Hepatitis E Virus Infection in Pediatric Oncology.

BACKGROUND: In the 2016 ESPGHAN recommendations on how to deal with hepatitis E virus infection in immunocompromised children, patients treated with chemotherapy were not specifically mentioned. OBSERVATIONS: Two teenagers treated with chemotherapy for acute leukemia and medulloblastoma, respectively, were diagnosed with hepatic cytolysis. After numerous investigations hepatitis E was found, limiting the good progress of the chemotherapy treatment. CONCLUSION: In the case of liver cytolysis in immunocompromised children treated with chemotherapy, hepatitis E virus infection has to be promptly diagnosed.

The importance of basonuclin 2 in adult mice and its relation to basonuclin 1.

BNC2 is an extremely conserved zinc finger protein with important functions in the development of craniofacial bones and male germ cells. Because disruption of the Bnc2 gene in mice causes neonatal lethality, the function of the protein in adult animals has not been studied. Until now BNC2 was considered to have a wider tissue distribution than its paralog, BNC1, but the precise cell types expressing Bnc2 are largely unknown. We identify here the cell types containing BNC2 in the mouse and we show the unexpected presence of BNC1 in many BNC2-containing cells. BNC1 and BNC2 are colocalized in male and female germ cells, ovarian epithelial cells, sensory neurons, hair follicle keratinocytes and connective cells of organ capsules. In many cell lineages, the two basonuclins appear and disappear synchronously. Within the male germ cell lineage, BNC1 and BNC2 are found in prospermatogonia and undifferentiated spermatogonia, and disappear abruptly from differentiating spermatogonia. During oogenesis, the two basonuclins accumulate specifically in maturing oocytes. During the development of hair follicles, BNC1 and BNC2 concentrate in the primary hair germs. As follicle morphogenesis proceeds, cells possessing BNC1 and BNC2 invade the dermis and surround the papilla. During anagen, BNC1 and BNC2 are largely restricted to the basal layer of the outer root sheath and the matrix. During catagen, the compartment of cells possessing BNC1 and BNC2 regresses, and in telogen, the two basonuclins are confined to the secondary hair germ. During the next anagen, the BNC1/BNC2-containing cell population regenerates the hair follicle. By examining Bnc2(-/-) mice that have escaped the neonatal lethality usually associated with lack of BNC2, we demonstrate that BNC2 possesses important functions in many of the cell types where it resides. Hair follicles of postnatal Bnc2(-/-) mice do not fully develop during the first cycle and thereafter remain blocked in telogen. It is concluded that the presence of BNC2 in the secondary hair germ is required to regenerate the transient segment of the follicle. Postnatal Bnc2(-/-) mice also show severe dwarfism, defects in oogenesis and alterations of palatal rugae. Although the two basonuclins possess very similar zinc fingers and are largely coexpressed, BNC1 cannot substitute for BNC2. This is shown incontrovertibly in knockin mice expressing Bnc1 instead of Bnc2 as these mice invariably die at birth with craniofacial abnormalities undistinguishable from those of Bnc2(-/-) mice. The function of the basonuclins in the secondary hair germ is of particular interest.

The zinc-finger protein basonuclin 2 is required for proper mitotic arrest, prevention of premature meiotic initiation and meiotic progression in mouse male germ cells.

Absence of mitosis and meiosis are distinguishing properties of male germ cells during late fetal and early neonatal periods. Repressors of male germ cell meiosis have been identified, but mitotic repressors are largely unknown, and no protein repressing both meiosis and mitosis is known. We demonstrate here that the zinc-finger protein BNC2 is present in male but not in female germ cells. In testis, BNC2 exists as several spliced isoforms and presumably binds to DNA. Within the male germ cell lineage, BNC2 is restricted to prospermatogonia and undifferentiated spermatogonia. Fetal prospermatogonia that lack BNC2 multiply excessively on embryonic day (E)14.5 and reenter the cell cycle prematurely. Mutant prospermatogonia also engage in abnormal meiosis; on E17.5, Bnc2(-/-) prospermatogonia start synthesizing the synaptonemal protein SYCP3, and by the time of birth, many Bnc2(-/-) prospermatogonia have accumulated large amounts of nonfilamentous SYCP3, thus appearing to be blocked at leptonema. Bnc2(-/-) prospermatogonia do not undergo proper male differentiation, as they lack almost all the mRNA for the male-specific methylation protein DNMT3L and have increased levels of mRNAs that encode meiotic proteins, including STRA8. Bnc2(-/-) prospermatogonia can produce spermatogonia, but these enter meiosis prematurely and undergo massive apoptotic death during meiotic prophase. This study identifies BNC2 as a major regulator of male germ stem cells, which is required for repression of meiosis and mitosis in prospermatogonia, and for meiosis progression during spermatogenesis. In view of the extreme evolutionary conservation of BNC2, the findings described here are likely to apply to many species.

