Quantification of Imatinib Plasma Levels in Patients with Chronic Myeloid Leukemia: Comparison Between HPLC–UV and LC–MS/MS

Measurement of imatinib plasma concentration can be useful to evaluate patient adherence to daily oral therapy, potential drug–drug interaction, treatment efficacy, and severe drug-related adverse events. The aim of this study was to correlate the two different blood level test methods, HPLC–UV and LC–MS/MS. We analyzed 162 plasma samples from patients treated with imatinib. We estimated the correlation between the two analytical methods on total data set and on five sets of patients grouped into different categories based on the drug-dose therapy. Finally, imatinib quantification was correlated with genetic data on the molecular response in monitoring follow-up of CML patients.     

Dopamine Signaling Leads to Loss of Polycomb Repression and Aberrant Gene Activation in Experimental Parkinsonism

Polycomb group (PcG) proteins bind to and repress genes in embryonic stem cells through lineage commitment to the terminal differentiated state. PcG repressed genes are commonly characterized by the presence of the epigenetic histone mark H3K27me3, catalyzed by the Polycomb repressive complex 2. Here, we present in vivo evidence for a previously unrecognized plasticity of PcG-repressed genes in terminally differentiated brain neurons of parkisonian mice. We show that acute administration of the dopamine precursor, L-DOPA, induces a remarkable increase in H3K27me3S28 phosphorylation. The induction of the H3K27me3S28p histone mark specifically occurs in medium spiny neurons expressing dopamine D1 receptors and is dependent on Msk1 kinase activity and DARPP-32-mediated inhibition of protein phosphatase-1. Chromatin immunoprecipitation (ChIP) experiments showed that increased H3K27me3S28p was accompanied by reduced PcG binding to regulatory regions of genes. An analysis of the genome wide distribution of L-DOPA-induced H3K27me3S28 phosphorylation by ChIP sequencing (ChIP-seq) in combination with expression analysis by RNA-sequencing (RNA-seq) showed that the induction of H3K27me3S28p correlated with increased expression of a subset of PcG repressed genes. We found that induction of H3K27me3S28p persisted during chronic L-DOPA administration to parkisonian mice and correlated with aberrant gene expression. We propose that dopaminergic transmission can activate PcG repressed genes in the adult brain and thereby contribute to long-term maladaptive responses including the motor complications, or dyskinesia, caused by prolonged administration of L-DOPA in Parkinson''s disease.     

Expression and Distribution of the Adrenomedullin System in Newborn Human Thymus

Adrenomedullin (AM) is a multifunctional peptide endowed with various biological actions mediated by the interaction with the calcitonin receptor-like receptor (CLR), which couples to the receptor activity-modifying proteins 2 or 3 (RAMP2 or RAMP3) to form the functional plasma membrane receptors AM1 and AM2, respectively. In this study, we investigated for the first time the expression and localization of AM, CLR, RAMP2 and RAMP3 in human thymic tissue from newborns and in primary cultures of thymic epithelial cells (TECs) and thymocytes. Immunohistochemical analysis of thymic tissue showed that both AM and RAMP2 are abundantly expressed in the epithelial cells of medulla and cortex, blood vessels and mastocytes. In contrast, RAMP3 could not be detected. In cultured TECs, double immunofluorescence coupled to confocal microscopy revealed that AM is present in the cytoplasmic compartment, whereas RAMP2 could be detected in the cytoplasm and nucleus, but not in the cell membrane. At variance with RAMP2, CLR was not only present in the nucleus and cytoplasm of TECs, but could also be detected in the cell membrane. The nuclear and cytoplasmic localizations of RAMP2 and CLR and the absence of RAMP2 in the cell membrane were confirmed by western-blot analysis performed on cell fractions. AM, RAMP2 and CLR could also be detected in thymocytes by means of double immunofluorescence coupled to confocal microscopy, although these proteins were not present in the whole thymocyte population. In these cells, AM and RAMP2 were detected in the cytoplasm, whereas CLR could be observed in the cytoplasm and the plasma membrane. In conclusion, our results show that the AM system is widely expressed in human thymus from newborns and suggest that both AM1 receptor components CLR and RAMP2 are not associated with the plasma membrane of TECs and thymocytes but are located intracellularly, notably in the nucleus.     

Human cytomegalovirus differentially controls B cell and T cell responses through effects on plasmacytoid dendritic cells

Plasmacytoid dendritic cells (PDCs), the main producers of type I IFN in response to viral infection, are essential in antiviral immunity. In this study, we assessed the effect of human CMV (HCMV) infection on PDC function and on downstream B and T cell responses in vitro. HCMV infection of human PDCs was nonpermissive, as immediate-early but not late viral Ags were detected. HCMV led to partial maturation of PDCs and up-regulated MHC class II and CD83 molecules but not the costimulatory molecules CD80 and CD86. Regardless of viral replication, PDCs secreted cytokines after contact with HCMV, including IFN-alpha secretion that was blocked by inhibitory CpG, suggesting an engagement of the TLR7 and/or TLR9 pathways. In the presence of B cell receptor stimulation, soluble factors produced by HCMV-matured PDCs triggered B cell activation and proliferation. Through PDC stimulation, HCMV prompted B cell activation, but only induced Ab production in the presence of T cells or T cell secreted IL-2. Conversely, HCMV hampered the allostimulatory ability of PDCs, leading to decreased proliferation of CD4(+) and CD8(+) T cells. These findings reveal a novel mechanism by which HCMV differentially controls humoral and cell-mediate immune responses through effects on PDCs.     

