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Am J Physiol Lung Cell Mol Physiol 295: L336-L347, 2008. First published May 30, 2008; doi:10.1152/ajplung.00059.2008
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Chemical conjugation of {Delta}F508-CFTR corrector deoxyspergualin to transporter human serum albumin enhances its ability to rescue Cl– channel functions

Caroline Norez,1,4 Matteo Pasetto,2 Maria Cristina Dechecchi,1 Erika Barison,2 Cristina Anselmi,2 Anna Tamanini,1 Federica Quiri,1 Luigi Cattel,3 Paolo Rizzotti,1 Franco Dosio,3 Giulio Cabrini,1,* and Marco Colombatti2,*

1Laboratory of Molecular Pathology, Laboratory of Clinical Chemistry and Hematology, University Hospital of Verona and 2Department of Pathology, Section of Immunology, University of Verona, Verona; 3Department of Science and Technology of Medicines, University of Turin, Turin, Italy; and 4Institut de Physiologie et Biologie Cellulaires, Université de Poitiers, Poitiers, France

Submitted 4 February 2008 ; accepted in final form 26 May 2008


   ABSTRACT
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 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The most common mutation of the cystic fibrosis (CF) gene, the deletion of Phe508, encodes a protein ({Delta}F508-CFTR) that fails to fold properly, thus mutated {Delta}F508-cystic fibrosis transmembrane conductance regulator (CFTR) is recognized and degraded via the ubiquitin-proteasome endoplasmic reticulum-associated degradation pathway. Chemical and pharmacological chaperones and ligand-induced transport open options for designing specific drugs to control protein (mis)folding or transport. A class of compounds that has been proposed as having potential utility in {Delta}F508-CFTR is that which targets the molecular chaperone and proteasome systems. In this study, we have selected deoxyspergualin (DSG) as a reference molecule for this class of compounds and for ease of cross-linking to human serum albumin (HSA) as a protein transporter. Chemical cross-linking of DSG to HSA via a disulfide-based cross-linker and its administration to cells carrying {Delta}F508-CFTR resulted in a greater enhancement of {Delta}F508-CFTR function than when free DSG was used. Function of the selenium-dependent oxidoreductase system was required to allow intracellular activation of HSA-DSG conjugates. The principle that carrier proteins can deliver pharmacological chaperones to cells leading to correction of defective CFTR functions is therefore proven and warrants further investigations.

cystic fibrosis; cystic fibrosis transmembrane conductance regulator; correctors; transporter protein; intracellular activation

STUDIES ON TRANSPORTERS such as the human multidrug resistance P-glycoprotein (26) and the cystic fibrosis transmembrane conductance regulator (CFTR) (6) suggest that processing mutation(s) interferes in folding by trapping the protein in a partially folded structure. The mutated protein is unstable and is targeted for degradation. Therefore, one approach to promote folding of processing mutants would be to stabilize the mutant protein during the folding process through the use of chemical or pharmacological chaperones (35). Indeed, small molecules can bring about environmental changes that promote folding of mutant proteins, thus reducing the onset and/or progression of the clinical phenotypes associated with disorders due to those mutants. Efforts to overcome these defects have led to the development of various interventions that successfully repair protein folding while precluding the proteins from either aggregation or degradation pathways (27).

A classic example of disease arising from a reduction or absence of functional protein as a result of defective CFTR protein is cystic fibrosis (CF). CFTR folding is facilitated by chaperones (Hsc70, Hsp90, and calnexin) in the cytosol and in the endoplasmic reticulum (ER). Chaperone release correlates with acquisition of a stable structure in the ER membrane. In contrast, unstable conformations of CFTR lead to polyubiquitination and degradation due to the intervention of a series of molecules recently defined as "CFTR interactome" (54). The deletion of the phenylalanine in position 508 ({Delta}F508-CFTR) is the most common mutation observed in CF patients, resulting in a protein that cannot be folded correctly and is thus retained in the ER (7). Restoring the {Delta}F508-CFTR defect was originally achieved using glycerol (42) and more recently with myo-inositol, betaine, or taurine (59). In spite of extensive investigation, including also high-throughput screening technologies, only a limited number of molecules have been shown to partially restore CFTR function in cells, including deoxyspergualin (DSG) (21), 4-phenylbutyrate (4-PBA) (39, 40), curcumin (14, 25, 33), CFTR corr-4a (36), VRT-325 (53), CFTRcor-325 (55), and the {alpha}1,2-glucosidase inhibitor miglustat (33). However, poor clinical performance of chemical and/or pharmacological correctors might be expected due to unfavorable pharmacokinetics or side effects or both (16). A potential strategy to increase the efficiency of CFTR maturation, to decrease the associated toxicity of the correctors and improve pharmacokinetics, may be to employ macromolecules as vehicles for the {Delta}F508-CFTR corrector. Albumin is a suitable carrier for cytostatic agents (15), and studies provided a further proof of concept for the development of albumin-binding, enzymatically cleavable prodrugs of anticancer drugs (12, 28, 56).

DSG, a stable synthetic analog of the natural product spergualin (52), has demonstrated potent {Delta}F508-CFTR corrector activity in vitro (21). It has been proposed that DSG can compete effectively for binding to HSP70 and HSP90 to promote trafficking of {Delta}F508-CFTR to the cell membrane (21). DSG is endowed with a primary (spermidinic) aminogroup allowing easy cross-linkage to carrier proteins (30). Thus, we have decided to select it as a reference molecule to investigate the feasibility of enhancing its {Delta}F508-CFTR correcting activity by cross-linkage to human serum albumin (HSA). The results described herein using a CF airway epithelial cell line bearing a defective CFTR prove that delivery of DSG in a complex with HSA results in a greater efficiency of {Delta}F508-CFTR correction.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
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 REFERENCES
 
Synthesis of 1-(2-pyridyl-dithio-propionammido)19-guanidino-11-hydroxy-4,9,12-triazanonadecane-10,13-dione (DSG-ss-PDP)

