Determination of endocrine disruptors and endogenic androgens and estrogens in rat serum by high-performance liquid chromatography-tandem mass spectrometry.

To simultaneously measure some targeted endocrine disruptors and several forms of sex hormones in rat serum, an accurate analytical procedure was developed. First, a comparison between a polymeric-based solid-phase extraction (SPE) and a micro-extraction by packed sorbent was performed to choose the optimal method to extract and concentrate the analytes: bisphenol A, atrazine, vinclozolin metabolite, testosterone, androstenedione, estrone, estradiol, estrone-sulfate and glucuronide and estradiol-sulfate and glucuronide. The analyses were then performed by high-performance liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) with electrospray ionisation in positive and negative modes. The protocol based on SPE was validated using the ICH/2005 guidelines. The validation demonstrated good performance in terms of linearity (R(2)>0.99), recovery (71-90%) and repeatability (relative standard deviation: 1-18%). The method was sensitive with LOQ comprised between 0.1 and 0.4 ng/ml for androgens and between 0.098 and 10.2 ng/ml for estrogens. The results obtained on the serum of rats exposed to the targeted endocrine disruptors showed the suitability of this analytical strategy.

[Image-guided radiotherapy: Overview of devices and practice in France in 2015]. / État des lieux des dispositifs et des pratiques de radiothérapie guidée par l'image en France en 2015.

Image-guided radiation therapy consists in acquiring in-room images to improve patient and mainly tumour set up accuracy. Many devices based on ionising or non-ionising radiations were designed in recent years. The use of such devices is of major importance in the management of patient radiotherapy courses. Those imaging sessions require to clearly define procedures in each radiotherapy department (image modality, acquisition frequency, corrective action, staff training and tasks). A quick review of the different existing image-guided radiation therapy devices is presented. In addition, the results of a French national survey about image-guided radiation therapy are presented: the survey is about both equipment and procedures. A total of 57 radiotherapy departments have participated, representing more than 160 treatment devices. About three linear accelerators out of four are equipped with an image-guiding device. The most common equipment is the CBCT system. Most centres have set up training sessions for the technicians to allow them to analyse online daily images. The management of in-room imaging dose is still under investigation, but many centres use an accounting scheme. While the devices are used to adjust the positioning of patients, in more than half of the centres, the practice had an impact on the choice of clinical and planning target volume margins. This survey led to an inventory in 2015, and could be renewed in some years.

[Image guided radiotherapy with the Cone Beam CT kV (Elekta): experience of the Léon Bérard centre]. / Radiothérapie guidée par l'image avec le Cone Beam CT kV (Elekta) : expérience du centre Léon-Bérard.

Image guide radiotherapy with the Cone Beam CT kV (CBCT-kV) developed by Elekta has been implemented at the centre Léon Bérard in November 2006. The treatment procedure is presented and detailed for prostate cancer IGRT and non small cell lung cancer (NSCLC) stereotactic radiotherapy (SRT). CBCT-kV is routinely used for SRT, selected paediatric cancers, all prostate carcinomas, primitive brain tumours and head and neck cancers that do not require nodes irradiation. Thirty-five to 40 patients are treated within a daily 11-hours period. The general procedure for 3D images acquisition and their analysis is described. The CBCT-kV permitted to identify about 10% of prostate cancer patients for whom a positioning with bone-based 2D images only would have led to an unacceptable dose distribution for at least one session. SRT is now used routinely for inoperable NSCLC. The easiness of implementing CBCT-kV imaging and its expected medical benefit should lead to a rapid diffusion of this technology that is also submitted to prospective and multicentric medico-economical evaluations.

Quantitative analysis of DHEA and androsterone in female urine: investigating the effects of menstrual cycle, oral contraception and training on exercise-induced changes in young women.

Dehydroepiandrosterone (DHEA) and its metabolite androsterone (A) are natural steroids secreted in high quantities in human body. To assess the influence of oral contraceptives, menstrual cycle phase, and also physical exercise (acute and chronic such as training) on these metabolites excretions, a collection of 28 female urine specimens was organized. A three-extraction-step method was developed, and the analyses were performed by gas chromatography-mass spectrometry using deuterated 19-noretiocholanolone as the internal standard. Sample hydration state was found to be of great importance for kinetic studies, as it directly influenced the concentrations. No influence of menstrual cycle and training was found for androsterone and DHEA. However, oral contraceptive intake lowered DHEA excretion in urine and A seems to be slightly affected by exercise.

