A peptide carrier for the delivery of biologically active proteins into mammalian cells.

The development of peptide drugs and therapeutic proteins is limited by the poor permeability and the selectivity of the cell membrane. There is a growing effort to circumvent these problems by designing strategies to deliver full-length proteins into a large number of cells. A series of small protein domains, termed protein transduction domains (PTDs), have been shown to cross biological membranes efficiently and independently of transporters or specific receptors, and to promote the delivery of peptides and proteins into cells. TAT protein from human immunodeficiency virus (HIV-1) is able to deliver biologically active proteins in vivo and has been shown to be of considerable interest for protein therapeutics. Similarly, the third alpha-helix of Antennapedia homeodomain, and VP22 protein from herpes simplex virus promote the delivery of covalently linked peptides or proteins into cells. However, these PTD vectors display a certain number of limitations in that they all require crosslinking to the target peptide or protein. Moreover, protein transduction using PTD-TAT fusion protein systems may require denaturation of the protein before delivery to increase the accessibility of the TAT-PTD domain. This requirement introduces an additional delay between the time of delivery and intracellular activation of the protein. In this report, we propose a new strategy for protein delivery based on a short amphipathic peptide carrier, Pep-1. This peptide carrier is able to efficiently deliver a variety of peptides and proteins into several cell lines in a fully biologically active form, without the need for prior chemical covalent coupling or denaturation steps. In addition, this peptide carrier presents several advantages for protein therapy, including stability in physiological buffer, lack of toxicity, and lack of sensitivity to serum. Pep-1 technology should be extremely useful for targeting specific protein-protein interactions in living cells and for screening novel therapeutic proteins.

A novel potent strategy for gene delivery using a single peptide vector as a carrier.

We have shown previously that a peptide, MPG, derived from the hydrophobic fusion peptide of HIV-1 gp41 and the hydrophilic nuclear localisation sequence of SV40 large T antigen, can be used as a powerful tool for the delivery of oligonucleotides into cultured cells. Now we extend the potential of MPG to the delivery of nucleic acids into cultured cells. In vitro, MPG interacts strongly with nucleic acids, most likely forming a peptide cage around them, which stabilises and protects them from degradation in cell culture media. MPG is non-cytotoxic, insensitive to serum and efficiently delivers plasmids into several different cell lines in only 1 h. Moreover, MPG enables complete expression of the gene products encoded by the plasmids it delivers into cultured cells. Finally, we have investigated the potential of MPG as an efficient delivery agent for gene therapy, by attempting to deliver antisense nucleic acids targeting an essential cell cycle gene. MPG efficiently delivered a plasmid expressing the full-length antisense cDNA of human cdc25C, which consequently successfully reduced cdc25C expression levels and promoted a block to cell cycle progression. Based on our results, we conclude that MPG is a potent delivery agent for the generalised delivery of nucleic acids as well as of oligonucleotides into cultured cells and believe that its contribution to the development of new gene therapy strategies could be of prime interest.

[Traumatic optic neuropathy: Report of 8 cases and review of the literature]. / Neuropathies optiques post-traumatiques : à propos de 8 cas et revue de la littérature.

Although underestimated, visual involvement is among the most frequent neurological complications of head trauma. There is no consensus in the management of these patients and visual recovery is uncertain. The goal of our study is to describe the clinical presentation and the clinical course of traumatic optic neuropathy in patients with head or maxillo-facial trauma. The clinical records of 8 patients, treated from November 2007 to March 2012, were reviewed in the department of ophthalmology (visual testing) of the university regional medical center in Lille. The most frequent cause of injury was traffic accidents. Unilateral optic neuropathy was observed in 6 cases, and bilateral in two cases, for a total of 10 eyes. Eight presented a significant visual loss<6/12. Improvement of visual acuity was achieved in 5 cases to 9/10 distance acuity without any medical or surgical treatment. One patient required surgical decompression, without improvement of visual acuity, and with persistent oculomotor disturbance and unreactive mydriasis. Traumatic optic neuropathy can cause profound visual acuity loss, especially if it is already significantly decreased on presentation.

Cell cycle dependent toxicity of an amphiphilic synthetic peptide.

The cytotoxic properties of an amphiphilic synthetic peptide are presented. Comparative analysis of proliferating, differentiated and confluent H9C2 adherent cells and L1210 cells in suspension shows a correlation between toxicity and cell stage (proliferating cells). Electrophysiological measurements on Xenopus laevis oocytes bathed in the peptide also demonstrated the induction of cationic currents, which is voltage and phosphate dependent. These results allow us to hypothesize that the observed toxicity is related to membrane hyperpolarization of proliferating cells at the G1/S cell cycle phase transition.

