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M Conese

Publications and source records attributed to M Conese.

At least 19 recordsLinked to original sources
PubMed2005

S-CMC-Lys-dependent stimulation of electrogenic glutathione secretion by human respiratory epithelium.

Glutathione (GSH) is one of the most important defense mechanisms against oxidative stress in the respiratory epithelial lining fluid. Considering that GSH secretion in respiratory cells has been postulated to be at least partially electrogenic, and that the mucoregulator S-carbocysteine lysine salt monohydrate (S-CMC-Lys) can cause an activation of epithelial Cl(-) conductance, the purpose of this study was to verify whether S-CMC-Lys is able to stimulate GSH secretion. Experiments have been performed by patch-clamp technique, by high-performance liquid chromatography (HPLC) assay, and by Western blot analysis on cultured lines of human respiratory cells (WI-26VA4 and CFT1-C2). In whole-cell configuration, after cell exposure to 100 microM S-CMC-Lys, a current due to an outward GSH flux was observed, which was inhibitable by 5-nitro-2-(3-phenylpropylamino)-benzoate and glibenclamide. This current was not observed in CFT1-C2 cells, where a functional cystic fibrosis transmembrane conductance regulator (CFTR) is lacking. Inside-out patch-clamp experiments (GSH on the cytoplasm side, Cl(-) on the extracellular side) showed the activity of a channel, which was able to conduct current in both directions: the single channel conductance was 2-4 pS, and the open probability (P(o)) was low and voltage-independent. After preincubation with 100 microM S-CMC-Lys, there was an increase in P(o), in the number of active channels present in each patch, and in the relative permeability to GSH vs Cl(-). Outwardly directed efflux of GSH could also be increased by protein kinase A, adenosine 5'-triphosphate, and cyclic adenosine monophosphate (cAMP) added to the cytoplasmic side (whole-cell configuration). The increased secretion of GSH observed in the presence of S-CMC-Lys or 8-bromoadenosine-3',5'-cyclic monophosphate was also confirmed by HPLC assay of GSH on a confluent monolayer of respiratory cells. Western blot analysis confirmed the presence of CFTR in WI-26VA4 cells. This study suggests that S-CMC-Lys is able to stimulate a channel-mediated GSH secretion by human respiratory cells: electrophysiological and pharmacological characteristics of this channel are similar to those of the CFTR channel.

Carbocysteine↗
PubMed2004

Gene therapy progress and prospects: episomally maintained self-replicating systems.

The use of nonviral gene therapy vectors has been hampered by low level of transfection efficiency and lack of sustained gene expression. Episomal self-replicating systems may overcome these hurdles through their large packaging capacity, stability and reduced toxicity. This article reviews three classes of episomal molecules that have been tested with possible therapeutic genes: (1) self-replicating circular vectors, containing the Epstein-Barr virus (EBV) elements oriP and EBNA1; (2) small circular vectors containing scaffold/matrix attachment regions (S/MARs) as cis-acting elements to maintain the episomal status of the vector; (3) chromosomal vectors, based on the functional elements of the natural chromosomes. The studies reported validate the use of episomal vectors to obtain stable and prolonged gene expression, although reveal some limitations that necessitate additional work.

Chromosomes, Artificial↗
PubMed2004

Lentivirus-mediated gene transfer to the respiratory epithelium: a promising approach to gene therapy of cystic fibrosis.

Gene therapy of cystic fibrosis (CF) lung disease needs highly efficient delivery and long-lasting complementation of the CFTR (cystic fibrosis transmembrane conductance regulator) gene into the respiratory epithelium. The development of lentiviral vectors has been a recent advance in the field of gene transfer and therapy. These integrating vectors appear to be promising vehicles for gene delivery into respiratory epithelial cells by virtue of their ability to infect nondividing cells and mediate long-term persistence of transgene expression. Studies in human airway tissues and animal models have highlighted the possibility of achieving gene expression by lentiviral vectors, which outlasted the normal lifespan of the respiratory epithelium, indicating targeting of a 'stem cell' compartment. Modification of the paracellular permeability and pseudotyping with heterologous envelopes are the strategies currently used to overcome the paucity of specific viral receptors on the apical surface of airway epithelial cells and to reach the basolateral surface receptors. Preclinical studies on CF mice, demonstrating complementation of the CF defect, offer hope that lentivirus gene therapy can be translated into an effective treatment of CF lung disease. Besides a direct targeting of the stem/progenitor niche(s) in the CF airways, an alternative approach may envision homing of hematopoietic stem cells engineered to express the CFTR gene by lentiviral vectors. In the context of lentivirus-mediated CFTR gene transfer to the CF airways, biosafety aspects should be of primary concern.

