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.     

Mechanisms of HIV-1 evasion to the antiviral activity of chemokine CXCL12 indicate potential links with pathogenesis

HIV-1 infects CD4 T lymphocytes (CD4TL) through binding the chemokine receptors CCR5 or CXCR4. CXCR4-using viruses are considered more pathogenic, linked to accelerated depletion of CD4TL and progression to AIDS. However, counterexamples to this paradigm are common, suggesting heterogeneity in the virulence of CXCR4-using viruses. Here, we investigated the role of the CXCR4 chemokine CXCL12 as a driving force behind virus virulence. In vitro, CXCL12 prevents HIV-1 from binding CXCR4 and entering CD4TL, but its role in HIV-1 transmission and propagation remains speculative. Through analysis of thirty envelope glycoproteins (Envs) from patients at different stages of infection, mostly treatment-naïve, we first interrogated whether sensitivity of viruses to inhibition by CXCL12 varies over time in infection. Results show that Envs resistant (RES) to CXCL12 are frequent in patients experiencing low CD4TL levels, most often late in infection, only rarely at the time of primary infection. Sensitivity assays to soluble CD4 or broadly neutralizing antibodies further showed that RES Envs adopt a more closed conformation with distinct antigenicity, compared to CXCL12-sensitive (SENS) Envs. At the level of the host cell, our results suggest that resistance is not due to improved fusion or binding to CD4, but owes to viruses using particular CXCR4 molecules weakly accessible to CXCL12. We finally asked whether the low CD4TL levels in patients are related to increased pathogenicity of RES viruses. Resistance actually provides viruses with an enhanced capacity to enter naive CD4TL when surrounded by CXCL12, which mirrors their situation in lymphoid organs, and to deplete bystander activated effector memory cells. Therefore, RES viruses seem more likely to deregulate CD4TL homeostasis. This work improves our understanding of the pathophysiology and the transmission of HIV-1 and suggests that RES viruses’ receptors could represent new therapeutic targets to help prevent CD4TL depletion in HIV+ patients on cART.     

Porcine D-amino acid oxidase: production of the biologically active enzyme in Escherichia coli

DNA molecules coding either for mature porcine D-amino acid oxidase or for truncated forms of the enzyme have been obtained by stepwise addition of synthetic oligonucleotides to a partial cDNA. Under the control of the lambda PL thermoregulatable promoter, these DNAs were respectively expressed in Escherichia coli as 36, 28 and 25 kilodalton polypeptides, specifically recognised by antibodies raised against the natural enzyme. None of the truncated proteins were biologically active whereas the mature recombinant species was able to hydrolyze D-alanine in vitro as efficiently as the natural product.     

Direct evidence for the clustered nature of complement receptors type 1 on the erythrocyte membrane

C receptors 1 (CR1) of human E are involved in the transport of C3b-coated immune complexes (IC) in the circulation. Many studies have suggested that the binding of IC to E is multivalent. This would require CR1 to be clustered on the cell membrane, but no direct evidence for such clustering is available. We studied the distribution of CR1 on human E by immunofluorescence and shadow-casting immuno-electron microscopy techniques with the use of a monoclonal anti-CR1 antibody followed by FITC- or gold-conjugated second antibodies, respectively. By immunofluorescence, CR1 appeared as small dots (clusters) on fixed and unfixed E prepared either at 4 degrees C or at 37 degrees C. In the same donor, the number of clusters varied extensively from cell to cell (e.g., 1 to 43 clusters/E for a donor with 520 CR1/cell), but the mean number of clusters per cell correlated significantly with the mean number of CR1/cell. These images contrasted with those obtained for Rhesus D (RhD) Ag used as controls (RhD Ag are known to be evenly distributed): only a faint uniform fluorescence was seen despite the presence of 10,000 antigenic sites. As determined by immunocytochemical method, more than 65% of the total gold particles were organized in clusters (2 to 15 gold particles/cluster) whether cells were prefixed or not. Quantitative determinations suggested that each gold particle corresponded to one CR1. The fraction of gold particles grouped into clusters of three or more receptors, the mean size of the clusters, and the maximal size of clusters correlated with the mean number of CR1 per cell. By contrast, RhD Ag were distributed homogeneously (less than 2% gold particles in clusters). These data are the first to demonstrate the preclustered nature of CR1 on E. Such distribution could explain the high binding efficiency of C3b-coated IC to E despite the low number of CR1 per cell.     