Characteristics and Their Clinical Relevance of Respiratory Syncytial Virus Types and Genotypes Circulating in Northern Italy in Five Consecutive Winter Seasons

In order to investigate the genetic diversity and patterns of the co-circulating genotypes of respiratory syncytial virus (RSV) and their possible relationships with the severity of RSV infection, we studied all of the RSV-positive nasopharyngeal samples collected from children during five consecutive winters (2009–2010, 2010–2011, 2011–2012, 2012–2013 and 2013–2014). The RSVs were detected using the respiratory virus panel fast assay and single-tube RT-PCR, their nucleotides were sequenced, and they were tested for positive selection. Of the 165 positive samples, 131 (79.4%) carried RSV-A and 34 (20.6%) RSV-B; both groups co-circulated in all of the study periods, with RSV-A predominating in all the seasons except for winter 2010–2011, which had a predominance of RSV-B. Phylogenetic analysis of the RSV-A sequences identified genotypes NA1 and ON1, the second replacing the first during the last two years of the study period. The RSV-B belonged to genotypes BA9 and BA10. BA9 was detected in all the years of the study whereas BA only desultorily. Comparison of the subjects infected by RSV-A and RSV-B types did not reveal any significant differences, but the children infected by genotype A/NA1 more frequently had lower respiratory tract infections (p<0.0001) and required hospitalisation (p = 0.007) more often than those infected by genotype A/ON1. These findings show that RSV has complex patterns of circulation characterised by the periodical replacement of the predominant genotypes, and indicate that the circulation and pathogenic role of the different RSV strains should be investigated as each may have a different impact on the host. A knowledge of the correlations between types, genotypes and disease severity may also be important in order to be able to include the more virulent strains in future vaccines.     

Development of Lentivirus-Based Reference Materials for Ebola Virus Nucleic Acid Amplification Technology-Based Assays

The 2013-present Ebola virus outbreak in Western Africa has prompted the production of many diagnostic assays, mostly based on nucleic acid amplification technologies (NAT). The calibration and performance assessment of established assays and those under evaluation requires reference materials that can be used in parallel with the clinical sample to standardise or control for every step of the procedure, from extraction to the final qualitative/quantitative result. We have developed safe and stable Ebola virus RNA reference materials by encapsidating anti sense viral RNA into HIV-1-like particles. The lentiviral particles are replication-deficient and non-infectious due to the lack of HIV-1 genes and Envelope protein. Ebola virus genes were subcloned for encapsidation into two lentiviral preparations, one containing NP-VP35-GP and the other VP40 and L RNA. Each reference material was formulated as a high-titre standard for use as a calibrator for secondary or internal standards, and a 10,000-fold lower titre preparation to serve as an in-run control. The preparations have been freeze-dried to maximise stability. These HIV-Ebola virus RNA reference materials were suitable for use with in-house and commercial quantitative RT-PCR assays and with digital RT-PCR. The HIV-Ebola virus RNA reference materials are stable at up to 37°C for two weeks, allowing the shipment of the material worldwide at ambient temperature. These results support further evaluation of the HIV-Ebola virus RNA reference materials as part of an International collaborative study for the establishment of the 1^st International Standard for Ebola virus RNA.     

Ontogenetic trajectories of chimpanzee social play: similarities with humans

Social play, a widespread phenomenon in mammals, is a multifunctional behavior, which can have many different roles according to species, sex, age, relationship quality between playmates, group membership, context, and habitat. Play joins and cuts across a variety of disciplines leading directly to inquiries relating to individual developmental changes and species adaptation, thus the importance of comparative studies appears evident. Here, we aim at proposing a possible ontogenetic pathway of chimpanzee play (Pan troglodytes) and contrast our data with those of human play. Chimpanzee play shows a number of changes from infancy to juvenility. Particularly, solitary and social play follows different developmental trajectories. While solitary play peaks in infancy, social play does not show any quantitative variation between infancy and juvenility but shows a strong qualitative variation in complexity, asymmetry, and playmate choice. Like laughter in humans, the playful expressions in chimpanzees (at the different age phases) seem to have a role in advertising cooperative dispositions and intentions thus increasing the likelihood of engaging in solid social relationships. In conclusion, in chimpanzees, as in humans, both play behavior and the signals that accompany play serve multiple functions according to the different age phases.