[Image-guided radiotherapy]. / Radiothérapie guidée par l'image.

Recent advances in radiation oncology are based on improvement in dose distribution thanks to IMRT and improvement in target definition through new diagnostic imaging such as spectroscopic or functional MRI or PET. However, anatomic variations may occur during treatment decreasing the benefit of such optimization. Image-guided radiotherapy reduces geometric uncertainties occurring during treatment and therefore should reduce dose delivered to healthy tissues and enable dose escalation to enhance tumour control. However, IGRT experience is still limited, while a wide panel of IGRT modalities is available. A strong quality control is required for safety and proper evaluation of the clinical benefit of IGRT combined or not with IMRT.

[The stereotactic body radiation therapy: initiation and clinical program]. / Principe et mise en oeuvre de la radiothérapie en conditions stéréotaxiques extracrânienne.

We fully describe an innovative radiotherapy technique called Stereotactic Body Radiation Therapy (SBRT), and explain how this technique is commonly used for clinical purpose at the anticancer center Léon-Bérard (Lyon, France). In this technique, a non-invasive stereotactic body frame is used to locate the tumor site with a great precision. This frame is combined with a system, which enables to track the respiratory motions (Active Breathing Control (ABC) or diaphragmatic compression (DC)) in order to reduce the treatment margins for organ motion due to breathing. Thus, the volume of normal tissues that will be irradiated is considerably reduced. The dosimetry is realized with 3 CT exams performed in treatment conditions. The 3D patient "repositioning" is done with a volume CT acquisition (kV) combined with orthogonal images (kV and MV). The SBRT requires a system to limit the organ motions. Although the ABC seems to be more fastidious for patient, it would enable to use smaller margins than with DC technique. Nevertheless, the ABC is not compatible with volume CT acquisitions, which considerably improve the patient repositioning. In conclusion, the quality of repositioning and the high level of conformation enable to deliver high equivalent doses (>100 Gy) in hypofractionated mode, without increasing the treatment toxicity. The SBRT employs the last technologic innovations in radiotherapy and is therefore considered as a new efficient tool for solid tumors treatment.

[Acute respiratory tract infections due to a human metapneumovirus in children: descriptive study and comparison with respiratory syncytial virus infections]. / Infections respiratoires aiguës à métapneumovirus humain chez l'enfant: études descriptive et comparative avec le virus respiratoire syncytial.

BACKGROUND: A new paramyxovirus, the human metapneumovirus was recently isolated. We report the first French cases collected between 2000 and 2002. MATERIAL AND METHODS: Samples were obtained from nasopharyngeal aspirates from children hospitalised for acute respiratory tract infection in hospitals of Caen and Flers in Basse-Normandie. Human metapneumovirus was studied by polymerase chain reaction on negative samples for respiratory syncytial virus, influenza A and B virus, parainfluenza (1, 2 and 3) virus, adenovirus, coronavirus and rhinovirus. Comparison between metapneumovirus virus and respiratory syncytial virus infections was done after matching sex, age and infection month. RESULTS: Twenty-six human metapneumovirus infections were identified. A comparative study of a matched group of children infected by respiratory syncytial virus found no significative difference for hospitalisation motive, clinical criteria and treatment. CONCLUSION: The human metapneumovirus is responsible for typical acute bronchiolitis in children.

[Rhinovirus and acute respiratory infections in hospitalized children. Retrospective study 1998-2000]. / Rhinovirus et infections respiratoires aiguës de l'enfant hospitalisé. Etude rétrospective de 1998 à 2000.

OBJECTIVES: Rhinoviruses are the most common aetiological agents of colds, but the frequency and the severity of other locations of the infection are not well known. This study describes the clinical aspects and the severity of rhinovirus infections in hospitalised children. METHODS: Isolation in culture and a RT-PCR were performed for the detection of rhinovirus in nasal aspirates from hospitalised children from September 1998 to October 2000. A group of 211 children found to be positive for rhinovirus was studied. RESULTS: Rhinovirus-infected children suffered from the following clinical syndromes: 60 (28.4%) upper airway infections, 81 (38.4%) bronchiolitis, 25 (11.9%) pneumonias and 12 (4.7%) acute attacks of asthma. Clinical symptoms were wheezing (32%), ronchi (37%) and 29% of children presented with acute distress respiratory syndrome; 40% of the available chest X-Ray were abnormal. Eight children were hospitalised in the intensive care unit and two children died. Twenty-five children (10.9%) had a nosocomial infection; a dual infection was observed in 19 cases (9%) with the following viruses: RSV (3), influenza (2) parainfluenza (8), adenovirus (2), enterovirus (4); 19 (9%) children had a secondary bacterial infection. Rhinoviruses were detected in nasal aspirates in 112 cases (53%) according to the culture and in the rhinovirus culture-negative samples in 99 cases (47%) according to the RT-PCR assay. CONCLUSION: After eliminating cases of bacterial or viral dual infections, the clinical aspects of rhinovirus infections in children are the following: upper respiratory tract infections (25.6%), bronchiolitis ou bronchitis (25.6%), pneumonia (6.2%), acute attack of asthma (5.7%). The virological diagnosis according to culture is mainly improved by molecular techniques.