Twenty microliters of triethylamine (0.14 mmol) were added to a solution of 150 mg NTK-01 (0.129 mmol as DSG hydrochloride) in 2 ml of anhydrous dimethylformamide. Then, 60 mg (0.19 mmol) of 3-(2-pyridyldithio) propionic acid N-hydroxysuccinimide ester (SPDP) dissolved in 1 ml of anhydrous dimethylformamide was added under stirring. After 2 h at room temperature, the solution was treated with ethyl acetate (50 ml) to precipitate lactose and then evaporated under reduced pressure. The results of a TLC in butanol were as follows: acetic acid:water:pyridine (6:2:4:4) Rf DSG 0.4, DSG-ss-PDP 0.65. The TLC was stained with the Sakaguchi reagent and then with 10% sulfuric acid. The residue was dissolved in 50 mM, pH 6.5, citrate buffer and transferred on a CM-Sepharose column (25 x 1.3 cm internal diameter). The column was washed with 0.1, 0.4, and then 1 M sodium chloride (50 ml each). The fractions positive to Sakaguchi reaction were collected and lyophilized. The residue was suspended in methanol, and excess sodium chloride was removed by means of a Sephadex LH-20 column (13.5 x 2.5 cm I.D.) eluted with methanol. The eluate was evaporated to dryness, and the white powder residue 1-(2-pyridyl-dithio-propionammido)19-guanidino-11-hydroxy-4,9,12-triazanonadecane-10,13-dione (32 mg, yield 42%) was stored at –20°C in 4-mg aliquots. Results were as follows: 1H NMR (CD3OD) {delta} 1.2–1.8 (14 H, m CH2), 2.0–2.6 (8H, m CH2, CO-CH2), 2.8–3.4 (6H, m NCH2, SCH2), 5.5 (1H, s CH), 7.2–7.6 (3H, m CH aromatic), 8.4 (1H, d CH-N aromatic). HR-MS ESI (M + 1) = 585.30.

Synthesis of 1-(maleimidohexanoic amido)19-guanidino-11-hydroxy-4,9,12-triazanonadecane-10,13-dione (DSG-MI)

Twenty microliters of triethylamine (0.14 mmol) were added to a solution of 150 mg NTK-01 (0.129 mmol as DSG hydrochloride) in 2 ml of anhydrous dimethylformamide. Then, 62 mg (0.20 mmol) of maleimidohexanoic acid N-succinimidyl ester, dissolved in 1 ml of anhydrous dimethylformamide, were added under stirring. After 3 h at room temperature, the solution was treated with ethyl acetate (50 ml) to precipitate lactose and then evaporated under reduced pressure. The results of a TLC in butanol were as follows: acetic acid:water:pyridine (6:2:4:4) Rf DSG 0.4, DSG-MI 0.59. The purification method was the same as reported for DSG-ss-PDP. Thirty milligrams of 1-(maleimidohexanoic ammido)19-guanidino-11-hydroxy-4,9,12-triazanonadecane-10,13-dione as white powder was obtained (yield 40%) and stored at –20°C. NMR analysis supplied the following results: 1H NMR (CD3OD) {delta} 1.2–1.8 (20 H, m CH2 DSG and hexanoyl chain), 2.0–2.6 (8H, m CH2-N, CO-CH2), 3.2–3.5 (6H, m NCH2, N-maleimido-CH2), 5.5 (1H, s CH), 6.8 (2H, m CH maleimido), 8.4 (1H, d CH-N aromatic). MS ESI (M + 1) = 581.37.

Synthesis of 1-amino-19-guanidino-11-(14C)methoxy-4,9,12-triazanonadecane-10,13-dione [11-methoxy-DSG] and 1-(2-pyridyl-dithio-propionammido)19-guanidino-11-(14C)methoxy-4,9,12-triazanonadecane-10,13-dione [11-methoxy-DSG-ss-PDP]

A mixture of 10 mg of DSG or 4 mg of DSG-ss-PDP in a microvial with rubber closure was added with 1 mCi 14C methanol (15 mCi/ml, 0.6 mCi/mmol; Vitrax, Placentia, CA) and 2.5 µl of 37% HCl. After complete dissolution, the mixture was stored at room temperature overnight. Methanol was removed by a gentle flow of nitrogen into a sealed vial filled with adsorbent charcoal, and the residue was washed once with ethanol plus 2.5 µl NaOH 0.5g/ml and then three times with ethanol. The purity of radiolabeled compound was checked by TLC eluted with butanol:acetic acid:water:pyridine (6:2:4:4) Rf 14C-11-methoxy-DSG 0.43, 14C-11-methoxy-DSG-ss-PDP 0.62. Radiochromatographic analyses were performed using a System 200 Imaging Scanner (Canberra Packard, Milan, Italy) and liquid scintillator analyzer (Packard 2500 TR; PerkinElmer, Waltham, MA). Labeling yield was 58% for DSG and 55% for DSG-ss-PDP. To validate this procedure, the same conditions were used to synthesize 11-methoxy-DSG and 11-methoxy-DSG-ss-PDP: 11-methoxy-DSG 1H NMR (CD3OD) 1.2–2.0 (12H, m CH2), 2.0–2.5 (4H, m CH2), 2.9–3.4 (10 H, NCH2), 3.4 [3H s (OCH3)], 5.3 (1H, s, CH). MS ESI (M + 1) = 402.31; 11-methoxy-DSG-ss-PDP 1H NMR (CD3OD) {delta} 1.2–1.8 (14 H, m CH2), 2.0–2.6 (8H, m CH2, CO-CH2), 2.8–3.5 (9H, m NCH2, SCH2, OCH3), 5.4 (1H, s CH), 7.2–7.6 (3H, m CH aromatic), 8.4 (1H, d CH-N aromatic). MS ESI (M + 1) = 599.31.

Determination of Guanidinium Groups (DSG-Containing Fractions) By Sakaguchi Reaction

The method is based on color reaction of guanidine derivatives with {alpha}-naphthol and sodium hypobromite in alkaline medium. The method described by Tomlinson and Viswanatha (51) was modified for the determination of guanidine residues in 96-well polystyrene plates. A solution of 0.1% {alpha}-naphthol in 50% ethanol, 10% KOH, 5% urea, and 5% potassium hypobromite was added to each well containing 0.5 ml of the solutions under study. After a 20-min incubation, the absorption of the reaction mixtures at 492 nm was measured on a Titertek (Flow Laboratories, McLean, VA) photometer for U-well microplates.