Animals↗
PubMed2002

Non-viral approach toward gene therapy of cystic fibrosis lung disease.

Since Cystic Fibrosis (CF) is an autosomal recessive disorder due to mutations in the CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) gene, studies towards a gene therapy approach to its treatment followed immediately upon the cloning of the gene. It was demonstrated that the insertion of a single copy of the wild-type gene restored the normal phenotype in CF cells in vitro. Encouraging results were obtained in many in vivo model systems (CF transgenic mice) involving viral as well as non-viral vectors, which demonstrated the recovery of CFTR function in the airways. These results constituted the basis for human studies. Of those with a non-viral approach, a total of seven clinical trials using cationic lipids have reported data on efficiency, efficacy and safety. An effective gene transfer approach for the treatment of CF lung disease is not however imminent: low transfection efficiency and poor maintenance of gene expression are so far the main obstacles on this therapeutic path. On the other hand, no important adverse effects have been documented and repeated administration in humans is possible. The understanding of tissue and cellular barriers is a prerequisite for the development of more efficient non-viral gene therapy protocols for CF patients. While cationic lipids have been shown to be blocked by the mucous airway barrier and not be able to transfect differentiated respiratory epithelial cells, a new class of non-viral vectors, cationic polymers, are endowed with chemical and biological properties that make them more efficient in mediating gene transfer than lipids. Cationic polymers, such as polyethylenimine, are promising vectors for CF lung gene therapy.

Animals↗
PubMed2001

PAI-1 inhibits urokinase-induced chemotaxis by internalizing the urokinase receptor.

PAI-1 (plasminogen activator inhibitor-1) binds the urokinase-type plasminogen activator (uPA) and causes its degradation via its receptor uPAR and low-density lipoprotein receptor-related protein (LRP). While both uPA and PAI-1 are chemoattractants, we find that a preformed uPA-PAI-1 complex has no chemotactic activity and that PAI-1 inhibits uPA-induced chemotaxis. The inhibitory effect of PAI-1 on uPA-dependent chemotaxis is reversed when uPAR internalization is inhibited by the 39 kDa receptor-associated protein or by anti-LRP antibodies. Under the same conditions, the uPA-PAI-1 complex is turned into a chemoattractant causing cytoskeleton reorganization and extracellular-regulated kinase/mitogen-activated protein kinases activation. Thus, uPAR internalization by PAI-1 regulates cell migration.

Active Transport, Cell Nucleus↗
PubMed2001

Human respiratory cells from nasal polyps as a model for gene transfer by non-viral cationic vectors.

The influence of cell differentiation and proliferation on cationic vector mediated gene transfer into the explant-outgrowth cell culture from nasal polyps was investigated. Respiratory cells were categorized into two groups based on the expression of cytokeratin filaments (CKs), which were used as differentiation markers. Outgrowths grown for 2 weeks expressed similar levels of CKs 14, 13 and 18 showing a de-differentiated phenotype, while outgrowths cultured for 4 weeks presented very high levels of CK 13, high CK 14 and low CK 18 expression and were squamous differentiated. De-differentiated cells presented higher proliferation indexes than squamous cells. Gene transfer levels, as evaluated using a quantitative reporter gene (firefly luciferase), were significantly higher in the 2- than in the 4-week-old outgrowths. Cationic vector transfected respiratory cells were located both proximally and distally to the explant, as shown by enzymatic staining of beta-galactosidase-positive cells. Respiratory cell outgrowths from nasal polyps can be considered a suitable model to study gene transfer protocols in vitro.

Biomarkers↗
PubMed2000

Biodistribution and transgene expression with nonviral cationic vector/DNA complexes in the lungs.