[Basonuclins and DISCO proteins: regulators of development in vertebrates and insects]. / Basonuclines et protéines DISCO: des régulateurs du développement des vertébrés et des insectes.

Basonuclins 1 and 2 are nuclear proteins specific to vertebrates. They have recently been shown to be the orthologs of the DISCO proteins of insects. All of these proteins have an essential function in embryonic development. It is likely that they are transcription factors with multiple gene targets, some of which are transcribed by DNA polymerase I, and others by polymerase II. It remains to be found which targets are common to the two basonuclins and even to the DISCO proteins, and why certain cell types possess either basonuclin 1 or basonuclin 2, while others possess both. The implication of the basonuclins in several human diseases adds to their interest as regulatory proteins.

Basonuclins and disco: Orthologous zinc finger proteins essential for development in vertebrates and arthropods.

Basonuclin 1 and the recently discovered basonuclin 2 are vertebrate proteins with multiple paired C(2)H(2) zinc fingers. It has long been known that the zinc fingers of basonuclin 1 closely resembled those of the Drosophila disconnected and discorelated proteins, two proteins essential for head development, but the relation between the basonuclins and the disco proteins has remained unclear because the putative function of basonuclin 1 in the control of keratinocyte growth potential appeared unrelated to that of disco and there was no resemblance between basonuclin 1 and Drosophila disco outside of the zinc fingers. The recent generation of a basonuclin-2 knockout has demonstrated that basonuclin 2 shares with disco a function in head development and the availability of new arthropod genome sequences has shown that the basonuclins are the vertebrate orthologs of the insect disco proteins. All these proteins are thought to be transcription factors, and it will have to be determined to what extent they share similar targets.

Determination of 3,4-methylenedioxymethamphetamine and its five main metabolites in rat urine by solid-phase extraction and high performance liquid chromatography with on line mass spectrometry.

The consumption of psychostimulant amphetamine-like drugs has increased significantly in recent years. Some MDMA metabolites are probably involved in the neurotoxicity and neurodegeneration caused by prolonged use rather than MDMA itself. We recently developed a method to analyze MDMA and its five main metabolites in rat plasma [7]. We have now fully validated this method to the quantification of these drugs in rat urine. We extracted MDMA and its metabolites with Oasis WCX cartridges, separated them on a Nucleodur C18 analytical column and quantified them by ion-trap mass spectrometry. Linearity was excellent: 12.5-1250ng/mL urine for HMA, HMMA, MDA and MDMA, 25-2500ng/mL for HHMA, and 150-7500ng/mL for HHA (r(2)>0.993 for all analytes). The lower limits of quantification were 12.5ng/mL urine for MDMA, MDA, HMA and HMMA, 25ng/mL for HHMA and 150ng/mL for HHA. Reproducibility was good (intra-assay precision=1.7-6.1%; inter-assay precision=0.6-5.7%), as was accuracy (intra-assay deviation=0.1-4.8%; inter-assay deviation=0.7-7.9%). Average recoveries were around 85.0%, except for HHMA (66.2%) and HHA (53.0%) (CV<8.3%). We also checked the stability of stock solutions and the internal standards after freeze-thawing and in the autosampler. Lastly, we measured the MDMA, MDA, HHMA, HHA, HMMA and HMA in urine samples taken over 24h from rats given subcutaneous MDMA.

Basonuclin 2 has a function in the multiplication of embryonic craniofacial mesenchymal cells and is orthologous to disco proteins.

Basonuclin 2 is a recently discovered zinc finger protein of unknown function. Its paralog, basonuclin 1, is associated with the ability of keratinocytes to multiply. The basonuclin zinc fingers are closely related to those of the Drosophila proteins disco and discorelated, but the relation between disco proteins and basonuclins has remained elusive because the function of the disco proteins in larval head development seems to have no relation to that of basonuclin 1 and because the amino acid sequence of disco, apart from the zinc fingers, also has no similarity to that of the basonuclins. We have generated mice lacking basonuclin 2. These mice die within 24 h of birth with a cleft palate and abnormalities of craniofacial bones and tongue. In the embryonic head, expression of the basonuclin 2 gene is restricted to mesenchymal cells in the palate, at the periphery of the tongue, and in the mesenchymal sheaths that surround the brain and the osteocartilagineous structures. In late embryos, the rate of multiplication of these mesenchymal cells is greatly diminished. Therefore, basonuclin 2 is essential for the multiplication of craniofacial mesenchymal cells during embryogenesis. Non-Drosophila insect databases available since 2008 reveal that the basonuclins and the disco proteins share much more extensive sequence and gene structure similarity than noted when only Drosophila sequences were examined. We conclude that basonuclin 2 is both structurally and functionally the vertebrate ortholog of the disco proteins. We also note the possibility that some human craniofacial abnormalities are due to a lack of basonuclin 2.