Production and characterization of site-directed antibodies against dermorphin and dermorphin-related peptides.

To detect and purify endogenous dermorphin-like molecules in mammalian tissues, an immunological approach was developed. Site-directed antibodies against synthetic dermorphin and related dermorphin peptides were produced. The immunogenic forms of dermorphin were selected to obtain antibodies recognizing different epitopes overlapping the whole dermorphin molecule. One of them specifically recognized the crucial "opioid message" (the N-terminal part of the molecule), which is required for a ligand to exert its full opioid activity. The validity of our immunological approach was analyzed by studying the dermorphin-related peptide distribution in Phyllomedusa sauvagei skin. The finding that tetrapeptide Y-A-G-F-OH was present in Phyllomedusa sauvagei extracts suggested that either the Tyr3-Pro6 peptidic bond may be relatively unstable or endogenous proteolytic enzymes present in Phyllomedusa skin may inactivate this peptidic bond.

Functional evaluation of normal and ischemic kidney by means of gadolinium-DOTA enhanced TurboFLASH MR imaging: a preliminary comparison with 99Tc-MAG3 dynamic scintigraphy.

The functional value of TurboFLASH MR imaging in the assessment of dynamic contrast enhancement and renal perfusion anomalies was evaluated in seven patients, who also underwent renal scintigraphy in baseline conditions. The basal renograms obtained from MAG-3 scintigraphy (mercapto acetyl triglycine, MAG3-S) and from Gd-DOTA-enhanced turboFLASH MRI were compared. After hydration, the protocol used consisted in breath-hold coronal turboFLASH acquisitions after IV bolus of Gd-DOTA (4 s every 20 s during 10 min) for MRI, and IV bolus of 370 MBq of 99mTc-MAG3 followed by 60 frames of 1 s and then 120 frames of 10 s for MAG3-S. Relative renal functions were computed for both methods by calculation of the integral of the uptake phase between the first and the second minute. Renograms exhibited 10 normal and 4 ischemic kidneys. There was a close correlation between the contrast enhancement of MRI and isotopic uptake in normal and ischemic kidneys. Global renograms of MRI correlated with MAG3-S (r = .82, p < .001) with similar curve shape and time to peak. Relative renal function of the right and left kidney were closely correlated in all patients (r = .98, p < .001), although there was a tendency for MR to overestimate MAG3-S evaluation in kidneys with severe basal dysfunction. Enhanced turboFLASH provides noninvasive assessment of renal perfusion in patients with renovascular disease. Accurate renograms are obtained with dynamic-enhanced MRI, but the relative renal function seems to be overestimated in low values of ischemic kidneys, and needs further comparative evaluation.

Pyrazinamide and pyrazinoic acid pharmacokinetics in patients with chronic renal failure.

Pharmacokinetics of pyrazinamide and its major metabolite, pyrazinoic acid, were assessed in 10 chronic uremic patients treated by maintenance hemodialysis in comparison with 10 normal subjects. All subjects ingested a single dose of 1 g of pyrazinamide, the patients receiving the drug immediately after the end of a dialysis session. Bioavailability of pyrazinamide was only slightly increased in patients, its dialysis extraction coefficient being 55.3%. In contrast, pyrazinoic acid has an elimination rate-dependent metabolism with a bioavailability markedly increased in patients and a dialysis extraction coefficient of 59.8%. These data may lead to recommendations of a reduction in the dosage of pyrazinamide in dialysis patients. However, administering the usual dosage of the drug at the end of each dialysis session seems preferable to the daily administration of a reduced dosage.

Changes in erythromycin pharmacokinetics induced by renal failure.

As erythromycin ototoxicity appears to be favored by renal insufficiency, its pharmacokinetics were assessed in chronic uremic patients treated by maintenance hemodialysis in comparison with normal subjects. Two groups of 8 patients each were studied, the first one on an interdialytic day, the second immediately after the end of an hemodialysis session. All subjects ingested a single dose of 1 gram of erythromycin ethylsuccinate. Times of peak serum concentration and biological half-lifes were similar in patients and in controls. Maximum serum concentrations and areas under the serum concentration time-curve were higher in patients than in controls whereas apparent oral clearances were lower in the former. The differences between the two groups of patients were not significant. These pharmacokinetic changes are suggestive of an enhanced bioavailability of erythromycin in chronic renal failure which might predispose uremics to the ototoxicity of the drug.