Biodistribution of nonviral cationic vector/DNA complexes was studied after systemic or intratracheal administration to the lungs and correlated with transgene expression. Intravenous injection in C57Bl/6 mice gave maximal and significant luciferase expression in the lungs with the cationic polymer PEI 22K/DNA complexes at the highest ratios of positive/negative charges versus DNA alone. While DOTAP/DNA complexes with high charge ratio determined lower but still significant luciferase activity versus uncomplexed DNA, GL-67A and PEI 25K mediated negligible luciferase expression. Labelled PEI 22K and DOTAP complexes were evenly distributed in the alveolar region, where GFP expression was revealed, while PEI 25K and GL-67A complexes were not detected, suggesting a different interaction of these complexes with the plasma membrane of endothelial cells. Following an intratracheal injection, the highest and significant levels of transfection were obtained with slightly positive PEI complexes as compared with DNA alone, whereas cationic lipid-based vectors, DOTAP and GL-67A, gave not significant luciferase activity. Both types of polyplexes gave similar levels of lung luciferase expression by targeting different airway cell populations. PEI 25K complexes determined high levels of GFP in the bronchial cells, confirming confocal data on fluorescent complexes internalization. PEI 22K complexes gave mainly high GFP signal in the distal tract of the bronchial tree, where tagged complexes were recovered. Fluorescent lipid complexes were found in aggregates in the lumen of bronchi totally (DOTAP) or partially (GL-67A) co-localizing with surfactant protein A. Results indicated that cationic polymers could overcome the surfactant barrier which inhibited airway cell transfection mediated by cationic lipids.

Animals↗
PubMed2000

Modification of transepithelial ion transport in human cultured bronchial epithelial cells by interferon-gamma.

Human bronchial epithelial cells were treated in vitro with interferon-gamma or tumor necrosis factor-alpha to assess their effect on transepithelial ion transport. Short-circuit current measurements revealed that Na(+) absorption was markedly inhibited by interferon-gamma (10-1,000 U/ml). The cystic fibrosis transmembrane conductance regulator was also downregulated by interferon-gamma as evident at the protein level and by the decrease in the cAMP-dependent current. On the other hand, interferon-gamma caused an increase of the current elicited by apical UTP application, which is due to the activity of Ca(2+)-dependent Cl(-) channels. Tumor necrosis factor-alpha caused few changes in ion transport. Transepithelial fluid transport was measured in normal and cystic fibrosis cells. At rest, both types of cells showed an amiloride-sensitive fluid absorption that was inhibited by interferon-gamma but not by tumor necrosis factor-alpha. Our results show that interferon-gamma alters the transepithelial ion transport of cultured bronchial cells. This effect may change the ion composition and/or volume of periciliary fluid.

Biological Transport↗
PubMed1999

Restoration of bacterial killing activity of human respiratory cystic fibrosis cells through cationic vector-mediated cystic fibrosis transmembrane conductance regulator gene transfer.

In vitro and in vivo studies have demonstrated that gene transfer of the CFTR (cystic fibrosis transmembrane conductance regulator) cDNA into human respiratory cells through nonviral vectors can occur safely and can be done repeatedly. Although functional evaluation of CFTR in cystic fibrosis (CF) patients enrolled in phase I clinical trials using cationic liposomes has shown a partial correction of nasal potential difference, a biological assay indicating a therapeutic relevance of CFTR gene transfer is still missing. Our aims were to study the induction of killing activity toward Pseudomonas aeruginosa (PA) in CF cells by cationic vector-mediated CFTR gene transfer and to use this assay as a therapeutic end point. Luciferase expression and GFP FACS analysis were used to evaluate the optimal vector and the efficiency of gene transfer into non-CF human respiratory cells growing from nasal polyp explants at the air-liquid interface. To prove that transgenic CFTR was expressed in CF cell cultures under the same experimental conditions, a specific RT-PCR was performed. Challenge of the outgrowths with a known amount of PA showed a bacterial clearance activity by non-CF respiratory cells, while in the case of CF cells it even resulted in bacterial growth. Cationic vector-mediated CFTR cDNA determined the recovery of bacterial clearance activity only under those conditions yielding 5% or more of GFP-positive cells. The results shown in this study might be helpful in considering cationic vectors as therapeutic nonviral vectors for transferring CFTR into human CF respiratory cells, as well as for restoring the bacterial killing activity defective in cystic fibrosis.