Introduction of Thiol Groups in HSA with Cleland's Reagent

Thirty-six milligrams of HSA (0.545 µmol) was dissolved in 3 ml of degassed PBS-EDTA buffer, 0.1 M, pH 7.0, and an aliquot of DTT (150 µl, 0.16 M, molar excess 44 in water) was added. After stirring at room temperature for 2.5 h, the mixture was purified by gel centrifugation on P6DG BioRad Gel. Thiol groups were determined by reaction with a solution of 3 mg/ml Ellman's reagent dissolved in PBS-EDTA buffer 0.2 M, pH 7.0 ({varepsilon} 412 = 14,200). The mean protein recovery was ~80%. The thiolated solution was used immediately for the preparation of DSG conjugates.

Synthesis of Albumin-DSG Conjugates

DSG was first modified by cross-linking to the heterobifunctional cross-linkers SPDP or maleimidohexanoic acid N-succinimidyl ester (MI) obtaining DSG-ss-PDP or DSG-MI (Fig. 1A). Synthesis of these compounds is further described in the Supplementary Materials and Methods (Supplemental data for this article are available online at the AJP-Lung web site).


Figure 1
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  		Fig. 1. Schematic flow-diagram illustrating the procedure followed for the synthesis of human serum albumin (HSA)-deoxyspergualin (DSG) conjugates. A: the primary amino group of DSG was reacted with the N-hydroxysuccinimido ester of 3-(2-pyridyldithio) propionic acid N-hydroxysuccinimide ester (SPDP) and MC-NHS to obtain a disulfide or a maleimido derivative. B: in the presence of excess of DTT, the disulfide bonds in HSA were reduced to cysteinyl residues, and these reacted with DSG-ss-PDP and DSG-MI derivatives to give disulfide- or thioether-linked conjugates. In a standard preparation, a mean (n) of 9–10 mol DSG/mol HSA was linked.

 
Preparation of albumin conjugates with DSG-ss-PDP and with 14C-11-methoxy-DSG-ss-PDP. A solution of HSA (10 mg/ml, 1 ml), containing 9.5–11.5 thiol groups, was reacted with a 20 molar excess of DSG-SS-PDP dissolved in 0.2 ml of methanol. The degree of derivatization was monitored by checking the amount of remaining thiol groups by Ellman reaction. After 120 min at room temperature, the mixture was centrifuged and then extensively dialyzed against PBS buffer. The average protein recovery was 65%. The same procedure was applied to prepare 14C-labeled DSG-HSA conjugates. For a standard preparation, 23 mg of HSA with an average of 10 thiol groups/molecule was reacted with 2.9 µCi of DSG derivative with a specific activity of 0.69 µCi/µmol. An average of 2.5 µCi were cross-linked to HSA. After purification, the radioactivity linked to HSA was evaluated by precipitation with 10% ammonium sulfate (~90% of the radioactivity in solution) and measured in a beta-scintillator. An average value of 1.6–1.8 µCi of radioactive conjugate was obtained in each preparation.

Preparation of albumin conjugates with DSG-MI. A solution of HSA (1 ml), containing an average of 9.5–11.5 thiol groups, was reacted with a 20 molar excess of DSG-MI dissolved in 0.2 ml of methanol. The degree of thiol alkylation was monitored by Ellman reagent. After 120 min at room temperature, the reaction was completed, and the mixture was centrifuged and then extensively dialyzed against PBS buffer. Protein recovery was 65%.

To determine the degree of DSG derivatization, HSA conjugates were analyzed and compared using the Lowry and Sakaguchi methods with a calibration curve of HSA and a calibration curve of L-arginine.

Cell Lines

For this study, we used the human bronchial epithelial cell lines NuLi-1 and CuFi-1, derived respectively from a non-CF or a CF patient homozygous for the {Delta}F508-CFTR mutation (58), a generous gift of A. Klingelhutz, P. Karp, and J. Zabner (Univ. of Iowa, Iowa City, IA). Cells were grown on collagen type VI-coated plastic dishes in serum-free BEGM by supplementing BEBM (Clonetics/Lonza, Walkersville, MD) with essential nutrients as described (58).

Polarized Cell Cultures

Cells were seeded at a density of 6 x 105 cells/cm2 onto collagen-coated Transwell polyester membranes (0.33 cm2, 0.4 µm pore size; Becton Dickinson, Franklin Lakes, NJ) in DMEM/Ham's F-12 (50:50) supplemented with nonheat-inactivated 5% FBS for 24 h. To obtain cell polarization, medium was replaced with DMEM/Ham's F-12 (50:50) supplemented with 2% Ultroser G (PALL Life Sciences, New York, NY). Cells were grown by using liquid-covered culture with 0.2 and 0.7 ml apical and basolateral medium volumes, respectively. Medium was replaced every 2 days. After 15 days, transepithelial resistance (TER) measurements were made by using chopstick-like electrodes with an EVOM voltohmmeter (WPI, Darmstadt, Germany). TER was calculated by correcting the resistance value measured for the surface area of the Transwell cell culture support.

Single Cell Measurement of CFTR Activity

CFTR function was assessed by single-cell fluorescence imaging, using the potential-sensitive probe, bis-(1,3-diethylthiobarbituric acid)trimethine oxonol [DiSBAC2 (3); Molecular Probes, Eugene, OR], as previously reported (37), with minor changes. Briefly, cells grown on coated, round, glass coverslips were washed in a Cl– containing solution (101 mM Na+, 114 mM Cl–, 5 mM K+, 2 mM Ca2+, 2 mM Mg2+, 50 mM mannitol, 5 mM glucose, 5 mM HEPES-Tris, pH 7.4), mounted in the perfusion chamber (Medical Systems, Greenvale, NY), and perfused for 10–15 min at 25°C with a Cl–-free solution (101 mM Na+, 106 mM gluconate, 14 mM acetate, 5 mM K+, 2 mM Ca2+, 2 mM Mg2+, 50 mM mannitol, 5 mM glucose, and 5 mM HEPES-Tris, pH 7.4) containing 100 nM DiSBAC2 (3). The time courses were performed at 25°C, and a baseline signal was acquired for 3 min before the addition of the stimulus. CFTR-dependent Cl– channel was stimulated by a cAMP-elevating cocktail (20 µM forskolin + 100 µM IBMX and 50 µM genistein) added to the chamber at the time indicated in the figures. The thiazolidinone CFTR inhibitor, CFTRinh-172, kindly provided by A. Verkman (Univ. of California, San Francisco, CA) (29), was added to a final concentration of 10 µM. Fluorescence was measured with a Nikon TMD inverted microscope through a Nikon Fluor x40 objective (Nikon Europe, Firenze, Italy). The signal was acquired with a Hamamatsu C2400-97 charge-coupled intensified video camera (Hamamatsu City, Japan) at a rate of 1 frame/30 s, with an integration time ranging from 0.1 to 1.0 s. Fluorescence coming from each single cell was analyzed by customized software (Spin, Vicenza, Italy). Results are presented as transformed data to obtain the percentage signal variation (Fx) relative to the time of addition of the stimulus, according to the equation: Fx = ([Ft – Fo]/Fo) x 100, where Ft and Fo are the fluorescence values at the time t and at the time of addition of the stimulus, respectively.

