[How a self-managed team can improve cohesion and communication during the patient's journey in a radiotherapy department: Experience of centre Jean-Bernard (Le Mans, France)]. / Comment une équipe autonome peut améliorer la cohésion et la communication autour de la prise en charge du patient en radiothérapie : expérience du centre Jean-Bernard.

Efficient communication between professionals is of upmost importance in improving treatment safety in a radiotherapy department, and is also necessary to enhance the quality of work life. Taking as example the organizations in industry, a self-managed team centred on patients with head and neck cancers treated with radiation has been implemented in 2018 in centre Jean-Bernard (Le Mans, France). After over a year's experience, a real benefice has been found and validates the plan to extend this model to other departments.

Immune tolerance induction in patients with IgA anaphylactoid reactions following long-term intravenous IgG treatment.

To date, there is very little information regarding the pathomechanism of IgA anaphylactoid reactions and the management of affected patients. Five adult patients with common variable immunodeficiency (CVID) and a history of anaphylactic reactions due to the administration of immunoglobulin preparations were studied. The activity of anti-IgA was determined by the gel agglutination technique using IgA-coated beads. Antibodies to IgA were detected in the serum of all five patients. Initially, IgA 'depleted' intravenous (i.v.) IgG preparations were infused carefully into the patients until the activity of anti-IgA was decreased significantly or became undetectable. Subsequently, unselected i.v. IgG preparations were infused, and the activity of anti-IgA was abolished in all cases. Intravenous IgG long-term administration results in tolerance induction in patients with IgA anaphylactoid reactions. This tolerance appears to be related to antibody blockage in the circulation and an inhibition of antibody production. Most importantly, IgA appears to play an important role in the treatment of CVID. Patients with IgA anaphylactoid reactions can be treated safely with IgA containing i.v. IgG preparations following tolerance induction.

A rapid gel agglutination test for the determination of fetomaternal haemorrhage.

Determination of fetomaternal haemorrhage (FMH) remains an area of difficulty. In most cases, prophylactic Rh immunoglobulin is usually administered to affected women without testing for foetal red blood cells (RBC). Here, we describe a new particle gel immunoassay (PaGIA) for the determination FMH (FMH-PaGIA). Superparamagnetic particles were coated with monoclonal anti-D and mixed with ethylenediaminetetraacetic acid-anticoagulated blood samples from D-negative pregnant women. The particles were isolated using a magnetic particle concentrator and then placed into the reaction chamber of a gel card. Agglutinated particles on top or dispersed through the gel matrix indicated the presence of D-positive cells. After the test was adapted to detect >or=0.3% D-positive RBC, randomly selected postpartum samples from 208 women were analysed in parallel with the Kleihauer-Betke test (KBT). In addition, all discrepancies were further analysed by flow cytometry. A total of 203 of the 208 postpartum samples were negative in both tests. One sample reacted positive with both assays. Two samples were strongly positive in the new FMH-PaGIA, but negative in the KBT. A serological re-examination revealed that both women were D positive. The KBT gave a false-positive result in two cases because of hereditary persistence of haemoglobin F. The new test is specific, easy to perform and can be done at any time in all laboratories.

Application of the particle gel agglutination system as a new check gel assay for PCR products.

In this study, we describe a simple and rapid agglutination test for the detection of PCR products prior to the application of specific hybridization by sequence-specific oligonucleotide typing. This test is based on the particle gel agglutination immunoassay, incorporating biotinylated primers and streptavidin particles. Visually detectable agglutination was only observed in samples which contained the specific amplification products. The results obtained by the new test were in accordance with those obtained by standard gel electrophoresis in all cases that have been tested to date.

A novel antigen-specific capture assay for the detection of platelet antibodies and HPA-1a phenotyping.