Cations↗
PubMed1999

Comparison between cationic polymers and lipids in mediating systemic gene delivery to the lungs.

Airway inflammation frequently found in congenital and acquired lung diseases may interfere with gene delivery by direct administration through either instillation or aerosol. Systemic delivery by the intravenous administration represents an alternative route of delivery that might bypass this barrier. A nonviral approach for transfecting various airway-derived cell lines in vitro showed that cationic polymers (PEI 22K and 25K) and lipids (DOTAP, GL-67/DOPE) are able to transfect with high efficiency the reporter genes firefly luciferase and E. coli lacZ. Notably, two properties predicted that cationic vectors would be useful for a systemic gene delivery approach to the lung: (1) transfection was not inhibited or increased when cells were incubated with cationic lipids or polymers in the presence of serum; and (2) cationic vectors protected plasmid DNA from DNase degradation. A single injection of DNA complexed to the cationic polymer PEI 22K into the tail vein of adult mice efficiently transfected primarily the lungs and to a lesser extent, heart, spleen, kidney and liver. The other vectors mediated lower to undetectable levels of luciferase expression in the lungs, with DOTAP > GL67/DOPE > PEI 25K > DOTMA/DOPE. A double injection protocol with a 15-min interval between the two doses of DOTAP/DNA complexes was investigated and showed a relevant role of the first injection in transfecting the lungs. A two log increase in luciferase expression was obtained either when the two doses were comprised of luciferase plasmid or when an irrelevant plasmid was used in the first injection. The double injection of luciferase/PEI 22K complexes determined higher transgene levels than a single dose, but a clear difference using an irrelevant plasmid as first dose was not observed. Using lacZ as a reporter gene, it was shown that only cells in the alveolar region, including type II penumocytes, stained positively for the transgene product.

Animals↗
PubMed1999

Retinoic acid stimulates plasminogen activator inhibitor 2 production by blood mononuclear cells and inhibits urokinase-induced extracellular proteolysis.

Retinoids have been shown to modulate several functions of mononuclear phagocytes. We investigated the in vitro effect of all-trans-retinoic acid (ATRA) on the production of two major fibrinolytic components, urokinase-type plasminogen activator (u-PA) and PA inhibitor 2 (PAI-2), by human blood mononuclear cells (MNC). ATRA caused a dose-dependent (range 0.01-10 microM) accumulation of PAI-2 antigen and activity into the cell culture medium, with a maximal increase (about 5-fold over control) at a concentration of 1-10 microM. Similarly, a dose-dependent increase in PAI-2 antigen was observed in cell extracts upon ATRA stimulation. Northern blot analysis showed a parallel increase in the amount of PAI-2 mRNA in ATRA-treated cells. Time-course experiments with 1 microM ATRA showed enhanced PAI-2 mRNA expression as early as 2 h, reaching a maximum at 4-6 h and then declining at 18-24 h, and a time-dependent increase in PAI-2 antigen in the cell culture medium. At variance with PAI-2, u-PA was not influenced by the drug. To establish whether ATRA-induced changes influenced the fibrinolytic process, we evaluated the effect of MNC stimulated with ATRA on u-PA-induced degradation of diluted plasma clots. ATRA-treated cells markedly inhibited clot lysis induced by low concentrations of u-PA. The effect was due to enhanced extracellular PAI-2 accumulation since it was observed with conditioned medium from ATRA-treated cells; it was abolished by the addition of neutralizing anti-PAI-2 antibodies and was negligible when single-chain t-PA was used instead of u-PA. Since monocyte/macrophage-mediated, plasminogen-dependent extracellular proteolysis has been proposed as an important mechanism of tissue damage in several inflammatory states, our findings might contribute to better explain the anti-inflammatory properties of retinoids.

Blotting, Northern↗
PubMed1997

Recycling of the urokinase receptor upon internalization of the uPA:serpin complexes.