In some instances, cells were pretreated for 24 h in the presence of a 200 nM concentration of the selenium-dependent oxidoreductase inhibitor S-triethylphosphine gold(I)-2,3,4,6-tetra-O-acetyl-1-thio-β-D-glucopyranoside (auranofin).

Measurement of CFTR Activity on Cell Populations by the Iodide Efflux Technique

Cl– channel activity was assessed in cell populations by measuring the rate of iodide (125I) efflux from cells as previously described (34). At the beginning of each experiment, cells were washed twice with efflux buffer containing 136.9 mM NaCl, 5.4 mM KCl, 0.3 mM KH2PO4, 0.3 mM NaH2PO4, 1.3 mM CaCl2, 0.5 mM MgCl2, 0.4 mM MgSO4, 5.6 mM glucose, and 10 mM HEPES, pH 7.4. Cells were incubated in efflux buffer containing Na125I (1 µCi Na125I/ml; NEN, Boston, MA) during 1 h at 37°C. Cells were then washed with efflux medium to remove extracellular 125I. The exit of intracellular 125I was determined by removing the medium with efflux buffer every 1 min for up to 10 min. The first three aliquots were used to establish a stable baseline in efflux buffer alone. A medium containing the appropriate drug was used for the remaining aliquots. Residual radioactivity was extracted with 0.1 N NaOH/0.1% SDS and measured in a gamma-spectrometer. The fraction of initial intracellular 125I exited during each time point was measured and time-dependent rates of 125I efflux calculated from: ln (125It1/125It2)/(t1 – t2) where 125It is the intracellular 125I at time t, and t1 and t2 successive time points. Curves were constructed by plotting efflux rates of 125I vs. time. All comparisons were based on maximal values for the time-dependent rates (k = peak rates, min–1) excluding the points used to establish the baseline (k basal, min–1) (34). Chloride current inhibitors were added 30 min before the beginning of the kinetic assay to ensure a complete inhibition of the iodide efflux stimulation.

Statistics

Results are expressed as means ± SE of n observations. Sets of data were compared with ANOVA or a Student's t-test. Differences were considered statistically significant when P < 0.05. *P < 0.05, **P < 0.01, ***P < 0.001. All statistical tests were performed using GraphPad Prism version 4.0 for Windows (Graphpad Software).

Chemicals

TS-TM calix[4]arene (5,11,17,23-tetrasulfonato-25,26,27,28-tetramethoxy-calix[4]arene), an inhibitor of outwardly rectifying Cl– channels, was generously provided by A. Singh and R. J. Bridges (Univ. of Pittsburgh, Pittsburgh, PA). DSG, formulated with lactose (66.7%) as NKT-01, was kindly supplied by Nippon Kayaku (Tokyo, Japan). 2-Iminothiolane hydrochloride (Traut's reagent), 5,5%-dithio-bis-(2-nitrobenzoic acid) (Ellman's reagent), and DTT (Cleland's reagent) were purchased from Pierce (Rockford, IL). S-triethylphosphine gold(I)-2,3,4,6-tetra-O-acetyl-1-thio-β-D-glucopyranoside (auranofin) was from ICN Biomedicals (Aurora, OH). Other reagents and solvents were obtained from Sigma-Aldrich (Milan, Italy).


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Design and Synthesis of HSA-Conjugated DSG

DSG was cross-linked to HSA by first reacting its primary amino group with the heterobifunctional cross-linking agents SPDP or MC-NHS (Fig. 1A) and then exploiting the cysteinyl residues of reduced HSA to obtain covalently linked HSA-DSG conjugates stabilized by a reducible disulfide (SPDP) or by a thioether bond (MC-NHS) (Fig. 1B). The purification procedure of cross-linker-modified DSG took advantage of the main physico-chemical properties of DSG (i.e., water solubility and basicity). To make sulfhydryl groups of HSA available for cross-linking to DSG, the protein was reduced by DTT treatment. An average of 10 thiol groups were exposed to the solvent without formation of protein aggregates, as evaluated by SDS-PAGE. By addition of derivatized DSG, the conjugation reaction took place, and after 120 min, ~90% of thiol groups were blocked, as determined by Ellman reagent. The suitability of the reaction conditions and the number of DSG molecules cross-linked/HSA molecule were confirmed using 14C-labeled 11-methoxy-DSG-ss-PDP for conjugation to HSA and then measuring the covalently linked radioactivity. After centrifugation and extensive dialysis, SDS-PAGE analysis showed no presence of high molecular aggregates (not shown).

