Gérard Toubeau

Molecular imaging of αvβ3 integrin expression in atherosclerotic plaques with a mimetic of RGD peptide grafted to Gd-DTPA†

AIMSnThe integrin alpha v beta3 is highly expressed in atherosclerotic plaques by medial and intimal smooth muscle cells and by endothelial cells of angiogenic microvessels. In this study, we have assessed non-invasive molecular magnetic resonance imaging (MRI) of plaque-associated alpha v beta3 integrin expression on transgenic ApoE-/- mice with a low molecular weight peptidomimetic of Arg-Gly-Asp (mimRGD) grafted to gadolinium diethylenetriaminepentaacetate (Gd-DTPA-g-mimRGD). The analogous compound Eu-DTPA-g-mimRGD was employed for an in vivo competition experiment and to confirm the molecular targeting. The specific interaction of mimRGD conjugated to Gd-DTPA or to 99mTc-DTPA with alpha v beta3 integrin was furthermore confirmed on Jurkat T lymphocytes.nnnMETHODS AND RESULTSnThe mimRGD was synthesized and conjugated to DTPA. DTPA-g-mimRGD was complexed with GdCl3.6H2O, EuCl3.6H2O, or with [99mTc(CO)3(H2O)3]+. MRI evaluation was performed on a 4.7 T Bruker imaging system. Blood pharmacokinetics of Gd-DTPA-g-mimRGD were assessed in Wistar rats and in c57bl/6j mice. The presence of angiogenic blood vessels and the expression of alpha v beta3 integrin were confirmed in aorta specimens by immunohistochemistry. Gd-DTPA-g-mimRGD produced a strong enhancement of the external structures of the aortic wall and of the more profound layers (possibly tunica media and intima). The aortic lumen seemed to be restrained and distorted. Pre-injection of Eu-DTPA-g-mimRGD diminished the Gd-DTPA-g-mimRGD binding to atherosclerotic plaque and confirmed the specific molecular targeting. A slower blood clearance was observed for Gd-DTPA-g-mimRGD, as indicated by a prolonged elimination half-life and a diminished total clearance.nnnCONCLUSIONnThe new compound is potentially useful for the diagnosis of vulnerable atherosclerotic plaques and of other pathologies characterized by alpha v beta3 integrin expression, such as cancer and inflammation. The delayed blood clearance, the significant enhancement of the signal-to-noise ratio, and the low immunogenicity of the mimetic molecule highlight its potential for an industrial and clinical implementation.

Galectin 7 (p53-induced gene 1): a new prognostic predictor of recurrence and survival in stage IV hypopharyngeal cancer.

BackgroundEighty percent of hypopharyngeal squamous cell carcinoma patients have advanced stages (III and IV) of the disease, and biological markers are required to predict high-risk head and neck squamous cell carcinoma patients in need of highly aggressive treatments after surgery to improve the survival rate. We analyzed the potential prognostic value of galectin 7 in a series of 81 stage IV hypopharyngeal SCCs because galectin 7 is an emerging marker involved in the epidermal development of pluristratified epithelia and in epidermal cell migration.MethodsThe immunohistochemical expression of galectin 7 was determined on a series of 81 stage IV hypopharyngeal SCCs and was compared with that of galectins 1 and 3.ResultsHigh levels of galectin 7 expression were associated with rapid recurrence rates and dismal prognoses in these 81 stage IV hypopharyngeal SCCs, a feature not observed with galectin 3 and one observed weakly, if at all, with galectin 1.ConclusionsThese data suggest that the immunohistochemical determination of galectin 7 expression in the case of high-risk hypopharyngeal cancers is a meaningful tool to identify patients who should benefit from aggressive postsurgical adjuvant therapy after surgery, including not only radiotherapy, but also chemotherapy.

Increased expression and altered intracellular distribution of adhesion/growth‐regulatory lectins galectins‐1 and ‐7 during tumour progression in hypopharyngeal and laryngeal squamous cell carcinomas

Aims: To examine the level of expression of the pleiotropic regulators galectins‐1 and ‐7 in relation to neoplastic progression of hypopharyngeal (HSCCs) and laryngeal (LSCCs) squamous cell carcinomas.