The GPI-anchored urokinase plasminogen activator receptor (uPAR) does not internalize free urokinase (uPA) but readily internalizes and degrades uPA:serpin complexes in a process that requires the alpha2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha2MR-LRP). This process is accompanied by the internalization of uPAR which renders it resistant to phosphatidylinositol-specific phospholipase C (PI-PLC). In this paper we show that during internalization of uPA:serpins at 37 degrees C, analysed by FACScan, immunofluorescence and immunoelectron microscopy, an initial decrease of cell surface uPAR was observed, followed by its reappearance at later times. This effect was not due to redistribution of previously intracellular receptors, nor to the surface expression of newly synthesized uPAR. Recycling was directly demonstrated in cell surface-biotinylated, uPA:PAI-1-exposed cells in which biotinylated uPAR was first internalized and subsequently recycled back to the surface upon incubation at 37 degrees C. In fact, uPAR was resistant to PI-PLC after the 4 degrees C binding of uPA:PAI-1 to biotinylated cells, but upon incubation at 37 degrees C PI-PLC-sensitive biotinylated uPAR reappeared at the cell surface. Binding of uPA:PAI-1 by uPAR, while essential to initiate the whole process, was, however, dispensable at later stages as both internalization and recycling of uPAR could be observed also after dissociation of the bound ligand from the cell surface.

Animals↗
PubMed1996

Removal of domain D2 or D3 of the human urokinase receptor does not affect ligand affinity.

The main ligand-binding determinant of the human urokinase receptor (uPAR) is located in the amino terminal domain D1, but when isolated this domain presents a 1500 fold lower affinity than the intact three-domain uPAR (D1D2D3). uPAR mutants missing either domain 2 (D1HD3) or domain 3 (D1D2) were expressed in murine LB6 cells and showed to be properly GPI-anchored to the cell surface. Binding assays with [125I]ATF demonstrated that these mutants possessed a normal (D1D2) or slightly reduced (D1HD3) affinity, indicating that a high ligand-affinity may be achieved by a combination of D1 with domain D2 or D3.

Amino Acid Sequence↗
PubMed1995

PMA-induced down-regulation of the receptor for alpha 2-macroglobulin in human U937 cells.

Transcription and expression of the urokinase (uPA) receptor (uPAR) are strongly stimulated by PMA. As for uPAR, the expression of alpha 2-MR is regulated by PMA in U937 cells. Ligand blotting experiments with the 39 kDa receptor-associated protein RAP, a ligand for alpha 2-MR, indicated that alpha 2-MR levels first increased and then decreased after PMA treatment. FACscan as well as immunoblotting analysis with alpha 2-MR-specific antibodies showed an identical trend: alpha 2-MR levels increased within the first day of treatment with PMA, decreased at later times, and totally disappeared by three days of treatment. The effect of PMA was not due to transcriptional down-regulation, as the alpha 2-MR mRNA level did not decrease at later times. Sensitivity of U937 cells to uPA-saporin, a toxin conjugate that reguires binding to uPAR for killing activity, was also markedly decreased. These results suggest that uPAR-mediated endocytosis depends on alpha 2-MR expression.

Base Sequence↗
PubMed1995

Blood and tissue fibrinolytic profiles in patients with colorectal carcinoma.

In patients with colorectal cancer, profound alterations of the plasminogen activator system have been described at the tumor level, but conflicting results have been obtained for fibrinolytic parameters in plasma. Components of the fibrinolytic system, including tissue-type and urokinase-type plasminogen activators and their inhibitors type 1 and 2, were measured in tissue and/or plasma from 41 patients with colorectal cancer and in 40 controls. Procoagulant activity of freshly isolated mononuclear cells (basal activity) and the procoagulant activity and fibrinolytic proteins produced by the cells after incubation for 18 h without exogenous stimulation were also evaluated. Malignant tissue extracts had significantly higher levels of urokinase-type plasminogen activator and plasminogen activator inhibitor-1, but lower levels of tissue-type plasminogen activator than normal mucosa. Plasminogen activator inhibitor-1 alone was higher in advanced (Dukes' stages C + D) than limited (B) tumors. Plasminogen activator inhibitor-2 was not different in malignant tissue and normal mucosa. Plasma levels of plasminogen activator inhibitor-1 antigen were significantly increased in cancer patients compared with controls, but there were no differences in tissue-type and urokinase-type plasminogen activator, in plasminogen activator inhibitor-2, and D-dimer levels. Intra-patient analysis revealed no significant correlation between tumor and plasma levels of plasminogen activators or type 1 inhibitor. Tissue-type plasminogen activator, but not the urokinase type or inhibitor type 1, was higher in venous than in arterial blood collected at the tumor site during surgery. Basal procoagulant activity of mononuclear cells and the procoagulant activity and inhibitor type-2 produced by the cells after short-term culture were comparable in patients and controls. These findings indicate that, at least in our patients with colorectal cancer, the profound changes occurring at tumor level are barely detectable in the blood. Thus, the clinical relevance of plasma fibrinolytic parameters, especially urokinase-type plasminogen activator antigen, as tumor markers in colorectal cancer remains to be established.