HSA-DSG Shows Greater {Delta}F508-CFTR Correcting Effects Than DSG Alone

{Delta}F508-CFTR correction by DSG and by HSA-DSG conjugates was assessed by single-cell fluorescence imaging, a method validated in evaluating CFTR function in respiratory cells after wt-CFTR gene transfer (37) and by screening of CFTR correctors and potentiators in our and other laboratories (10, 53). The effects of the two compounds under study (i.e., HSA-DSG conjugates and free DSG) on CFTR functions were evaluated by the fluorescent probe DiSBAC2 (3) in both CF (i.e., CuFi-1) and non-CF (i.e., NuLi-1) airway epithelial cell lines. As shown in Fig. 2A, a sharp increase of fluorescence was recorded in NuLi-1 cells after activation of protein kinase A [induced by a cAMP-elevating cocktail: 20 µM forskolin (Fsk) + 100 µM IBMX], followed by addition of the CFTR potentiator genistein (20), confirming the expression of functional CFTR in NuLi-1 cells (58) (Fig. 2A). As expected, no changes were instead observed in the CF CuFi-1 cells bearing a defective {Delta}F508-CFTR (58). The decrease of the fluorescence trace observed after addition of CFTRinh-172 (29) further confirmed the specificity of the CFTR-mediated fluorescence variation measured in this functional assay. Remarkably, only pretreatment of CuFi-1 cells with the higher concentration of DSG (50 µg/ml) resulted in a clear-cut activation of function, whereas lower concentrations of DSG yielded little or no rescue of CFTR function. When CuFi-1 cells were pretreated with HSA-DSG for 24 h, they displayed a cAMP-responsive Cl– channel activity comparable with that obtained with 50 µg/ml DSG alone (Fig. 2C). Moreover, the correction effect plateaued already using the lowest HSA-DSG dosage (i.e., 5 µg/ml) (Fig. 2C). In CuFi-1 cells treated with HSA alone (in concentrations equivalent to HSA-DSG), we could detect no rescue of CFTR activity (data not shown). The difference of the maximal level of correction obtained in HSA-DSG corrected cells vs. DSG-corrected cells can be explained by an increase of the number of corrected cells. The percentage of responsive cells observed with free DSG (5 µg/ml) was only 12% but increased to 21% at a DSG concentration of 50 µg/ml (Fig. 2D). Conversely, no differences were observed between cells corrected with 5 µg/ml or with 50 µg/ml HSA-DSG. Moreover, we observed an increase of the maximal number of corrected cells when CuFi-1 CF cells were treated with HSA-DSG (30%) with respect to cells treated with free DSG (21%) (Fig. 2D). Thus, even if CF cells were treated with a higher concentration of DSG (50 µg/ml) compared with HSA-DSG (5 µg/ml), the maximal number of corrected cells remained lower. Thus, exposure of {Delta}F508-CFTR expressing cells to DSG or HSA-DSG resulted in functional correction of the trafficking defect of the mutant protein in CuFi-1 cells, also demonstrating that HSA-bound DSG is pharmacologically active.


Figure 2
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  		Fig. 2. Restoration of {Delta}F508-CFTR function by DSG and HSA-DSG. A: functional evaluation of CFTR by DiSBAC2 (3) assay in NuLi-1 cells. Cells grown on round, glass coverslips were mounted on the perfusion chamber and perfused with Cl–-free solution containing DiSBAC2 (3) to allow the equilibration of the dye within cell membranes. Fluorescence from each single cell was then analyzed. Typical time courses are shown. Data represent the means (±SE) of the relative fluorescence collected from all the cells of the field (from 8 to 20). Forskolin (Fsk)- and IBMX activated Cl– efflux. The CFTR potentiator, genistein, has a large response in NuLi-1 cells, inhibited by CFTRinh-72. No activation of CFTR function was detected in CuFi-1 cells. B and C: functional evaluation of CFTR by DiSBAC2 (3) assay in CuFi-1 cells treated with DSG alone (B) or with HSA-DSG (C). In treated CuFi-1 cells, the CFTR potentiator, genistein, has a large response, inhibited by CFTRinh-72. D: cells were treated with DSG or HSA-DSG at the concentrations shown, and then the percentage of responsive cells in the whole population was determined. Histograms report the percentage ± SE of cells showing increased fluorescent emission measured in 10 experiments. Statistical significance is *P < 0.05, **P < 0.01; ns, not significant.

 
The Presence of HSA Enhances the Corrector Effect of DSG

To analyze in further detail the correcting effect of HSA-DSG and to compare the efficiency of correction brought about by free DSG and by HSA-DSG, we next performed experiments using the iodide efflux technique. In this assay, a rapid change in the rate of iodide efflux upon stimulation is indicative of the presence of active CFTR molecules at the plasma membrane (34). Figure 3A shows the results of assays conducted to investigate the CFTR-dependent iodide efflux in CuFi-1 cells treated or not. No iodide efflux could be stimulated by a cAMP-cocktail in {Delta}F508-CFTR-expressing cells, as expected. Following treatment with DSG (24 h, 5 µg/ml), a cAMP-cocktail could stimulate the iodide efflux (kpeak – kbasal = 0.046 ± 0.011 min–1) to a level twofold lower than after HSA-DSG treatment (kpeak – kbasal = 0.108 ± 0.016 min–1) (Fig. 3, A and B). Again, treatment with HSA alone in equivalent concentrations to that present in HSA-DSG conjugates could not allow rescue of defective CFTR function nor could we observe any CFTR rescue when the two compounds (i.e., HSA and DSG) were premixed and added to the cells without prior cross-linkage at DSG concentrations corresponding to those of the chemically conjugated samples (50 µg/ml) (Fig. 3B). The half-maximal effective concentration (EC50) needed for {Delta}F508-CFTR rescue occurred, after a 24-h treatment, at 5.4 µM DSG and at 768 nM HSA-DSG (Fig. 3C). A higher maximal potency of correction with HSA-DSG (kpeak – kbasal = 0.130 ± 0.005 min–1) compared with free DSG (kpeak – kbasal = 0.106 ± 0.005 min–1) was observed (Fig. 3C) consistently with the results obtained in the fluorescence assay (see above paragraph).


Figure 3
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  		Fig. 3. Correction of the Cl– channel activity of {Delta}F508-CFTR in CuFi-1 cells treated with DSG or HSA-DSG. Channel activity was measured by iodide efflux as described in MATERIALS AND METHODS. A: the presence of forskolin (20 µM) and genistein (50 µM) is indicated by the horizontal bar. Data represent the mean (±SE) of 4 experiments. B: histograms showing the percent maximal Cl– channel activation. Statistical significance is *P < 0.05 and **P < 0.01; ns, not significant. C: concentration dependence of the iodide efflux rates for cells treated with HSA-DSG or DSG alone at the DSG concentrations indicated. D: histograms showing the percent maximal Cl– channel activation for the experimental conditions indicated below each bar. Cocktail, mix of 100 nM TS-TM calix[4]arene and 500 µM DIDS; calix, 100 nM TS-TM calix[4]arene; DIDS, 500 µM DIDS; glib, 100 µM glibenclamide; CFTRihn-172, 100 µM CFTRihn-172; basal, no treatment. Statistical significance is ***P < 0.001; ns, not significant.