BACKGROUND AND OBJECTIVES: The antigen-specific assays currently used for the laboratory investigation of platelet antibodies and antigens are technically complex and cannot be used in most routine laboratories. Here, we describe a simple antigen-specific capture assay (ASCA) for the detection of serum platelet antibodies and for human platelet antigen-1a (HPA-1a) phenotyping. MATERIALS AND METHODS: For the detection of platelet antibodies, platelets from healthy blood donors were incubated with biotinylated monoclonal antibodies to platelet glycoprotein complexes (GP), then solubilized and mixed with superparamagnetic streptavidin particles. Serum samples from patients with autoimmune thrombocytopenia (n = 39), from patients with platelet alloantibodies (6 HPA-1a, 1 HPA-2b, 1 HPA-3a, 6 HPA-5b), and from healthy blood donors (n = 70), were tested. All serum samples from the patients were investigated in parallel by the indirect monoclonal antibody-specific immobilization of platelet antigen assay (MAIPA). For HPA-1a phenotyping, superparamagnetic particles were coated with a monoclonal antibody to HPA-1a and mixed with diluted whole blood samples from healthy blood donors (n = 139), who had previously been genotyped for platelet alloantigens. Results The indirect MAIPA detected autoantibodies in 18%, and the direct MAIPA in 50% of patients tested. In contrast, the new ASCA demonstrated positive results in 77% of patients. All tested alloantibodies reacted positive by the ASCA, and all serum samples from healthy blood donors were negative. The results of HPA-1a phenotyping were in concordance with those of genotyping in all cases. CONCLUSION: In our opinion, the ASCA is easy to perform and much more sensitive than the currently available antigen-specific assays for the detection of platelet antibodies.

Human platelet antigen genotyping by using sequence-specific primers and the particle gel agglutination assay.

BACKGROUND AND OBJECTIVES: Polymerase chain reaction using sequence-specific primers (PCR-SSP) is currently the most widely used technique for human platelet antigen (HPA) genotyping. Here, we describe a novel particle gel-agglutination technique for simplified visualization of the amplified products. MATERIALS AND METHODS: Biotinylated primers were used to amplify HPA-1, -2, -3, -4, -5, -6, and -15, and the PCR products were incubated with streptavidin particles. Fluorescein isothiocyanate (FITC)-labelled primers [amplifying a fragment of the human growth hormone (HGH) gene] and anti-FITC-coated particles were used as internal controls. Agglutination of the particles in or on top of the gel indicated specific amplification. A total of 100 samples from blood donors was tested by using this new technique and a standard PCR-SSP protocol. RESULTS: The use of biotinylated sequence-specific primers resulted in PCR products that agglutinated streptavidin particles, and the FITC-labelled HGH primers led to agglutination of anti-FITC-coated particles. Negative reactions were clearly distinguishable from positive reactions. The results of the particle gel agglutination method were in concordance with those of the electrophoretic visualization in all cases tested. CONCLUSIONS: The new particle agglutination method is reliable and easy to use.

Prospective assessment of a new polymerase chain reaction target (STEVOR) for imported Plasmodium falciparum malaria.

The diagnostic value of a polymerase chain reaction (PCR)-based method for amplifying a new target of repeated genes (STEVOR) in Plasmodium falciparum was prospectively assessed on samples from 210 febrile patients returning from areas endemic for malaria. This method is capable of detecting 0.01 parasites in one microliter of blood. Plasmodium falciparum STEVOR PCR confirmed the results of the thin- and thick-film direct examination method but identified Plasmodium falciparum in four patients in whom direct examination was inconclusive at the species level. Moreover, PCR was positive in two patients with a negative direct examination. Thus, Plasmodium falciparum STEVOR PCR had 100% sensitivity and specificity and could be used in selected parasitology laboratories when expert advice is required.

Platination of the (T2G4)4 telomeric sequence: a structural and cross-linking study.