Adult↗
PubMed1995

The urokinase/urokinase-receptor system and cancer invasion.

u-PA binds with high affinity to its specific GPI-anchored receptor on the cell surface. The binding has at least two important consequences: (1) it enhances the rate of plasminogen activation on the cell surface; and (2) it focuses the u-PA proteolytic activity at the leading front of migrating cells. Several recent findings suggest that surface-bound u-PA is essential for the invasive ability of tumour cells, even if a picture is emerging indicating a concerted action with other proteases, like collagenases and cathepsin B (Kobayashi et al, 1992; Ossowski, 1992; Schmitt et al, 1992; (Danø et al, 1994). In some tumours, e.g. colon, breast and skin cancer, in situ hybridization studies have given an insight into the u-PA/u-PAR tumour biology showing a complex interplay between stromal and cancer cells Danø et al, 1994). u-PA, u-PAR, and PAI-1 tumour content are now well established prognostic factor in breast cancer. This body of knowledge could be used for theurapeutic purposes. For example, a large study with 671 patients has allowed the identification of node-negative patients which, according to their u-PA levels, would need adjuvant therapy (Foekens et al, 1992). Many other tumours, especially colorectal cancer, expect a direct clinical evaluation of u-PA, u-PAR and serpins as prognostic factors.

Animals↗
PubMed1995

alpha-2 Macroglobulin receptor/Ldl receptor-related protein(Lrp)-dependent internalization of the urokinase receptor.

The GPI-anchored urokinase plasminogen activator receptor (uPAR) does not internalize free urokinase (uPA). On the contrary, uPAR-bound complexes of uPA with its serpin inhibitors PAI-1 (plasminogen activator inhibitor type-1) or PN-1 (protease nexin-1) are readily internalized in several cell types. Here we address the question whether uPAR is internalized as well upon binding of uPA-serpin complexes. Both LB6 clone 19 cells, a mouse cell line transfected with the human uPAR cDNA, and the human U937 monocytic cell line, express in addition to uPAR also the endocytic alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (LRP/alpha 2-MR) which is required to internalize uPAR-bound uPA-PAI-1 and uPA-PN-1 complexes. Downregulation of cell surface uPAR molecules in U937 cells was detected by cytofluorimetric analysis after uPA-PAI-1 and uPA-PN-1 incubation for 30 min at 37 degrees C; this effect was blocked by preincubation with the ligand of LRP/alpha 2-MR, RAP (LRP/alpha 2-MR-associated protein), known to block the binding of the uPA complexes to LRP/alpha 2-. MR. Downregulation correlated in time with the intracellular appearance of uPAR as assessed by confocal microscopy and immuno-electron microscopy. After 30 min incubation with uPA-PAI-1 or uPA-PN-1 (but not with free uPA), confocal microscopy showed that uPAR staining in permeabilized LB6 clone 19 cells moved from a mostly surface associated to a largely perinuclear position. This effect was inhibited by the LRP/alpha 2-MR RAP. Perinuclear uPAR did not represent newly synthesized nor a preexisting intracellular pool of uPAR, since this fluorescence pattern was not modified by treatment with the protein synthesis inhibitor cycloheximide, and since in LB6 clone 19 cells all of uPAR was expressed on the cell surface. Immuno-electron microscopy confirmed the plasma membrane to intracellular translocation of uPAR, and its dependence on LRP/alpha 2-MR in LB6 clone 19 cells only after binding to the uPA-PAI-1 complex. After 30 min incubation at 37 degrees C with uPA-PAI-1, 93% of the specific immunogold particles were present in cytoplasmic vacuoles vs 17.6% in the case of DFP-uPA. We conclude therefore that in the process of uPA-serpin internalization, uPAR itself is internalized, and that internalization requires the LRP/alpha 2-MR.

Animals↗