 
Finally, we determined the effect of a range of Cl– channel blockers on the cAMP-stimulated iodide efflux in HSA-DSG-treated cells. Glibenclamide is a nonspecific inhibitor of CFTR channels (45, 47). The thiazolidinone compound CFTRinh-172 has been recently developed as a specific CFTR blocker with no significant inhibitory action on other chloride channels (29). DIDS and TS-TM calix[4]arene are instead inhibitors of outwardly rectifying Cl– channels but not of CFTR (45, 47). In HSA-DSG-treated CuFi-1 cells stimulated by cAMP, the efflux was fully inhibited by 100 µM CFTRinh-172 and 100 µM glibenclamide but was not affected by 500 µM DIDS or by 100 nM TS-TM calix[4]arene (Fig. 3D). Similar results were obtained in CuFi-1 cells treated with DSG alone (not shown). Thus, the pharmacological profile of inhibition obtained by us is in agreement with the expected signature of CFTR (45, 47), demonstrating the identity of rescued cAMP-dependent {Delta}F508-CFTR protein after DSG or HSA-DSG treatment.

In conclusion, the above data demonstrated that the presence of HSA in the HSA-DSG complexes increased the corrector effect of DSG in a manner that 10-fold lower DSG amounts were sufficient to obtain a correction effect comparable to higher free DSG dosages.

HSA Protects DSG From Degradation

DSG can be modified and inactivated by polyamine oxidases present in the fetal bovine serum (50). Degradation/inactivation of DSG by serum- and/or tissue-dependent mechanisms could explain the high in vivo dosages needed to achieve a therapeutic effect (32); this, together with a maximum tolerated dose being close to the therapeutic dose, represents a limitation to in vivo applications. These observations can be generalized to almost any corrector identified to date for which the level of correction of {Delta}F508-CFTR may be too low to be of therapeutic value and prompted us to test if linkage to HSA can stabilize DSG in the medium. We thus evaluated CFTR channel activity in CuFi-1 cells cultured in the presence of refreshed treatment or not by measuring the iodide efflux. As illustrated in Fig. 4A, DSG treatment for 24 h resulted in minimal restoration of CFTR activity (kpeak – kbasal = 0.045 min–1), whereas DSG pretreatment for 48 and 72 h did not result in any detectable correction of defective CFTR function. Conversely, when the treatment with DSG was refreshed every 24 h, the correction of {Delta}F508-CFTR was increased already after 48 h (kpeak – kbasal = 0.075 min–1) and was maintained to higher levels up to 72 h of treatment (kpeak – kbasal = 0.080 min–1) (Fig. 4A). Interestingly, whereas DSG was degraded/inactivated within 48 h if no new compound was added to the culture medium, the correction induced by HSA-DSG was maintained after 48 h of treatment to a level similar to 24 h of treatment (kpeak – kbasal = 0.108 min–1) (Fig. 4B) with no need of new HSA-DSG additions. Only after 72 h of continuous treatment with HSA-DSG, the correction afforded began to decrease (kpeak – kbasal = 0.05 min–1) if no new compound was included in the medium.


Figure 4
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  		Fig. 4. Effect of multiple successive additions of DSG or HSA-DSG to the culture medium on the {Delta}F508-CFTR response. Function of {Delta}F508-CFTR was evaluated by measuring the iodide efflux as in Fig. 3. CuFi-1 cells were treated once with DSG (A) or HSA-DSG (B) for the indicated times (left) or treated every 24 h with DSG or HSA-DSG for the times indicated at right. Histograms show the Cl– channel activation as a measure of iodide efflux as described in MATERIALS AND METHODS. Data represent the mean (±SE) of 4 experiments. Statistical significance is *P < 0.05; ns, not significant.

 
Our data confirm and extend previous observations (50) that DSG is indeed degraded in cell media but also reveal that HSA protects DSG from degradation/inactivation.

{Delta}F508-CFTR Correcting Activity of HSA-DSG Requires Intracellular Activation

DSG is linked to HSA by way of a chemical cross-linking involving the primary amino group of DSG that is needed for function. We therefore investigated whether HSA-DSG conjugates required intracellular manipulation and processing by cell-dependent mechanisms by comparing the correcting effects of disulfide-stabilized HSA-DSG (i.e., HSA-PDP-DSG) and of the thioether-stabilized HSA-DSG (i.e., HSA-MI-DSG). As shown in Fig. 5A, no correction was afforded by the nonreducible HSA-MI-DSG conjugates. Thus it appears that only the use of disulfide-based HSA-DSG conjugates could lead to intracellularly functional DSG molecules. The requirement for disulfide reduction by intracellular oxidoreductases in the activation of HSA-DSG conjugates was therefore further investigated.


Figure 5
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  		Fig. 5. Crucial role of selenium-dependent oxidoreductases in the intracellular activation of HSA-transported DSG. The role of intracellular mechanisms of HSA-DSG reduction and activation was evaluated by single-cell analysis using the fluorescent probe DiSBAC2(3) (A and B) and by iodide efflux assays (C). A: CuFi-1 cells were treated with either HSA-PDP-DSG (25 µg/ml) or HSA-MI-DSG (25 µg/ml) for 48 h, and then function of {Delta}F508-CFTR was evaluated as in Fig. 2 by recording the fluorescent emission of single cells. B: CuFi-1 cells were treated with HSA-PDP-DSG (HSA-DSG; 5 µg/ml) or free DSG (5 µg/ml) in the presence or in the absence of the selenium-dependent oxidoreductase auranofin (200 mM) for 48 h, and then the function of {Delta}F508-CFTR was evaluated. C: the function of {Delta}F508-CFTR was evaluated in CuFi-1 cells by the iodide efflux technique. Cells were treated for 48 h with HSA-DSG (5 µg/ml) or free DSG (5 µg/ml) in the presence or in the absence of the selenium-dependent oxidoreductase auranofin (200 mM). Histograms report the iodide efflux rates under the different conditions assayed.

 
We previously found that heterodimeric disulfide stabilized toxins and their antibody conjugates require functional intracellular selenium-dependent oxidoreductases to become activated and exert their cytotoxic effects in target cells (4). The major component of this family of oxidoreductases is thioredoxin reductase (TrxR), which is involved in most cellular reactions regulating the redox equilibrium in the cell (3). In an effort to understand the role of complexed HSA on the corrector effect of HSA-DSG and the possible mechanism(s) of activation involved in its correcting effects, we therefore performed experiments in the presence of auranofin, an irreversible alkylating agent of selenium that is necessary for the reductase activity of TrxR. Pretreatment of cells by auranofin prevents the reduction of the disulfide bonds by TrxR (4). As shown in Fig. 5, B and C, we observed no {Delta}F508-CFTR correction by HSA-DSG in the presence of auranofin, demonstrating that functional selenium-dependent oxidoreductases are indeed required to allow HSA-DSG to deploy its corrector function. This also demonstrates that activation of HSA-DSG takes place within the cell and that no breakdown of the conjugates occurs in the extracellular medium or at the cell surface. As expected, the corrector effect of DSG was not affected by pretreatment with auranofin (Fig. 5C).

