Rabies Virus Envelope Glycoprotein Targets Lentiviral Vectors to the Axonal Retrograde Pathway in Motor Neurons

Rabies pseudotyped lentiviral vectors have great potential in gene therapy, not least because of their ability to transduce neurons following their distal axonal application. However, very little is known about the molecular processes that underlie their retrograde transport and cell transduction. Using multiple labeling techniques and confocal microscopy, we demonstrated that pseudotyping with rabies virus envelope glycoprotein (RV-G) enabled the axonal retrograde transport of two distinct subtypes of lentiviral vector in motor neuron cultures. Analysis of this process revealed that these vectors trafficked through Rab5-positive endosomes and accumulated within a non-acidic Rab7 compartment. RV-G pseudotyped vectors were co-transported with both the tetanus neurotoxin-binding fragment and the membrane proteins thought to mediate rabies virus endocytosis (neural cell adhesion molecule, nicotinic acetylcholine receptor, and p75 neurotrophin receptor), thus demonstrating that pseudotyping with RV-G targets lentiviral vectors for transport along the same pathway exploited by several toxins and viruses. Using motor neurons cultured in compartmentalized chambers, we demonstrated that axonal retrograde transport of these vectors was rapid and efficient; however, it was not able to transduce the targeted neurons efficiently, suggesting that impairment in processes occurring after arrival of the viral vector in the soma is responsible for the low transduction efficiency seen in vivo, which suggests a novel area for improvement of gene therapy vectors.     

Radioprotection après injection thérapeutique de [131I]-mIBG : données préalables à l’ouverture du protocole MIITOP-0607

IntroductionThe MIITOP-0607 protocol, studying the efficiency of administration of topotecan and myelosupressive [¹³¹I]-mIBG therapy in children affected by neuroblastoma, needed to assess irradiation risks on staff and family of children to obtain the agreement of the Autorité de sûreté nucléaire (ASN). Our aim was to quantify irradiation of the staff during preparation of the mIBG and to assay the irradiation and contamination of the accompanying persons.Patient and methodsRadiation exposure of the staff was measured during the preparation, transport and administration of the first treatment. Salivary and urinary excretions were monitored well as the atmospheric radioactivity. Radiation exposure and contamination of the accompanying persons were also measured.ResultsFinger dose of 3 mSv and whole body dose of 50 μSv were estimated for preparation of an 11.1 GBq syringe. Irradiation from urinary activity can be as low as 100 μSv if a dedicated device is used. Salivary excretion decreased rapidly during the first 24 hours. Atmospheric contamination always remained below 25 Bq m^?3. Total irradiation of the accompanying persons is about 2.35 mSv for the two consecutive injections (9,3 and 11,1 GBq). Internal contamination occurred only once and corresponded to a 27 μSv whole body irradiation and 670 μSv thyroid irradiation.ConclusionThis study shows the safety of [¹³¹I]-mIBG treatments using high activities. The involved dose is not negligible but seems to be acceptable in the specific paediatric oncology context if radioprotection instructions are met and if optimization of protocols is performed.     

Internalization of the human insulin receptor. The insulin-independent pathway.

Internalization of the human insulin receptor requires the activation by insulin of the intrinsic kinase of the receptor. However, even in the absence of kinase activation, insulin receptors slowly enter the cells. In the present study, we addressed the question of this insulin-independent pathway of internalization. To that end, we traced insulin receptor internalization with a monoclonal antibody (mAb 83-14) directed against the alpha-subunit of the human insulin receptor. Internalization of this antibody was followed in Chinese hamster ovary (CHO) cells transfected with either normal (CHO.HIRC2) or kinase-deficient (CHO.A1018) human insulin receptors. The internalization rate of 125I-mAb 83-14 was comparable in CHO cells expressing kinase-active or kinase-inactive receptors and was similar to that observed for 125I-insulin in CHO.A1018 cells. Moreover, in CHO.HIRC2 cells, the internalization of 125I-mAb 83-14 was identical with that of its 125I-Fab fragments. Thus, mAb 83-14 represents an appropriate tool to study the constitutive internalization of the insulin receptor. Internalization of insulin receptors tagged with 125I-mAb 83-14 was unaffected by cytochalasin B, which excluded a macropinocytotic process. By contrast, internalization was sensitive to hypertonia, which abrogates clathrin-coated pits-mediated endocytosis. The implication of clathrin-coated pits in this internalization process was directly demonstrated by quantitative electron microscopic autoradiography, which showed that 125I-mAb 83-14 present on the nonvillous domain of the cell surface preferentially associate with clathrin-coated pits at all time points.     