Impact of tissues heterogeneities on monitor units calculation and ICRU dose point: analysis of 30 cases of prostate cancer treated with 18-MV photons after three-dimensional planning.

PURPOSE: Analysis of dose specification of tissue heterogeneities. METHODS AND MATERIALS: Three-dimensional dose distribution analyses of 30 patients with localized prostate cancer were reviewed with and without tissue heterogeneity correction. The number of monitor units for each portal entrance (more than 300 different fields) was calculated and the impact of targeting and number of portal entrances was also integrated. RESULTS: The presence of gas in the rectum induces an overdosage of 0.6%, pubic bone induces an underdosage of -1.5%, and femoral heads are responsible for 6% underdosage. For the treatment as a whole, the underdosage is correlated with targeting techniques and weighting of each portal entrance (range, -0.5% to -3.2%). CONCLUSION: Dose calculation must take into account tissue heterogeneities and more precise guidelines for dose prescription are mandatory for further intercomparison.

A study of dose delivery in small segments.

PURPOSE: Use of conformal therapy techniques increases the number of beams used in order to obtain better conformation of the treated volume to the planning target volume. As the number of beams increases, the number of monitor units (MU) for each beam decreases. In this work we have studied, the influence of low MU on dose and homogeneity. METHODS AND MATERIALS: To study the field symmetry and flatness, films were irradiated. The "dose" for each field was always 60 MU; but it was divided into different segment sizes: 2 segments of 30 MU, 3 segments of 20 MU, and so on up to 12 segments of 5 MU. After being developed, films were scanned and analyzed using a densitometer. The measurements were carried out for three X-ray energies: 6 MV, 10 MV, and 18 MV. RESULTS: Each measurement was repeated twice for each energy, and the results were equal. The means of the symmetry and flatness values obtained for each energy are lower than the commonly accepted limits. CONCLUSION: The dose delivered by adding small segments is equivalent to the dose delivered by a conventional segment with our Philips Linacs SL15 and SL20.

Neuronal propagation of HSV1 from the oral mucosa to the eye.

PURPOSE: To identify possible neuronal pathways leading to herpetic ocular disease after primary oral infection in mice. METHODS: The SC16 strain of herpes simplex virus (HSV)-1 (10(6) plaque-forming units) was injected into the mucocutaneous border of the left upper lip. Animals were killed 2 to 10 days postinoculation (DPI). Spread of the virus in neural structures was studied by immunochemistry. RESULTS: HSV1 first replicated at the site of inoculation and then at the superior cervical ganglion (at 2 DPI). The trigeminal ganglion and the facial nerve fibers were infected by 4 DPI. Infection of the ciliary body and iris occurred at 6 DPI, together with several brain stem nuclei belonging to the autonomic or sensory pathways. Between 8 and 10 DPI, the neural infection gradually cleared up, except for the ipsilateral sympathetic ganglion, and ipsilateral keratitis appeared in some animals. CONCLUSIONS: The pattern of viral dissemination in this mouse model suggests that infection of iris and ciliary body results from transfer of virus in the superior cervical ganglion from sympathetic neurons innervating the lip to neighboring neurons innervating the anterior uvea. Later, zosteriform spread of virus from the trigeminal system may have contributed to the clinical and histologic findings.

Neuronal pathways for the propagation of herpes simplex virus type 1 from one retina to the other in a murine model.