HSA-DSG Corrects Polarized Cells

The CFTR protein is generally polarized to the apical plasma membrane of many epithelial cells in vivo (9, 11), and distribution at the apical membrane is preserved in polarized cells in culture (22). Having demonstrated the greater {Delta}F508-CFTR correcting effects of HSA-DSG compared with equimolar amounts of free DSG in CuFi-1 cells grown on conventional tissue culture plastic supports, we asked whether correction can be achieved in a more physiological respiratory epithelial cell model and whether the route of administration of the correcting treatment (i.e., from the apical side or from the basolateral side) may influence the outcome of the treatment itself, indicating a preferential route of administration of the conjugate. Cultures of polarized CuFi-1 cells were therefore established following a described protocol allowing a high transepithelial resistance. HSA-DSG or free DSG were then added to the cells from the top (apical side) or from the bottom (basolateral side) chambers of the Transwell inserts or from both compartments at the same time, and iodide efflux was taken as a measure of {Delta}F508-CFTR function. Importantly, HSA-DSG conjugates displayed a greater correcting effect than free DSG also in polarized cell cultures, as illustrated in Fig. 6, and similar correction was obtained by adding the DSG-conjugate either at the apical or at the basolateral sites.


Figure 6
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  		Fig. 6. Restoration of {Delta}F508-CFTR function by DSG and HSA-DSG in polarized cell cultures. CuFi-1 cells were grown on Transwell inserts up to polarization, as confirmed by transepithelial resistance measurement, and function of {Delta}F508-CFTR was evaluated by measuring the iodide efflux as described in MATERIALS AND METHODS. DSG, HSA-DSG conjugate, or solvent (untreated) was added 24 h before measuring iodide efflux at the apical, basolateral (BL), or both sides of the Transwell insert. The iodide efflux calculated from basal and stimulated values, as reported in Figs. 3 and 4, is reported as the mean (±SE) of 3 experiments. Statistical significance indicated with horizontal line between the indicated bars is not significant.

 

   DISCUSSION
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In the present report, we have demonstrated that the correction of {Delta}F508-CFTR functions by HSA-transported DSG in human airway CF epithelial cells is greater than that obtained by equimolar amounts of free unconjugated DSG. This holds true both in nonpolarized and in polarized cells. Indeed, conjugation of DSG to HSA leads to an increase of the number of corrected cells as demonstrated by the lower half-maximal concentrations needed by HSA-DSG to exert its function. Obviously, HSA-DSG does not represent a new class of corrector molecules, but only a more efficient way of intracellular delivery of a pharmacologically active compound, because upon intracellular reduction HSA-DSG conjugates release their components, in particular free DSG, which can exert its function as an effector molecule once it becomes available to interact with its intracellular targets. Thus, HSA acts as a carrier protein to increase the efficiency of intracellular delivery of DSG. Intracellular processing and release of free functional DSG from HSA-DSG conjugates is, however, required to produce pharmacologically relevant correction of defective CFTR in airway epithelial cells.

That intracellular activation of HSA-transported DSG is crucially dependent on the disulfide-reducing activity of selenium-dependent oxidoreductases was demonstrated by inhibition of {Delta}F508-CFTR rescue in the presence of the selective blocker auranofin and by the observation that a nonreducible thioether-linked HSA-DSG conjugate yields a nonfunctional HSA-DSG molecule. In fact, only the reducible cross-linker SPDP, but not the thioether-based cross-linker MC-NHS, allows intracellular release of active DSG with a fully functional primary (spermidinic) group. This is in accordance with studies by Maeda and coworkers (30) who demonstrated that alkylation or acylation of the primary amino group of DSG resulted in a loss of function. Additionally, DSG simply derivatized with the SPDP cross-linker (DSG-PDP) is functionally unable to effect any rescue of {Delta}F508-CFTR activity in treated cells (not shown), likely because the DSG-active group (30) is hindered by derivatization with a nonaccessible disulfide bond that is unavailable to intracellular oxidoreductases because of lack of substrate recognition. The disulfide holding together HSA and DSG might instead be more easily accessible to intracellular oxidoreductases. Thus, intracellularly delivered DSG needs to be released from HSA, and chemical bonds need to be cleaved from its active group to become fully functional. It can be hypothesized that among the various members of the oxidoreductases family, TrxR is the one mainly involved in the intracellular activation of HSA-delivered DSG. Indeed, TrxR is inhibited by lower concentrations of auranofin (i.e., 50–500 nM, 200 nM in our assays) than other members of the family (e.g., glutaredoxin and its analogs), which are instead inhibited at much higher auranofin concentrations (4, 18). Moreover, TrxR is almost ubiquitously distributed within the cell, whereas other members are compartmentalized within specific cell districts, e.g., glutaredoxin is found in the cytoplasm and nucleus (19, 23), protein disulfide isomerase (PDI) is found in the plasma membrane, in secretory vesicles, in the Golgi apparatus, and most abundantly in the ER lumen (1). TrxR might therefore be readily available in almost any intracellular compartment reached by incoming HSA-DSG conjugates to effect the release of DSG from its carrier HSA. However, TrxR may not itself be responsible for disulfide reduction but may instead activate other members of the oxidoreductase family (e.g., thioredoxin, PDI) acting in turn on the disulfide bond between HSA and DSG. Regulation of HSA-DSG activation by intracellular oxidoreductases also provides a molecular switch to modulate the effects of the conjugates that might be exploitable also for in vivo treatments.

