcome measures to assess response to therapy in CF patients.
Pediatric Pulmonology 08/2005; 40(1):15-21. · 2.53 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Cystic fibrosis (CF) airway becomes colonized with only a limited number of bacterial pathogens. It is of paramount importance to establish in vitro and in vivo models to better understand bacterial-host interactions under CF-like conditions. In this article, in vitro methods suitable to study Pseudomonas aeruginosa (Pa) and Staphylococcus aureus (Sa) adherence to and uptake by airway epithelial cells are described. Acute and chronic respiratory infection models, which have been used in CF transgenic mice and mimic human CF lung pathology, are also taken into consideration.
Journal of Cystic Fibrosis 09/2004; 3 Suppl 2:197-201. · 3.19 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The pathological hallmark of cystic fibrosis (CF) chronic inflammatory response is the massive neutrophil influx into the airways. This dysregulated neutrophil emigration may be caused by the abnormal secretion of chemoattractants by respiratory epithelial cells and polarised lymphocyte T-helper response. Neutrophils from CF patients have a different response to inflammatory mediators than neutrophils from normal subjects, indicating that they are primed in vivo before entering the CF airways. CF neutrophils secrete more myeloperoxidase and elastase, mobilise less opsonin receptors and release less L-selectin than non-CF neutrophils. Moreover, they show altered cytokine production and a dysregulated chemotaxis response. Laboratory studies now suggest that CFTR is involved in regulating some neutrophil functions and indicate that altered properties of CF neutrophils may depend on genetic factors. Current gene therapy approaches are targeted to the respiratory epithelium, but many hurdles oppose an efficient and efficacious CFTR gene transfer. The possibility of CFTR gene therapy-based approach targeting CF neutrophils at the hematopoietic stem cell level is discussed.
Journal of Cystic Fibrosis 10/2003; 2(3):129-35. · 3.19 Impact Factor