Herpetic retinitis in humans is characterized by a high frequency of bilateral localization. In order to determine the possible mechanisms leading to bilateral retinitis, we studied the pathways by which herpes simplex virus type 1 (HSV-1) is propagated from one retina to the other after intravitreal injection in mice. HSV-1 strain SC16 (90 p.f.u.) was injected into the vitreous body of the left eye of BALB/c mice. Animals were sacrificed 1, 2, 3, 4 and 5 days post-inoculation (p.i.). Histological sections were studied by immunochemical staining. Primary retinitis in the inoculated eye (beginning 1 day p.i.) was followed by contralateral retinitis (in the uninoculated eye) starting at 3 days p.i. Infected neurons of central visual pathway nuclei (lateral geniculate nuclei, suprachiasmatic nuclei and pretectal areas) were detected at 4 days p.i. Iris and ciliary body infection was minimal early on, but became extensive thereafter and was accompanied by the infection of connected sympathetic and parasympathetic pathways. The pattern of virus propagation over time suggests that the onset of contralateral retinitis was mediated by local (non-synaptic) transfer in the optic chiasm from infected to uninfected axons of the optic nerves. Later, retinopetal transneuronal propagation of the virus from visual pathways may have contributed to increase the severity of contralateral retinitis.

Phage-displayed and soluble mouse scFv fragments neutralize rabies virus.

A phage-display technology was used to produce a single-chain Fv antibody fragment (scFv) from the 30AA5 hybridoma secreting anti-glycoprotein monoclonal antibody (MAb) that neutralizes rabies virus. ScFv was constructed and then cloned for expression as a protein fusion with the g3p minor coat protein of filamentous phage. The display of antibody fragment on the phage surface allows its selection by affinity using an enzyme-linked immunosorbent assay (ELISA); the selected scFv fragment was produced in a soluble form secreted by E. coli. The DNA fragment was sequenced to define the germline gene family and the amino-acid subgroups of the heavy (VH) and light (VL) chain variable regions. The specificity characteristics and neutralization capacity of phage-displayed and soluble scFv fragments were found to be identical to those of the parental 30AA5 MAb directed against antigenic site II of rabies glycoprotein. Phage-display technology allows the production of new antibody molecule forms able to neutralize the rabies virus specifically. The next step could be to engineer and produce multivalent and multispecific neutralizing antibody fragments. A cocktail of multispecific neutralizing antibodies could contain monovalent, bivalent or tetravalent scFv fragments, for passive immunoglobulin therapy.

Mapping of monoclonal antibody epitopes of the rabies virus P protein.

Thirty-six monoclonal antibodies (MAbs) specific for the rabies virus P phosphoprotein were obtained from mice immunized with recombinant P (PV strain) produced in E. coli. All MAbs reacted against the corresponding rabies virus protein by ELISA and by Western blot analysis and revealed the presence of cytoplasmic inclusions in rabies virus infected cells. The epitopes of seven MAbs were mapped by testing their reactivity with protein fragments expressed from deletion mutants in transfected cells. Western blotting, immunoprecipitation and immunofluorescence assays were performed. These MAbs recognized epitopes in different domains of the P protein: 60% were directed against a region lying between residues 83-172 suggesting a major antigenic determinant of the rabies virus P protein in this region. Most of the antigenic sites appeared to be composed of linear epitopes. These MAbs will be useful as tools to dissect structural and functional properties of the rabies virus P protein.

[Conformational radiotherapy with multi-leaf collimators: one year experience at the Leon-Berard Centre]. / Mise en oeuvre d'une radiothérapie conformationnelle avec collimateur multilames: un an d'expérience au centre Léon-Bérard.

Taking advantage of the renewal of a linear accelerator, the Radiation Therapy Department of the Centre Léon Bérard implemented, in collaboration with Philips Systèmes Médicaux, a conformal therapy set-up procedure using CT-scan for 3D treatment planning and a multileaf collimator that allows achievement of numerous irregular-shaped beams via the multileaf preparation system. The various elements of this equipment make possible well defined and structured procedures for treatment planning with different steps and essential tools used by this technique. We describe the means used and indicate future improvements that will lead to automation in order to provide good quality assurance, better security and substantial time saving. During the first year, 115 patients were treated with this new technique. They presented with central nervous system tumors (32 patients), lung cancer (29 patients), prostate cancer (20 patients), paranasal sinus tumors (14 patients) and tumors located in other sites (13 patients with soft sarcoma, hepato-bilary tumor, etc).

Immunodominant epitopes defined by a yeast-expressed library of random fragments of the rabies virus glycoprotein map outside major antigenic sites.

