ABCB1 and cytochrome P450 genotypes and phenotypes: influence on methadone plasma levels and response to treatment.

BACKGROUND AND OBJECTIVE: The in vivo implication of various cytochrome P450 (CYP) isoforms and of P-glycoprotein on methadone kinetics is unclear. We aimed to thoroughly examine the genetic factors influencing methadone kinetics and response to treatment. METHODS: Genotyping for CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP3A4, CYP3A5, ABCB1, and UGT2B7 polymorphisms was performed in 245 patients undergoing methadone maintenance treatment. To assess CYP3A activity, the patients were phenotyped with midazolam. RESULTS: The patients with lower CYP3A activity presented higher steady-state trough (R,S)-methadone plasma levels (4.3, 3.0, and 2.3 ng/mL x mg for low, medium, and high activity, respectively; P = .0002). As previously reported, CYP2B6*6/*6 carriers had significantly higher trough (S)-methadone plasma levels (P = .0001) and a trend toward higher (R)-methadone plasma levels (P = .07). CYP2D6 ultrarapid metabolizers presented lower trough (R,S)-methadone plasma levels compared with the extensive or intermediate metabolizers (2.4 and 3.3 ng/mL x mg, respectively; P = .04), whereas CYP2D6 poor metabolizer status showed no influence. ABCB1 3435TT carriers presented lower trough (R,S)-methadone plasma levels (2.7 and 3.4 ng/mL . mg for 3435TT and 3435CC carriers, respectively; P = .01). The CYP1A2, CYP2C9, CYP2C19, CYP3A5, and UGT2B7 genotypes did not influence methadone plasma levels. Only CYP2B6 displayed a stereoselectivity in its activity. CONCLUSION: In vivo, CYP3A4 and CYP2B6 are the major CYP isoforms involved in methadone metabolism, with CYP2D6 contributing to a minor extent. ABCB1 genetic polymorphisms also contribute slightly to the interindividual variability of methadone kinetics. The genetic polymorphisms of these 4 proteins had no influence on the response to treatment and only a small influence on the dose requirement of methadone.

Infection of B cells with hepatitis C virus for the development of lymphoproliferative disorders in patients with chronic hepatitis C.

Infection with hepatitis C virus (HCV) is associated with lymphoproliferative disorders, represented by essential mixed cryoglobulinemia and B-cell non-Hodgkin's lymphoma, but the pathogenic mechanism remains obscure. HCV may infect B cells or interact with their cell surface receptors, and induce lymphoproliferation. The influence of HCV infection of B cells on the development of lymphoproliferative disorders was evaluated in 75 patients with persistent HCV infection. HCV infection was more prevalent (63% vs. 16%, 14%, or 17% P < 0.05 for each), and HCV RNA levels were higher (3.35 +/- 3.85 vs. 1.75 +/- 2.52, 2.15 +/- 2.94 or 2.10 +/- 2.90 log copies/100 ng, P < 0.01 for each) in B cells than CD4(+), CD8(+) T cells or other cells. Negative-strand HCV RNA, as a marker of viral replication, was detected in B cells from four of the 75 (5%) patients. Markers for lymphoproliferative disorders were more frequent in the 50 patients with chronic hepatitis C than the 32 with chronic hepatitis B, including cryoglobulinemia (26% vs. 0%, P < 0.001), low CH(50) levels (48% vs. 3%, P = 0.012), and the clonality of B cells (12% vs. 0%, P < 0.01). By multivariate analysis, HCV RNA in B cells was an independent factor associated with the presence of at least one marker for lymphoproliferation (odds ratio: 1.98 [95% confidence interval: 1.36-7.24], P = 0.027). Based on the results obtained, the infection of B cells with HCV would play an important role in the development of lymphoproliferative disorders.

Matching stages of heavy-ion collision models

Heavy-ion reactions and other collective dynamical processes are frequently described by different theoretical approaches for the different stages of the process, like initial equilibration stage, intermediate locally equilibrated fluid dynamical stage, and final freeze-out stage. For the last stage, the best known is the Cooper-Frye description used to generate the phase space distribution of emitted, noninteracting particles from a fluid dynamical expansion or explosion, assuming a final ideal gas distribution, or (less frequently) an out-of-equilibrium distribution. In this work we do not want to replace the Cooper-Frye description, but rather clarify the ways of using it and how to choose the parameters of the distribution and, eventually, how to choose the form of the phase space distribution used in the Cooper-Frye formula. Moreover, the Cooper-Frye formula is used in connection with the freeze-out problem, while the discussion of transition between different stages of the collision is applicable to other transitions also. More recently, hadronization and molecular dynamics models have been matched to the end of a fluid dynamical stage to describe hadronization and freeze-out. The stages of the model description can be matched to each other on space-time hypersurfaces (just like through the frequently used freeze-out hypersurface). This work presents a generalized description of how to match the stages of the description of a reaction to each other, extending the methodology used at freeze-out, in simple covariant form which is easily applicable in its simplest version for most applications.