Impairment of Lysosome Pinocytic Vesicle Fusion in Rat-kidney Proximal Tubules After Treatment With Gentamicin At Low-doses

Gentamicin, an aminoglycoside antibiotic, is known to accumulate within the kidney cortex and to elicit nephrotoxic reactions due to the necrosis of proximal tubules. Female Sprague-Dawley rats, treated for 9 days with gentamicin at a low dose (10 mg/kg ip, once a day), were used to determine the fate of the antibiotic within the proximal tubular cells and its effects on the functional properties of the lysosomes. The analysis of the lysosomes by isopyknic equilibration in sucrose gradient (density 1.10-1.24 g/ml) revealed that gentamicin remains associated with these organelles (marker enzymes sulfatase B and cathepsin B) throughout the treatment duration. Gentamicin treatment markedly decreased the buoyant density of the lysosomes. As was shown by electron microscopic examination of the subcellular fractions collected from the sucrose gradient, the shift of the lysosomes toward lower densities was a result of overloading with undegraded phospholipids (myelin-like figures). The effect of gentamicin on the functional properties of the lysosomes was examined by using horseradish peroxidase (HRP) as a marker of endocytic activity and of the processing by tubular cells of exogenous proteins. Treatment with gentamicin did not significantly modify the intracortical accumulation of HRP, which was estimated to be 2.2% of the amount injected. HRP was shown by isopyknic equilibration to be mostly associated with the lysosomes. This was confirmed by electron microscopic examination of proximal tubular cells after cytochemical demonstration of HRP with diaminobenzidine and H2O2. In rats not exposed to gentamicin, more than half of the lysosomes contained HRP activity. In animals treated with gentamicin, one-third of the lysosomes that retained a normal appearance exhibited HRP activity. In contrast, lysosomes overloaded with phospholipids (identified by the presence of myelin-like figures) were very seldom labeled with HRP activity. Taken altogether, the present observations suggest that the alterations induced by gentamicin treatment impair their ability to fuse with incoming endocytic vesicles.

Magnetic resonance imaging of inflammation with a specific selectin-targeted contrast agent.

E‐selectin‐targeted contrast enhancement of blood vessels in inflamed tissues was investigated with a new contrast agent, Gd‐DTPA‐B(sLex)A, which was recently obtained by grafting a synthetic mimetic of sialyl‐Lewisx, an E‐selectin ligand, onto Gd‐DTPA. The pharmacokinetics, biodistribution, and potential to image inflammation by MRI of this E‐selectin‐targeted contrast agent were evaluated. The inhibition (by 15–34%) produced by Gd‐DTPA‐B(sLex)A on Sialyl Lex‐PAA‐biotin binding to E‐selectin confirmed the specific interaction of the new contrast agent with this adhesion molecule. Gd‐DTPA‐B(sLex)A was tested at a dose of 0.1 mmol/kg b.w. on mice and rats in a fulminant hepatitis model induced by the co‐administration of D‐galactosamine and E. coli lipopolysaccharide. A significant and prolonged contrast enhancement between blood vessels and liver parenchyma was obtained in pathological conditions, which attests to the specificity of the agent for E‐selectin. The prolonged vascular residence (48.9 min in hepatitis vs. 29.8 min in healthy animals), as evidenced by the pharmacokinetic characterization, suggests that Gd‐DTPA‐B(sLex)A interacts with the specific receptors expressed during inflammation. The biodistribution of the compound indicates its retention in inflamed liver by both specific mechanisms and nonspecific accumulation due to the necrotic lesions. The same mechanisms are invoked to account for its retention in the spleen. Magn Reson Med 53:800–807, 2005.

Kidney Tissue-repair After Nephrotoxic Injury - Biochemical and Morphological Characterization

Evidence has started to accumulate that tissue injury due to toxic compounds also elicits tissue repair reactions, especially in the kidney, but also possibly in other organs such as the liver. This article reviews current data concerning the repair of kidney cortex after nephrotoxin-induced tubular necrosis. The article mainly concentrates on the cellular proliferation involved in this phenomenon and discusses some aspects of its mechanism

Galectins as modulators of tumor progression in head and neck squamous cell carcinomas.

Head and neck squamous cell carcinomas (HNSCCs) remain a significant cause of morbidity worldwide. Biological therapies able to induce and/or upregulate antitumor immune responses could represent a complementary approach to conventional treatments for patients with HNSCC because, despite advances in surgery, radiotherapy, and chemotherapy, the overall survival rates for these patients have not changed over recent decades. Galectins are involved in the control of cell proliferation, cell death, and cell migration and in the modulation of various functions of the immune system. In this context, galectin‐1 is known to protect HNSCCs from the immune system. The present review details the involvement of galectins in HNSCC biology and suggests a number of approaches to reduce the levels of expression of galectin‐1 in HNSCCs, with the aim of improving the efficiency of HNSCC immunotherapy.