CNS delivery via adsorptive transcytosis.

Adsorptive-mediated transcytosis (AMT) provides a means for brain delivery of medicines across the blood-brain barrier (BBB). The BBB is readily equipped for the AMT process: it provides both the potential for binding and uptake of cationic molecules to the luminal surface of endothelial cells, and then for exocytosis at the abluminal surface. The transcytotic pathways present at the BBB and its morphological and enzymatic properties provide the means for movement of the molecules through the endothelial cytoplasm. AMT-based drug delivery to the brain was performed using cationic proteins and cell-penetrating peptides (CPPs). Protein cationization using either synthetic or natural polyamines is discussed and some examples of diamine/polyamine modified proteins that cross BBB are described. Two main families of CPPs belonging to the Tat-derived peptides and Syn-B vectors have been extensively used in CPP vector-mediated strategies allowing delivery of a large variety of small molecules as well as proteins across cell membranes in vitro and the BBB in vivo. CPP strategy suffers from several limitations such as toxicity and immunogenicity--like the cationization strategy--as well as the instability of peptide vectors in biological media. The review concludes by stressing the need to improve the understanding of AMT mechanisms at BBB and the effectiveness of cationized proteins and CPP-vectorized proteins as neurotherapeutics.

Covalent modifications of antitetanus F(ab')2 fragments with natural and synthetic polyamines and their effects on the antibody endocytosis in cultured HL60 cells.

For antibody therapeutics to succeed when intracellular target molecules are involved, a strategy must be applied to increase the delivery of antibodies into cells to reach their targets. Antibody cationization by chemical conjugation of a polyamine could be one such strategy. Both natural polyamines with increasing net charge valencies (putrescine, PUT; spermidine, SPD; and spermine, SPM) and a synthetic polyamine (hexamethylenediamine, HMD) can be used to cationize antibodies, but no comparison of the respective effects of these polyamines on intracellular delivery of antibodies has been performed yet. This study describes the covalent modification of antitetanus F(ab') 2 with these four polyamines using different reaction conditions, and compares the effects of these modifications on antibody interaction with cultured HL60 cells. The cationized antibodies retained > or =80% of the binding activity of the unmodified F(ab') 2 with regard to tetanus toxin, as measured by an antigen-binding capture enzyme immunoassay. This same method was used to quantify the amount of cell-associated F(ab') 2 following incubation with HL60 cells. Cationization was shown to enhance cell interaction of the F(ab') 2 : the higher the number of coupled polyamine molecules, the greater the amount of antibody associated with the cells. Moreover, coupling the F(ab') 2 to the SPD and SPM polyamines had greater effect on cell interaction than coupling the F(ab') 2 to the PUT and HMD diamines. Internalization of the cationized antibodies by the HL60 cells was demonstrated by confocal microscopy. This technique also showed that SPD and SPM were more effective than PUT and HMD in terms of intracellular delivery of the F(ab') 2 . It follows from all these results that electrostatic interaction involving charge density plays a predominant role in the endocytic transport mechanism of the F(ab') 2 modified with these polyamines. However, coupling the F(ab') 2 to SPM and SPD yielded the same maximum effects in terms of cell interaction, although coupling SPM was expected to increase the antibody net charge valency more than coupling SPD. This finding suggests that the effective global charge for the cell interaction and uptake of polyamine-modified antibodies does not simply correspond to the addition of the ionizable amine functions on the coupled polyamines, and that other factors may come into play.

[Introduction of platelet additive solutions in transfusion practice. Advantages, disadvantages and benefit for patients]. / Introduction en pratique transfusionnelle des concentrés de plaquettes en solution de conservation. Avantages, inconvénients, et intérêt pour les patients.