Étude du phénomène de l’induction de facteurs bactériolytiques chez les lépidoptères: Inhibition de la production du lysozyme chez les larves deGalleria mellonella [Lep.: Pyralidae] par l’Actinomycine D et la Cycloheximide

Résumé L’introduction de corps étrangers dans les larves de lépidoptères entra?ne une augmentation parfois considérable du taux de lysozyme dans l’hémolymphe. L’efficacité inductrice de diverses substances varie dans de larges mesures. L’action de l’Actinomycine D et de la Cycloheximide sur la production du lysozyme induit a été recherchée chez des larves deGalleria mellonella L. recevant un stimulus peu spécifique (chlorure de sodium) ou plus spécifique (Micrococcus luteus vivants). L’action de ces inhibiteurs sur la quantité de lysozyme présente dans l’hémolymphe 24 h après induction, indique que les variations du taux de l’enzyme sont sous le contr?le d’un mécanisme unique indépendant de la spécificité des stimuli inducteurs. L’augmentation du taux de lysozyme chez les larves deG. mellonella induites, fait suite à une synthèse protéique et non à la libération d’enzyme préformé.

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Summary Foreign bodies introduced into lepidopteran larvae induce an increase, often considerable, in amount of lysozyme in the hemolymph. The efficiency of various substances to elicit the release of lysozyme in the larvae varies to a great extent. The effect of Actinomycin D and Cycloheximide introduced in theGalleria mellonella L. larvae stimulated to produce lysozyme by injection of saline or liveMicrococcus luteus was studied. The action of these metabolic inhibitors on the quantity of lysozyme in the hemolymph, 24 h after injection of the inducers, showed that the variations of the amount of the enzyme are under the control of only one mechanism which is not affected by the nature of the inducers. The increase in lysozyme amount of the hemolymph of the inducedG. mellonella larvae is due to the protein synthesis; it does not result from the release of a preformed enzyme as it was demonstrated in other animal species.

     

Expression of Cytoplasmic Antigens Linked to Orthokeratosis During the Development of Parakeratosis in Newborn Mouse Tail Epidermis

Abstract. An unusual example of two different types of epidermal differentiation occurs side by side in adult mouse tail. The neonate shows only orthokeratotic differentiation, but develops parakeratotic scales in precise patterns over the first two weeks of life, retaining orthokeratotic differentiation at the necks of hair follicles. Certain human IgG antibodies from patients receiving bone marrow transplants bind only to cytoplasmic components of orthokeratotic epithelium. As markers of orthokeratotic differentiation in indirect immunofluorescent studies, these antibodies allowed the timing and location of the change in epidermal differentiation. A specific loss of 'orthokeratotic' antigens in the natural switch from orthokeratosis to parakeratosis was demonstrated. The sequence of the orthokeratotic antigens disappearance suggested that the switch to parakeratotic differentation occurred at a supra-basal level.     

Discovery and structure-guided drug design of inhibitors of 11β-hydroxysteroid-dehydrogenase type I based on a spiro-carboxamide scaffold

Spiro-carboxamides were identified as inhibitors of 11β-hydroxysteroid-dehydrogenase type 1 by high-throughput screening. Structure-based drug design was used to optimise the initial hit yielding a sub-nanomolar IC₅₀ inhibitor (0.5 nM) on human 11β-HSD1 with a high binding efficiency index (BEI of 32.7) which was selective against human 11β-HSD2 (selectivity ratio > 200000).     

Pleiotrophin cellular localization in nerve regeneration after peripheral nerve injury.

Pleiotrophin (PTN) is a member of the family of heparin-binding growth factors that displays mitogenic activities and promotes neurite outgrowth in vitro. In vivo, PTN is widely expressed along pathways of developing axons during the late embryonic and early postnatal period. Although the level of PTN gene expression is very low during adulthood, activation of the gene may occur during recovery from injury and seems to play an important role in tissue regeneration processes. In this study, we investigated whether PTN was involved in the regenerative process of injured peripheral nerves. To refer localization of the fluorescent markers to myelinated axons, we developed a specific computer tool for colocalization of fluorescence images with phase contrast images. Immunohistochemical analysis showed PTN in different types of nonneural cells in distal nerve segments, including Schwann cells, macrophages, and endothelial cells, but not in axons. Schwann cells exhibited PTN immunoreactivity as early as 2 days after injury, whereas PTN-positive macrophages were found 1 week later. Strong PTN immunoreactivity was noted in endothelial cells at all time points. These findings support the idea that PTN participates in the adaptive response to peripheral nerve injury. A better understanding of its contribution may suggest new strategies for enhancing peripheral nerve regeneration.