We found that cross-linkage to HSA increases the efficacy of DSG effects lowering the concentration of DSG needed to achieve rescue of defective CFTR function. It has been reported that diverse therapeutic compounds can be efficiently transported and delivered by complexing with albumin and that albumin may have a protective effect towards the carried molecule (13, 28, 31, 56), therefore it can be suggested that HSA protects DSG from degradation by the polyamine oxidase present in the culture medium. For example, it was demonstrated (28) that albumin binds low-molecular-weight molecules, including proteins and peptides, which then acquire longer half-life. The longer half-life of HSA-transported DSG and its resistance to degradative effects may thus result also in vivo in larger amounts of DSG available to target cells and positively affect the intracellular mechanisms leading to defective CFTR rescue. In addition, we have also consistently observed a 30% higher uptake of HSA-DSG vs. free DSG using radiolabeled DSG (not shown). Thus, a greater uptake of HSA-transported DSG may also contribute to an overall higher efficacy of HSA-DSG conjugates compared with DSG used alone.

Albumin and its chemical derivatives can be taken up by a variety of different receptors expressed at the surface of various cell types (43, 44); in particular, scavenger receptors may mediate high affinity uptake and intracellular trafficking of albumins that are conformationally modified either by surface adsorption to colloidal gold particles or by treatment with formaldehyde or maleic anhydride or by cross-linkage to methotrexate (15, 44), resulting in rapid internalization and lysosomal degradation of modified albumins. Cross-linkage to DSG might have produced sufficient conformational changes in HSA for it to be perceived as "modified" by such receptors. Lysosomal escape of internalized and degraded HSA-DSG complexes or fragments thereof might then supply intracellularly available amounts of pharmacologically active DSG sufficient to bring about defective CFTR rescue. Alternatively, HSA-DSG complexes may enter the cell by fluid phase macro/micropinocytosis, which may favor accumulation of protein-transported compounds. Whatever the mechanisms of HSA-DSG uptake and trafficking, they seem to be equally operative in both nonpolarized and polarized airway cell cultures and allow a higher concentration of intracellular DSG than the administration of free DSG. Thus, rescue of defective CFTR function in vivo could be attained regardless of the route of administration of the correcting treatment.

The properties of HSA-DSG conjugates make them potential candidates for in vivo applications in humans. Indeed, HSA is already widely used as a building material in microsphere and nanosphere preparations in drug and gene delivery (8); it is not immunogenic, and it is biodegradable. However, it is common knowledge that chemical modification of proteins may create new epitopes or expose previously hidden cryptic epitopes. Thus cross-linking of DSG or other correctors to HSA might indeed elicit an immune response against the administered conjugates. This, however, represents an unlikely event. Like gut mucosa, the airway mucosa is also maintained in a tolerogenic state by specialized immunological mechanisms to avoid deleterious responses to harmless antigens; in the absence of coadministered adjuvants, the inoculation of antigens would result more likely in tolerance than in immune response (2). DSG is a known immunosuppressant already in use in the clinics for the treatment of several human diseases; therefore, its application in the airway mucosa as a component of HSA-based conjugates would be even more unlikely to induce local immune responses (17). Additionally, as a major component of serum, HSA circumvents the problems often encountered with positively charged complexes in vivo, such as rapid opsonization by serum proteins (48). This could be also relevant in the airways, as it was found that extracellular components of the bronchoalveolar lavage fluid (i.e., complement fragments) could be opsonized on gene vectors (polyplexes) and compromise successful gene delivery (38). In CF, the target respiratory cells are layered with a tenacious viscous mucus, which may pose a significant barrier to successful corrector delivery to the lung (24, 41, 46). Nevertheless, delivery of CFTR gene in the airways of CF patients was improved by conjugated polyethylenimine (PEI) in PEI-albumin complexes (5). It was also reported that in animal models, drug-albumin conjugates accumulated preferentially in tumors (49) as well as in chronically inflamed areas (57) resulting in sizable therapeutic effects. Therefore, HSA-DSG complexes could reach target cells at therapeutically effective concentrations. Clinical use of DSG results in plasma levels of between 10 and 20 µg/ml at therapeutic doses (32). Therefore, pharmacologically relevant activity of DSG would be expected to occur at levels of 10–20 µg/ml or less. The dose-response determined by us for DSG showed a nonmaximal activity for cells treated with these concentrations. Conjugation to HSA allows us instead to obtain a maximal correction at DSG concentrations lower than 10 µg/ml and a greater resistance to degradation/inactivation processes. The demonstration obtained in polarized cells that the apical membrane is not a barrier to the intracellular uptake of this protein-conjugate cargo provides the rationale for testing intraluminal delivery in preclinical studies in the lung of animal models, and that its entry is unlikely due to compartmentalized cell entry mechanisms, thus reinforcing the concept that HSA-DSG and similar compounds are potentially also useful for in vivo treatments.

In conclusion, the use of correctors conjugated to carrier proteins may represent an advantageous strategy for improving the maturation efficiency of functionally defective CFTR processing mutants. Results obtained by us with DSG warrant further studies with other classes of corrector molecules, provided they display the properties required to allow chemical conjugation to carrier proteins without affecting their correcting activity. Indeed, because the pathways on which {Delta}F508 defects lie are distinct (16), it is likely that simultaneous use of different classes of correctors affecting any single pathway will act synergistically and achieve a greater therapeutic effect. Moreover, transporter proteins following different routes of cell entry could also be used to concentrate the appropriate corrector molecules in different subcellular compartments to allow more efficient targeting of molecules involved in CFTR maturation and expression.


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This work was partially supported by grants from Fondazione Cariverona (Progetti Bando 2004), by MIUR (PRIN 2005), and by Italian Cystic Fibrosis Research Foundation (#1/2006) (to M. Colombatti), and Fondazione Cariverona-Bando 2005-Malattie rare e della povertà (to G. Cabrini).


   ACKNOWLEDGMENTS
 
We are grateful to Nikkon Kayaku (Tokyo, Japan) for generously supplying DSG, to A. Klingelhutz, P. Karp, and J. Zabner (Univ. of Iowa) for providing Nuli-1 and CuFi-1 cells, and to A. S. Verkman (Univ. of California San Francisco) for providing the CFTR inhibitor, CFTRinh-172. C. Norez is on leave of absence from Institut de Physiologie et Biologie Cellulaires, Université de Poitiers, Poitiers, France.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. Colombatti, Dept. of Pathology, Section of Immunology, Univ. of Verona, c/o Policlinico G. B. Rossi, P.le L.A. Scuro, 10, 37134 Verona, Italy (e-mail: marco.colombatti{at}univr.it)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* Giulio Cabrini and Marco Colombatti share senior authorship. Back


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