Effects of 2 different prior endurance exercises on whole-body fat oxidation kinetics: light vs. heavy exercise.

This study aimed to compare the effects of 2 different prior endurance exercises on subsequent whole-body fat oxidation kinetics. Fifteen men performed 2 identical submaximal incremental tests (Incr2) on a cycle ergometer after (i) a ∼40-min submaximal incremental test (Incr1) followed by a 90-min continuous exercise performed at 50% of maximal aerobic power-output and a 1-h rest period (Heavy); and (ii) Incr1 followed by a 2.5-h rest period (Light). Fat oxidation was measured using indirect calorimetry and plotted as a function of exercise intensity during Incr1 and Incr2. A sinusoidal equation, including 3 independent variables (dilatation, symmetry and translation), was used to characterize the fat oxidation kinetics and to determine the intensity (Fat(max)) that elicited the maximal fat oxidation (MFO) during Incr. After the Heavy and Light trials, Fat(max), MFO, and fat oxidation rates were significantly greater during Incr2 than Incr1 (p < 0.001). However, Δ (i.e., Incr2-Incr1) Fat(max), MFO, and fat oxidation rates were greater in the Heavy compared with the Light trial (p < 0.05). The fat oxidation kinetics during Incr2(Heavy) showed a greater dilatation and rightward asymmetry than Incr1(Heavy), whereas only a greater dilatation was observed in Incr2(Light) (p < 0.05). This study showed that although to a lesser extent in the Light trial, both prior exercise sessions led to an increase in Fat(max), MFO, and absolute fat oxidation rates during Incr2, inducing significant changes in the shape of the fat oxidation kinetics.

Developmental changes in the heavy subunit of neurofilaments in the corpus callosum of the cat.

In the corpus callosum of the cat, the heavy subunit of neurofilaments (NFH) can be demonstrated with the monoclonal antibody NE14, as early as P11, not at P3, and only in a few axons. At P18-19 and more markedly at P29, many more callosal axons have become positive to NE14 and this is similar to what is found in the adult. In contrast, callosal axons become positive to the neurofilament antibody SMI-32 only between P29 and P39 and remain positive in the adult. Treatment with alkaline phosphatase prevents axonal staining with NE14, but results in SMI-32 staining of a few callosal axons as early as P11, but not at P3. Between P11 and P19 the number of axons stained with SMI-32 after alkaline phosphatase treatment increases, in parallel with that of axons stained with NE14. Thus NE14 appears to recognize a phosphorylated form of NFH, while SMI-32 appears to recognize an epitope of NFH which is either masked by phosphate or inaccessible until between P29 and P39, unless the tissue is treated with alkaline phosphatase. These two forms of NFH appear towards the end of the period of massive developmental elimination of callosal axons. They are also synchronous with changes in the spacing of neurofilaments quantified in a separate ultrastructural study. These cytoskeletal changes may terminate the juvenile-labile state of callosal axons and allow further axial growth of the axon.

Fitness to drive and cannabis: Experimental and real-life case study validation of two blood THCCOOH thresholds to distinguish occasional users from heavy smokers

Introduction: THC-COOH has been proposed as a criterion to help to distinguish between occasional from regular cannabis users. However, to date this indicator has not been adequately assessed under experimental and real-life conditions. Methods: We carried out a controlled administration study of smoked cannabis with a placebo. Twenty-three heavy smokers and 25 occasional smokers, between 18 and 30 years of age, participated in this study [Battistella G et al., PloS one. 2013;8(1):e52545]. We collected data from a second real case study performed with 146 traffic offenders' cases in which the whole blood cannabinoid concentrations and the frequency of cannabis use were known. Cannabinoid levels were determined in whole blood using tandem mass spectrometry methods. Results: Significantly high differences in THC-COOH concentrations were found between the two groups when measured during the screening visit, prior to the smoking session, and throughout the day of the experiment. Receiver operating characteristic (ROC) curves were determined and two threshold criteria were proposed in order to distinguish between these groups: a free THC-COOH concentration below 3 μg/L suggested an occasional consumption (≤ 1 joint/week) while a concentration higher than 40 μg/L corresponded to a heavy use (≥ 10 joints/month). These thresholds were successfully tested with the second real case study. The two thresholds were not challenged by the presence of ethanol (40% of cases) and of other therapeutic and illegal drugs (24%). These thresholds were also found to be consistent with previously published experimental data. Conclusion: We propose the following procedure that can be very useful in the Swiss context but also in other countries with similar traffic policies: If the whole blood THC-COOH concentration is higher than 40 μg/L, traffic offenders must be directed first and foremost toward medical assessment of their fitness to drive. This evaluation is not recommended if the THC-COOH concentration is lower than 3 μg/L. A THC-COOH level between these two thresholds can't be reliably interpreted. In such a case, further medical assessment and follow up of the fitness to drive are also suggested, but with lower priority.