The telomeric sequence (T(2)G(4))(4) was platinated in aqueous solutions containing 50 mM LiClO(4), NaClO(4), or KClO(4). The identification of the guanines which reacted with [Pt(NH(3))(3)(H(2)O)](2+) revealed that the same type of folding exists in the presence of the three cations and that the latter determine the relative stabilities of the G-quadruplex structures in the order K(+) > Na(+) >> Li(+). The tri-ammine complex yielded ca. 40--90% of adducts, mono- and poly-platinated, bound to 4 guanines out of the 16 guanines in the sequence, in the decreasing amounts G9 > G15 >> G3 > G21. The formation of these adducts was interpreted with a G-quadruplex structure obtained by restrained molecular dynamics (rMD) simulations which confirms the schematic model proposed by Williamson et al. [(1989) Cell 59, 871--880]. The bifunctional complexes cis- and trans-[Pt(NH(3))(2)(H(2)O)(2)](2+) also first reacted with G9 and G15 and gave cross-linked adducts between two guanines, which did not exceed 5% each of the products formed. Both the cis and trans isomers formed a G3-G15 platinum chelate, and the second also formed bis-chelates at both ends of the G-quadruplex structure: G3-G15/G9-G21 and G3-G15/G9-G24. The rMD simulations showed that the cross-linking reactions by the trans complex can occur without disturbing the stacking of the three G-quartets.

Hormonal induced lactation in transgenic goats.

The aim of this study was to hormonally induce lactation in prepubertal, nulliparous, and male goats both transgenic and non-transgenic. Analysis of milk quality, recombinant protein expression levels, total amount of recombinant protein produced, and the affect on long-term reproductive capability was assessed. Fifty-one goats (Saanen, Alpine, and Toggenburg), male and non-pregnant females, 2-31 months of age, either non-transgenic or transgenic were evaluated with a total of 10 transgenes (constructs) represented. Animals were given estradiol (0.25 mg/kg, i.m.) and progesterone (0.75 mg/kg, i.m.) on days 1, 3, 5, 7, 9, 11 and 13, while prednisilone (0.4 mg/kg, i.m.) was administered on days 14-16 with mammary massage occurring daily from day 5 onward. Forty of 51 animals, (36 of 38 females and 4 of 13 males) produced milk with total volumes in the 30-day experiment, ranging from 20 microl to 530 mls per day, or approximately 500 microl to 6.8 liters total. Milk composition was analyzed for various parameters (total protein, fat content, total solids and somatic cell count) with no significant differences found between induced and natural milk. Expression levels of recombinant proteins from transgenic animals that were analyzed during the induced lactation, and subsequently during normal lactations, were found to have no significant differences. Total amount of recombinant protein produced was evaluated at different expression levels with no statistical significance seen. While over 90% of the females placed in the regimen became pregnant, there was a correlation between increased age at time of induction and an increase in number of breedings, or reproductive cycles needed to establish a pregnancy after induction. For males, 100% placed in the regimen settled females after hormonal induction of lactation. Semen quality was evaluated prior to, during, and after hormonal treatments. Semen volume and sperm number did not differ; however, for a small percentage of males, there was a decrease in sperm and post thaw motility after hormonal treatments. These levels returned to normal within 4-5 weeks. Subsequent natural lactations showed total milk volumes within breed standards. These findings indicate that hormonal induction of lactation in the caprine species is a viable alternative to pregnancy for initiating lactation and milk production, does not adversely impact reproductive performance long-term, and can benefit the early assessment of recombinant proteins produced in a transgenic founder program.

Probing the mechanism of an Mn2+-dependent ribozyme by means of platinum complexes.