Nineteen yeast colonies secreting rabies virus glycoprotein (G) peptides immunoreactive with polyclonal anti-rabies virus sera were selected from a random expression library. The peptides, around 80 amino acids long, spanned amino acids 54-494 of the G protein. These peptides, together with two constructions including, respectively, immunodominant sites II and III, were analysed for their immunoreactivity with 40 anti-G protein monoclonal antibodies (MAbs) composed of 12 MAbs that reacted with SDS-treated protein in Western blot under reducing conditions (WB+) and 28 representative MAbs that did not react after denaturation (WB-). This last category represents 98% of anti-rabies virus G MAbs. None of the WB- MAbs bound peptides. Of the 12 WB+ MAbs, one bound two peptides situated before the transmembrane domain of the protein and six bound peptides overlapping a region situated between amino acids 223 and 276. These six MAbs define a new antigenic region that would be considered 'immunodominant' if the peptide strategy had been used to study the antigenicity of the protein; however, this region is only recognized by about 1% of our MAbs. Three of these WB+ MAbs had significant neutralizing activity; two were used for the selection of antigenic mutants (MAR mutants). Some mutants had a substitution within the region delimited by the peptides, confirming the pertinence of both the peptide and escape mutant approaches. However, a few mutants had a substitution outside the peptide-delimited region, suggesting that remote mutation(s) could affect epitope accessibility in the native protein.

Monoclonal antibodies which recognize the acidic configuration of the rabies glycoprotein at the surface of the virion can be neutralizing.

Around 15% of our anti-glycoprotein monoclonal antibodies (MAbs) failed to neutralize the infectivity of the rabies virus during a 1-hr incubation at room temperature. In previous studies, we have demonstrated that it is possible to induce a massive conformational change of the glycoprotein population by incubating the virus at acidic pH. The conformational change is reversible and consequently viral infectivity is not affected by transient exposure at acidic pH. The proportion of glycoproteins in acidic or neutral configuration depends on the pH which means that even at neutral pH some glycoproteins transiently adopt the acidic configuration and vice versa. Here we report that some of our nonneutralizing MAbs recognize the acidic form of the glycoprotein at the virion surface. After incubation of the virus at pH 6.4, most glycoproteins are in the acidic configuration. Further 1-hr incubation with these MAbs at the same pH resulted in more immunoglobulins being attached to the virus and consequently neutralization was induced. It was also possible to induce neutralization with the same MAbs by incubation at neutral pH for a longer period or at a higher temperature. Mutants resistant to neutralization by these MAbs could be selected. Mutations confering resistance to neutralization were not localized in previously described antigenic sites and did not modify these sites at distance. They had no effect on the pathogenic power of the virus. Either they are situated in the epitope or they modify the epitope, so that it is no longer recognized by the antibody on the acidic configuration of the protein. Alternatively, these mutations may stabilize the protein in its neutral configuration. In addition, these experiments confirm our previous finding that neutralization requires the fixation of a large number of immunoglobulins on the virus, irrespective of the region of the protein recognized by the antibody.

Vaccination against rabies: construction and characterization of SAG2, a double avirulent derivative of SADBern.

A double avirulent mutant was isolated from the SADBern strain of rabies virus by two successive selection steps using neutralizing anti-glycoprotein monoclonal antibodies. Both mutations affect the triplet coding for amino acid 333 of the glycoprotein. The resulting virus, called SAG2, has a glutamate coded by GAA in position 333 instead of an arginine. This new codon differs by two nucleotides from all the arginine triplets. SAG2 is avirulent in adult mice by intracerebral and intramuscular routes and it protects mice against a challenge by the CVS strain. This double mutant is still avirulent after three successive passages in suckling mouse brain or after ten successive cycles of multiplication in cell culture. Because it is protective and genetically stable, SAG2 represents an improvement on SAG1 which is already used for oral vaccination of foxes in Switzerland and France. It could also be a candidate for oral vaccination of dogs against rabies.

SAG-2 oral rabies vaccine.

The live modified rabies virus vaccine strain SAG-2 was selected from SADBerne in a two step process employing anti-rabies glycoprotein monoclonal antibodies. The first two nucleotides coding for the amino acid in position 333 of the rabies glycoprotein are mutated. Arginine at position 333, which is associated with rabies pathogenicity, was substituted first by lysine and then by glutamic acid. The two nucleotide differences at position 333 in SAG-2 to any of six possible arginine triplets translated into excellent genetic stability and apathogenicity for adult mice, foxes, cats and dogs. The vaccination of foxes and dogs by the oral route provided protection against a lethal challenge with rabies virus.