Resistance to low temperature photoinhibition is not associated with isolated thylakoid membranes of winter rye

In vivo measurements of chlorophyll a fluorescence indicate that cold-hardened winter rye (Secale cereale L. cv Musketeer) develops a resistance to low temperature-induced photoinhibition compared with nonhardened rye. After 7.2 hours at 5°C and 1550 micromoles per square meter per second, the ratio of variable fluorescence/maximum fluorescence was depressed by only 23% in cold-hardened rye compared with 46% in nonhardened rye. We have tested the hypothesis that the principal site of this resistance to photoinhibition resides at the level of rye thylakoid membranes. Thylakoids were isolated from cold-hardened and nonhardened rye and exposed to high irradiance (1000-2600 micromoles per square meter per second) at either 5 or 20°C. The photoinhibitory response measured by room temperature fluorescence induction, photosystem II electron transport, photoacoustic spectroscopy, or [¹⁴C]atrazine binding indicates that the differential resistance to low temperature-induced photoinhibition in vivo is not observed in isolated thylakoids. Similar results were obtained whether isolated rye thylakoids were photoinhibited or thylakoids were isolated from rye leaves preexposed to a photoinhibitory treatment. Thus, we conclude that increased resistance to low temperature-induced photoinhibition is not a property of thylakoid membranes but is associated with a higher level of cellular organization.     

The endosomal compartment of rat hepatocytes. Its characterization in the course of [125I]insulin internalization

When freshly isolated hepatocytes are incubated with [125I]insulin in the presence of the microtubule-disrupting agent colchicine, internalization of the labelled hormone is not significantly altered. However, the drug limits the endocytosis of the labelled material to a peripheral band of cytoplasm extending 1 micron beyond the plasma membrane. Both in the presence and absence of colchicine, internalized [125I]insulin preferentially associates with clear vesicles (endosomes) and lysosome-like structures, but the relative amount of labelled material associated with clear vesicles is higher in the presence of the drug than in its absence. An inverse pattern is observed for the lysosome-like structures. As demonstrated by cytochemical methods, clear vesicles do not contain the lysosomal enzyme aryl sulfatase. Moreover, colchicine induces an increase of the clear vesicle diameter without affecting their frequency, while it perturbs multivesicular bodies and dense bodies in an opposite way by increasing their frequency without affecting their size. By reducing and/or delaying the fusion between internalized endocytotic vesicles and lysosomes, colchicine allows better characterization of the endosomal compartment of isolated rat hepatocytes and allows it to be distinguished from other compartments, such as multivesicular bodies and the Golgi apparatus.     

Carbonic anhydrase in the rat salivary glands: distribution and ultrastructural localization

Rat salivary glands were studied by Hanson's method to specify the ultrastructural localization of carbonic anhydrase (CA). Two different procedures were used: 1) The embedding of the tissues in water-soluble resins, followed by the incubation of the resin sections on the medium. 2) The embedding in epon-araldite of previously incubated frozen sections. Light and electron microscopy were used to observe the distribution and the ultrastructural localization of the cobalt precipitate. In parotid and mandibular glands, CA was localized in the secretion granules and the hyaloplasma of the secretory endpieces. The enzyme was also detected on the basal and lateral membranes of the striated duct cells in the three glands. In the convoluted granular duct cells of the mandibular gland CA was found in the hyaloplasma only. In the sublingual gland, CA was localized in the hyaloplasma of the serous crescents and no activity was detected in the mucous tubules. As regards the localization of the enzyme in the granules of the secretory endpieces of parotid and mandibular glands, it appears that CA has to be considered as a secretory product of these cells; this localization is consistent with the presence of the enzyme in rat saliva.     

Photoacoustic spectroscopy of Anacystis nidulans. I. Effect of sample thickness on the photoacoustic signal

A photoacoustic spectroscopy study of the cyanobacteria Anacystis nidulans has been undertaken. It is demonstrated, by using a filter deposition technique, that the photoacoustic signal intensity becomes progressively saturated as the thickness of the algal layer is increased. This saturation effect originates mostly from the limited optical penetration of the sample and distorts the photoacoustic spectrum from its true shape. A theoretical model is proposed to explain these results, and practical means to obviate the limitations of this spectroscopic technique are suggested.     

Photoacoustic spectroscopy of Anacystis nidulans. II. Characterization of pigment holochroms and thermal deactivation spectrum

The photoacoustic spectrum of Anacystis nidulans recorded at room temperature is qualitatively similar to low-temperature absorption or fluorescence excitation spectra. The bands of pigment holochroms are well resolved compared to room-temperature absorption spectra. The thermal deactivation spectrum obtained by extrapolating acoustic data for an infinitely thin sample indicates that the photosynthetic efficiency decreases from phycocyanin to chlorophyll a and carotenoids.