The smallest ribozyme system known is the pentanucleotide GAAACp, which is specifically cleaved by Mn2+, in the presence of poly(U), generating guanosine 2',3'-cyclic phosphate and AAACp. A plausible mechanism has been proposed, involving the participation of two Mn2+ with structural and catalytic roles, the first one cross-linking the two N7 atoms of G1 and A4, and the other binding to the N7 atom of A2 and activating the 2'-OH group of G1 [Kazakov, S. & Altman, S. (1992) Proc. Natl Acad. Sci. USA 89, 7939-7943]. In the present work, we have utilized the high affinity of Pt(II) complexes for N7 atoms of purines in an attempt to form a stable active ribozyme by replacing the structural Mn2+ by Pt2+. We thus replaced the proposed kinetically labile G1N7-Mn2+-A4N7 cross-link by an inert N7-trans-Pt(NH3)(2)(2+)-N7 cross-link. In a complementary investigation, the N7 atoms of the individual purines of GAAACp were selectively blocked by a Pt(NH3)(3)(2+) residue to determine which of the N7 sites participate in the Mn2+-mediated cleavage. Other N7-Pt(II)-N7 crosslinks were also investigated. Accordingly, we have prepared four monoadducts, each bearing the Pt(NH3)(3)(2+) residue on one of the purines and a series of chelates of trans-Pt(NH3)(2)(2+) and cis-Pt(NH3)(2)(2+) and have tested them for Mn2+-induced cleavage. Binding of Pt(NH3)(3)(2+) to G1 or A4 did not alter the efficiency of the specific cleavage between G1 and A2 catalyzed by Mn2+/poly(U), whereas cross-linking of G1 and A4 by trans-Pt(NH3)(2)(2+) inhibited it completely. Hence, a cross-link between G1 and A4 is not required for the site-specific cleavage. Binding of Pt(NH3)(3)(2+) to A2 or A3 strongly inhibits the G1/A2 cleavage, suggesting that these bases are likely to be involved in manganese coordination in the cleaving complex. A site-specific Mn2+-dependent cleavage between A4 and C5 was observed for the G1-A4 and G1-A3 adducts cross-linked by trans-Pt(NH3)(2)(2+), the G1-A2 adduct cross-linked by cis-Pt(NH3)(2)(2+), and the three monoadducts bearing the Pt(NH3)(3)(2+) residue on G1, A2 or A3; poly(U) did not exert any influence on this cleavage.

Differentiation of females in Sergentomyia sensu stricto (Diptera: Psychodidae) using scanning electron microscopy of pharyngeal armatures.

Scanning electron microscopy of external ornamentation and internal armature of the pharynx was used to identify females of Sergentomyia sensu stricto. Five species from the eastern Mediterranean basin were compared; S. minuta clearly was separated from species of the fallax-group. Within the fallax-group, S. fallax was distinguished readily by its heart-shaped pharynx and the difference in armature between the dorsal and lateral plates.

Hexanal phenylhydrazone is a mechanism-based inactivator of soybean lipoxygenase 1.

Hexanal phenylhydrazone (1; 70:30 E:Z mixture) at micromolar concentration irreversibly inactivates soybean lipoxygenase 1 (L-1) in the presence of dioxygen. L-1 catalyzes the oxidation of 1 into its alpha-azo hydroperoxide 2 [C5H11CH(OOH)N = NC6H5]. 2 is an efficient inactivator of L-1. The aerobic reaction between 1 and L-1 follows a branched pathway leading to the release of 2 into the medium or to L-1 inactivation. The respective parameters corresponding to this inactivation by the (E)-1 and (Z)-1 isomers are Ki = 0.25 and 0.40 microM and kinact = 0.8 and 2.1 min-1. Linoleic acid protection agrees with a mechanism-based inactivation process. The oxidation of a minimum of 13 +/- 3 molar equiv of 1 is required for complete L-1 inactivation, but up to 70 equiv is necessary in the presence of a very large excess of 1. The inactivation is actually the result of two pathways: one is due to a reaction of 2 as soon as it is formed at the active site (20%); the other is due to 2 released into the medium and coming back to the active site (80%). The inactivation is accompanied by the oxidation of 1.8 +/- 0.8 methionine residues of the protein into the corresponding sulfoxide. The inactivated L-1 is electron paramagnetic resonance (EPR) silent with an effective magnetic moment of mu = 5.0 +/- 0.1 Bohr magnetons corresponding to an S = 2 spin state. An inactivation mechanism is proposed on the basis of EPR and magnetic susceptibility data obtained from the anaerobic and aerobic reactions of L-1 with 1 and 2.