Reframing video gaming and internet use addiction : empirical cross-national comparison of heavy use over time and addiction scales among young users

BACKGROUND AND AIMS: Evidence-based and reliable measures of addictive disorders are needed in general population-based assessments. One study suggested that heavy use over time (UOT) should be used instead of self-reported addiction scales (AS). This study compared UOT and AS regarding video gaming and internet use empirically, using associations with comorbid factors. DESIGN: Cross-sectional data from the 2011 French Survey on Health and Consumption on Call-up and Preparation for Defence-Day (ESCAPAD), cross-sectional data from the 2012 Swiss ado@internet.ch study and two waves of longitudinal data (2010-13) of the Swiss Longitudinal Cohort Study on Substance Use Risk Factors (C-SURF). SETTING: Three representative samples from the general population of French and Swiss adolescents and young Swiss men, aged approximately 17, 14 and 20 years, respectively. PARTICIPANTS: ESCAPAD: n =22 945 (47.4% men); ado@internet.ch: n =3049 (50% men); C-SURF: n =4813 (baseline + follow-up, 100% men). MEASUREMENTS: We assessed video gaming/internet UOT ESCAPAD and ado@internet.ch: number of hours spent online per week, C-SURF: latent score of time spent gaming/using internet] and AS (ESCAPAD: Problematic Internet Use Questionnaire, ado@internet.ch: Internet Addiction Test, C-SURF: Gaming AS). Comorbidities were assessed with health outcomes (ESCAPAD: physical health evaluation with a single item, suicidal thoughts, and appointment with a psychiatrist; ado@internet.ch: WHO-5 and somatic health problems; C-SURF: Short Form 12 (SF-12 Health Survey) and Major Depression Inventory (MDI). FINDINGS: UOT and AS were correlated moderately (ESCAPAD: r = 0.40, ado@internet.ch: r = 0.53 and C-SURF: r = 0.51). Associations of AS with comorbidity factors were higher than those of UOT in cross-sectional (AS: .005 ≤ |b| ≤ 2.500, UOT: 0.001 ≤ |b| ≤ 1.000) and longitudinal analyses (AS: 0.093 ≤ |b| ≤ 1.079, UOT: 0.020 ≤ |b| ≤ 0.329). The results were similar across gender in ESCAPAD and ado@internet.ch (men: AS: 0.006 ≤ |b| ≤ 0.211, UOT: 0.001 ≤ |b| ≤ 0.061; women: AS: 0.004 ≤ |b| ≤ 0.155, UOT: 0.001 ≤ |b| ≤ 0.094). CONCLUSIONS: The measurement of heavy use over time captures part of addictive video gaming/internet use without overlapping to a large extent with the results of measuring by self-reported addiction scales (AS). Measuring addictive video gaming/internet use via self-reported addiction scales relates more strongly to comorbidity factors than heavy use over time.

Stabilization of fluoroindate glasses by magnesium fluoride and other heavy metal fluorides

Their extended transparency in the IR makes them attractive for use as optical fibers for CO laser power delivery and optical amplification. This paper firstly describes the spectacular stabilizing effect of MgF2 on the binary system InF3-BaF2. The investigation of the InF3-BaF2-MgF2 system led to samples up to 5mm in thickness. Further optimization of this system was achieved by incorporation of limited amounts of other fluorides and resulted in increased resistence to devitrification. The second approach of this work was concerned to the investigation of the pseudo-ternary system InF3-GdF3-GaF3 at constant concentrations of ZnF2-SrF2-BaF2-NaF. Several compositions were studied in this system. The samples presented a better thermal stability when compared to other families of fluoride glasses. Therefore, these glasses seem to be very promising for the fabrication of special optical fibers. Thermal data are reported.