[Pheochromocytoma, first manifestation of Von Hippel-Lindau disease: a possibility to be considered]. / Phéochromocytome, première manifestation de la maladie de von Hippel-Lindau: une éventualité à connaître.

Von Hippel-Lindau (VHL) disorder is an autosomal dominant disease characterized by the almost constant development of hemangioblastomas in the central nervous system (cerebellum, spinal cord and retina). In addition, various types of tumors including renal cell carcinomas, pancreatic cysts and pheochromocytomas are frequently observed in VHL gene carriers. Linkage of the VHL locus to the RAF-1 oncogene on the short arm of chromosome 3 (3p25-26) has been recently reported. Pheochromocytoma is of particular interest because of the risk of inaugural malignant hypertensive crisis but especially because of a great degree of interfamily variability (from 0 to 92% of affected members in previously reported large kindreds). We have studied a French series of 25 pheochromocytoma (11 males, 14 females) in VHL affected patients. Twenty pheochromocytoma (80%) occurred in a familial context, whereas 5 (20%) were consistent with "apparent sporadic cases". The mean age at pheochromocytoma diagnosis was 27 years (5-55 years). Bilateral tumours have been documented in 13 cases (52%). The prevalence of pheochromocytoma revealing VHL was 14 out 25 (56%). In these cases, VHL diagnosis was considered up to 25 years later. In 6 cases (2 deceased) pheochromocytoma was the only manifestation of VHL. Thus, search for VHL must be systematic in the presence of pheochromocytoma, in the interest of the patients themselves and of potential at-risk family members (prevention of hypertensive crisis linked to latent tumours). Basic check-up (neurological and somatic examination, ophthalmoscopy, familial inquiry) may be completed with cerebral CT scan or MRI and abdominal ultrasonography followed, if positive or doubtful, by abdominal MRI or selective angiography.

[Vesical amyloidosis. Apropos of a case and a review of the literature]. / L'amylose vésicale. A propos d'une observation et revue de la littérature.

A case of primary localized amyloidosis of the bladder in a 60 years old male is described. The initial symptom was hematuria. Cystoscopy revealed a redish angioma-like lesion on the trigone, and the diagnosis was pointed out with biopsy, complementary data, and a close follow-up during two years. In the literature we found 47 cases of primary localized amyloidosis of the bladder and 10 others cases of amyloidosis of the bladder which were part of generalized amyloid disease. The interest of the Wright's special staining method is underlined specifying, on light microscopic study, the AA or AL type of the amyloid disease.

[Fibrillary glomerulopathies]. / Glomérulopathies fibrillaires.

Fibrillary glomerulonephritis is characterized by the presence, mainly in the mesangium, of fibrils demonstrated by electron microscopy of the renal parenchyma. There are several varieties of this disease, depending on the size and, above all, the composition of these fibrils. Thus, fibrillary glomerulonephritis in which the fibrils are made of the normal glomerular basement membrane components can be distinguished from a more frequent variety in which the fibrils are composed of circulating proteins deposited in glomeruli. For a long time, amyloid glomerular nephropathy has been the sole representative of the latter variety, but we now know that, beside renal amyloidosis, there is a non-amyloid fibrillary glomerulonephritis the fibrils of which are made of monoclonal or non-monoclonal immunoglobulins. If it were confirmed that the principal member of this variety, described as immunotactoid glomerulopathy, is frequently associated with monoclonal gammapathy, it would represent, together with amyloidosis and light chain deposition, another variety of glomerular lesions which may occur in monoclonal gammapathy.

[Components of the classical complement pathway in systemic lupus erythematosus]. / Les composants de la voie classique du complément au cours du lupus érythémateux disséminé.

Measurements of complement components in sera from patients with systemic lupus erythematosus (SLE) and some of their relatives indicated that decreased levels of CH50, C4 and C2 were mostly related to a genetic deficiency at one or both of the loci coding for C4, at least in those patients in whom decreased C4 levels were associated with normal C1 hemolytic activity. C4 deficiency is either isolated or associated with complement activation. In some patients with C4 deficiency, complement activation could only be demonstrated by measuring plasma level of the C3 cleavage fragment, C3a des Arg. Decreased concentration and/or hemolytic activity of C4 and C2 in SLE cannot be used to assess the activity of the disease.