Platelet additive solutions (PAS) have been developed since the years 1980. However, decisive improvements have been made in the last five years, leading nowadays to several PAS available for transfusion practice. Few compounds are present in PAS, with the intention of controlling platelet metabolic alterations and activation that occur during storage: acetate, which is a substrate for the tricarboxylic acid cycle, enables to maintain oxidative metabolism, is present in all PAS; a buffer effect is required to prevent the progressive pH fall during storage, and is obtained either with sodium phosphate or gluconate; platelet activation is controlled by citrate, and in the latest PAS, by magnesium and potassium. It is important to note that whatever the PAS used, it is mandatory to maintain a final concentration of 20-40% of plasma, mainly in order to ensure glucose availability. The use of PAS leads to a more rationalized blood processing, as it provides an additional volume of plasma available for plasma fractionation, it contributes to standardization of blood components, and it is part of at least one pathogen reduction process. The expected benefit for patient is the reduction of adverse reactions related to plasma. There is already evidence that the incidence of allergic adverse reactions is reduced. In the case of other less frequent adverse reactions such as transfusion related acute lung injury (TRALI) or haemolytic reaction due to minor ABO incompatibility, only a long-term follow-up through haemovigilance organization will be informative.

Liquid chromatographic-electrospray ionization mass spectrometric quantitative analysis of buprenorphine, norbuprenorphine, nordiazepam and oxazepam in rat plasma.

A liquid chromatographic-mass spectrometric method with electrospray ionization is presented for the simultaneous determination of buprenorphine, nordiazepam and their pharmacologically active metabolites, norbuprenorphine and oxazepam, in rat plasma. The drugs were extracted from plasma by liquid-liquid extraction and chromatographically separated using a gradient elution of aqueous ammonium formate and acetonitrile. Following electrospray ionization, the analytes were quantified in the single ion storage mode. The assay was validated according to current acceptance criteria for bioanalytical method validation. It was proved to be linear from 0.7 to 200 ng/ml plasma for buprenorphine, 1.0 to 200 ng/ml for norbuprenorphine, 2.0 to 200 ng/ml for nordiazepam, and from 5.0 to 200 ng/ml for oxazepam. The average recoveries of buprenorphine, norbuprenorphine, nordiazepam and oxazepam were 89, 39, 88 and 82%, respectively, with average coefficients of variation ranging from 1.8 to 14.3%. The limits of quantitation for these drugs were 0.7, 1.0, 2.0 and 5.0 ng/ml, respectively, with associated precisions within 17% and accuracies within +/-18% of the nominal values. Both the intra- and inter-assay precision values did not exceed 11.3% for the four analytes. Intra- and inter-assay accuracies lay within +/-15% of the nominal values. The validated method was applied to the determination of buprenorphine, norbuprenorphine, nordiazepam and oxazepam in plasma samples collected from rats at various times after intravenous administration of buprenorphine and nordiazepam.

Development and validation of an antigen-binding capture ELISA for native and putrescine-modified anti-tetanus F(ab')2 fragments for the assessment of the cellular uptake and plasma kinetics of the antibodies.

Cationization is a strategy to enhance the permeability of antibodies to physiological membranes for potential therapeutic and diagnostic applications of these proteins, with one of its crucial points being the retention of antigen binding activity. Here, we describe the cationization of horse polyclonal anti-tetanus F(ab')(2) fragments and the development and validation of an ELISA for quantitative measurements of the binding activity of the native and cationized F(ab')(2) in cell lysates and rat plasma samples, assessing the cellular uptake and plasma kinetics of these antibodies, respectively. The method used tetanus anatoxin coated on microtitre plates as capture antigen to bind sample or standard F(ab')(2), the amount of antibody binding being quantified using, first, a secondary biotinylated anti-horse antibody/streptavidin-alkaline phosphatase complex in situ and then a measurement of the substrate product. Cationization of the F(ab')(2) was performed with putrescine at pH 4.5 using soluble carbodiimide as carboxyl activator. The average substitution ratio was determined at 3 putrescine molecules per F(ab')(2) molecule. The cationized F(ab')(2) retained roughly 80% of the initial antigen binding activity and was stable over a 1 year period of storage at -20 degrees C. The ELISA validation data showed that the method was linear for both the native and cationized F(ab')(2) using Hanks' balanced saline solution with 0.2% bovine serum albumin as assay diluent for the cell lysate samples. The useful F(ab')(2) concentration range was 2.5-25 ng/ml and the limit of quantification was 2.5 ng/ml. With rat blank plasma used as assay diluent for the rat plasma samples the useful F(ab')(2) concentration range was 3.5-25 ng/ml and the limit of quantification was 3.5 ng/ml. Specific requirements for the limits of quantification were fulfilled: precision < or =20% CV and accuracy within +/-20% of the nominal values. Intra- and inter-assay coefficients of variation were within 9% and accuracies within +/-10% of the nominal values. The validated method was applied to the study of the cellular uptake of native and cationized anti-tetanus F(ab')(2) in an HL 60 cell model, and of plasma kinetics after i.v. administration to rats.