Magnetic Resonance Molecular Imaging of Vascular Cell Adhesion Molecule-1 Expression in Inflammatory Lesions Using a Peptide-Vectorized Paramagnetic Imaging Probe

The vascular cell adhesion molecule-1 (VCAM-1) has distinct roles in inflammatory cell recruitment to the damaged vessel wall. In the present work, a cyclic heptapeptide phage displayed library was screened in vitro during four rounds of biopanning. On the basis of Kd and IC50 values, a peptide (encoded as R832) was selected for in vitro and in vivo validation. After conjugation to Gd-DOTA, VCAM-1 imaging was assessed by MRI on a model of T cell mediated hepatitis, induced in mice by concanavalin A. On histological samples, the location of biotinylated R832 (R832-Bt) around liver veins in hepatitis resembles the pattern of MRI enhancement. Gd-DOTA-R832 was then assessed on ApoE(-/-) mice and produced an important signal enhancement of the aortic wall, while R832-Bt interacted with morphologic structures comparable to those marked by anti-VCAM-1 antibody. In conclusion, the in vitro and in vivo evaluation of peptide R832 suggests a specific interaction with the targeted biomolecule. Its conjugation to imaging reporters could assist the diagnosis of inflammatory diseases.

Tubular Injury and Regeneration in the Rat Kidney Following Acute Exposure to Gentamicin: A Time-Course Study

Aminoglycoside antibiotics act as nephrotoxic drugs, inducing a lysosomal phospholipidosis and necrotic lesions essentially in convoluted proximal tubules. Previous studies have demonstrated that tubular injury caused by these compounds elicits a process of renal tissue repair (tubular regeneration) involving an increase of cell turnover in tubular epithelium. The present study was performed in order to: (i) achieve further insight into the temporal relationship between aminoglycoside-induced phospholipidosis, tubular necrosis, and tubular regeneration; and (ii) approach the control of tubular regeneration after nephrotoxin-induced insult. To investigate the latter point, we examined by immunocytochemistry the intrarenal distribution of epidermal growth factor (EGF) during tubular regeneration. Five groups of female Sprague-Dawley rats (n = 5) were treated for 4 days with gentamicin i.p. at a daily dose of 50 mg/kg delivered in 2 injections per day. Sham-treated animals (n = 5) received an equivalent amount of vehicle (0.9% NaCl) according to the same protocol. Groups of treated rats, and controls, were terminated 16 h (day 1), 4 days, 7 days, 14 days, and 21 days after the end of gentamicin administration. One hour prior to necropsy, each animal was given an i.p. injection of 40 mg 5-bromo-2-deoxyuridine (BrdU) for the immunocytochemical demonstration of S-phase cells, using an anti-BrdU monoclonal antibody. Renal tissue was processed for light microscopy analysis, namely: a computer-aided morphometry of lysosomes in proximal tubular cells, a single-blind evaluation of gentamicin-induced tubular injury, the measurement of cell proliferation by immunocytochemical detection of BrdU-labeled nuclei, the demonstration of EGF-like immunoreactive material in renal tissue by using anti-rat EGF antiserum and immunogold-silver staining. As revealed by the morphometry of lysosomes in proximal tubular epithelium, the degree of gentamicin-induced phospholipidosis was maximum at day 1 (relative area occupied by lysosomes was increased 25-fold over mean control value) and declined thereafter. In contrast, tubular necrosis reached a peak 4 days after the end of drug administration. In proximal tubular epithelium, the stimulation of cell turnover associated with tubular regeneration showed a peak at day 7 (15-fold the mean control value). Tubular regeneration was also accompanied by mild interstitial hyperplasia. Three weeks after treatment with gentamicin, morphological evidence of drug-induced injury had disappeared due to the tissue repair process, except for the occasional presence of small hyperplastic foci in renal cortex interstitium. In both treated animals and controls, EGF immunoreactivity as revealed by immunocytochemical staining was associated with distal tubules (renal cortex and outer medulla).(ABSTRACT TRUNCATED AT 400 WORDS)

Potential amyloid plaque-specific peptides for the diagnosis of Alzheimer's disease.

Amyloid plaques (AP) represent one of the main molecular hallmarks of Alzheimers disease (AD). In order to develop new AP-specific contrast agents for AD molecular imaging, the phage display technology was used to identify peptides specific to amyloid-beta (A beta(42)). A random disulfide constrained heptapeptide phage display library was screened against A beta(42). After biopanning, 72 phage clones were isolated and their binding affinity to A beta(42) was evaluated by enzyme-linked immunosorbent assay (ELISA). The final library was enriched in two peptide sequences. The K(d) of candidate phage clones for binding to A beta(42) are in the picomolar range. The binding affinity for A beta(42) of two selected peptides was confirmed by ELISA, and the specific interaction with AP was validated by immunohistochemistry on brain sections. The preliminary MRI in vivo study, which was performed with a peptide functionalized contrast agent on AD transgenic mouse, showed encouraging results. To conclude, low molecular weight peptides presenting a specific affinity for A beta(42) were identified by phage display. As specific carriers, they have a real potential for molecular imaging of AD thanks to AP binding.