Development and validation of a gas chromatography-mass spectrometry method for the simultaneous determination of buprenorphine, flunitrazepam and their metabolites in rat plasma: application to the pharmacokinetic study.

Buprenorphine (BUP), a synthetic opioid analgesic, is frequently abused alone, and in association with benzodiazepines. Fatalities involving buprenorphine alone seem very unusual while its association with benzodiazepines, such as flunitrazepam (FNZ), has been reported to result in severe respiratory depression and death. The quantitative relationship between these drugs remain, however, uncertain. Our objective was to develop an analytical method that could be used as a means to study and explore, in animals, the toxicity and pharmacological interaction mechanisms between buprenorphine, flunitrazepam and their active metabolites. A procedure based on gas chromatography-mass spectrometry (GC-MS) is described for the simultaneous analysis of buprenorphine, norbuprenorphine (NBUP), flunitrazepam, N-desmethylflunitrazepam (N-DMFNZ) and 7-aminoflunitrazepam (7-AFNZ) in rat plasma. The method was set up and adapted for the analysis of small plasma samples taken from rats. Plasma samples were extracted by liquid-liquid extraction using Toxi-tubes A. Extracted compounds were derivatized with N,O-bis-(trimethylsilyl)trifluoroacetamide (BSTFA), using trimethylchlorosilane (TMCS) as a catalyst. They were then separated by GC on a crosslinked 5% phenyl-methylpolysiloxane analytical column and determined by a quadrupole mass spectrometer detector operated under selected ion monitoring mode. Excellent linearity was found between 0.125 and 25 ng/microl plasma for BUP, 0.125 and 12.5 ng/microl for NBUP and N-DMFNZ, 0.125 and 5 ng/microl for FNZ, and between 0.025 and 50 ng/microl for 7-AFNZ. The limit of quantification was 0.025 ng/microl plasma for 7-AFNZ and 0.125 ng/microl for the four other compounds. A good reproducibility (intra-assay CV=0.32-11.69%; inter-assay CV=0.63-9.55%) and accuracy (intra-assay error=2.58-12.73%; inter-assay error=0.83-11.07%) were attained. Recoveries were 71, 67 and 81%, for BUP, FNZ and N-DMFNZ, respectively, and 51% for NBUP and 7-AFNZ, with CV ranging from 5.4 to 13.9%, and were concentration-independent. The GC-MS method was successfully applied to the pharmacokinetic study of BUP, NBUP, FNZ, DMFNZ and 7-AFNZ in rats, after administration of BUP and FNZ.

Glycosylation study of the major genetic variants of human alpha1-acid glycoprotein and of their pharmacokinetics in the rat.

Human alpha1-acid glycoprotein (AAG) is a mixture of at least two genetic variants, the A variant and the F1 and/or S variant or variants, which are encoded by two different genes. AAG is also an extensively glycosylated protein which possesses five N-linked glycans exhibiting substantial heterogeneity in their structures. The first objective of this study was to investigate the glycosylation of the two major gene products of AAG, i.e. the A variant and a mixture of the F1 and S variants (F1*S). To this end, we combined a chromatographic method for the fractionation of the AAG variants with a lectin-binding assay to characterise the glycosylation of purified glycoproteins. Secondly, because the oligosaccharides can influence the disposition of AAG, a kinetic study of the AAG variants was carried out in the rat. After intravenous administration of whole human AAG, the separation and quantification of the AAG variants in plasma was performed by application of specific methods by isoelectric focusing and immunonephelometry. The binding studies carried out on a panel of lectins showed significant differences in the lectin-binding characteristics of the separated F1*S and A variants, accounting for differences in the degree of branching of their glycan chains and substitution with sialic acid and fucose. The plasma concentration-time profiles of the F1*S and A variants were biphasic, and only small differences were observed between the variants for their initial and terminal half-lives, clearance and distribution volume. This indicates that the structural differences between the two AAG gene products do not affect their pharmacokinetics in the rat. Specific drug transport roles have been previously demonstrated for the F1*S and A variants, calling for further investigations into their effects on the disposition of drugs they bind in plasma. The present study shows that such investigations are possible without being complicated by kinetic differences between these variants.