Quantitative evidence for neurofilament heavy subunit aggregation in motor neurons of spinal cords of patients with amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS), a neurodegenerative disease of unknown etiology, affects motor neurons leading to atrophy of skeletal muscles, paralysis and death. There is evidence for the accumulation of neurofilaments (NF) in motor neurons of the spinal cord in ALS cases. NF are major structural elements of the neuronal cytoskeleton. They play an important role in cell architecture and differentiation and in the determination and maintenance of fiber caliber. They are composed of three different polypeptides: light (NF-L), medium (NF-M) and heavy (NF-H) subunits. In the present study, we performed a morphological and quantitative immunohistochemical analysis to evaluate the accumulation of NF and the presence of each subunit in control and ALS cases. Spinal cords from patients without neurological disease and from ALS patients were obtained at autopsy. In all ALS cases there was a marked loss of motor neurons, besides atrophic neurons and preserved neurons with cytoplasmic inclusions, and extensive gliosis. In control cases, the immunoreaction in the cytoplasm of neurons was weak for phosphorylated NF-H, strong for NF-M and weak for NF-L. In ALS cases, anterior horn neurons showed intense immunoreactivity in focal regions of neuronal perikarya for all subunits, although the difference in the integrated optical density was statistically significant only for NF-H. Furthermore, we also observed dilated axons (spheroids), which were immunopositive for NF-H but negative for NF-M and NF-L. In conclusion, we present qualitative and quantitative evidence of NF-H subunit accumulation in neuronal perikarya and spheroids, which suggests a possible role of this subunit in the pathogenesis of ALS.

Élaboration d’un anticorps chimère anti-gp350 comme traitement prophylactique éventuel des syndromes lymphoprolifératifs B chez les greffés

Le virus Epstein-Barr (VEB) est fortement associé au développement de syndromes lymphoprolifératifs (SLP) en greffe pédiatrique. Ce virus a la capacité d’immortaliser les lymphocytes B et de provoquer leur prolifération incontrôlée chez l’hôte immunodéprimé. Plusieurs études démontrent que le cycle lytique du virus jouerait un rôle primordial dans la genèse des SLP en produisant des particules virales pouvant infecter les cellules B adjacentes. Chez un individu immunodéprimé, ces cellules B nouvellement infectées peuvent donner naissance à une expansion lymphocytaire. Le projet présenté dans ce mémoire fait partie d’un programme de recherche visant à élucider le rôle de l’infection productive par le VEB dans le développement des SLP. L’objectif précis de ce projet est de développer un anticorps monoclonal chimère contre la glycoprotéine gp350 du VEB dans le but de neutraliser le virus et d’ainsi prévenir son entrée dans les cellules B. Notre laboratoire a construit une version chimère de l’anticorps monoclonal murin 72A1, lequel se lie à la gp350 et bloque l’infection. Les premiers essais ont révélé la présence de chaînes non fonctionnelles (aberrantes) dans l’hybridome produisant l’anticorps 72A1. La construction de la chaîne légère authentique est maintenant complète alors que celle de la chaîne lourde est toujours en cours. Le processus de caractérisation de l’anticorps chimère inclura des essais de cytotoxicité à médiation cellulaire dépendante des anticorps (ADCC). Dans cette optique, une lignée cellulaire exprimant de façon stable la gp350 a été établie. Notre anticorps chimère anti-gp350 pourrait éventuellement être utilisé comme thérapie préventive chez les greffés présentant un risque élevé de SLP en empêchant l’infection des cellules B adjacentes.

Influence du chondroïtine sulfate (CS) sur l’activité et l’expression de plusieurs isoformes du cytochrome P450 et de la NADPH P450 réductase

Le CS fait partie de la famille des SYSADOA (SYmptomatic Slow Acting Drugs for OsteoArthritis) et est utilisé par les patients avec de l’ostéoarthrose de façon chronique pour ses propriétés anti-inflammatoires. Étant donné que ces patients reçoivent d’autres médicaments, il était intéressant de documenter les effets du CS sur le cytochrome P450 et la NADPH-réductase (NADPH). Pour cette étude, deux modèles ont été utilisés: des lapins témoins (LT) et des lapins avec une réaction inflammatoire (LRI) afin de diminuer l’activité et l’expression du CYP. Six groupes contenant chacun cinq lapins ont été utilisés: un groupe sans CS et deux groupes qui ont pris oralement dans l’eau approximativement 20.5 mg/kg/jour de CS pendant 20 et 30 jours; les lapins des trois groupes restants ont pris du CS comme décrit plus haut, mais ont reçu 5 ml sous-cutanées de térébenthine afin de produire une réaction inflammatoire aseptique (RIA) deux jours avant leur sacrifice, c’est-à-dire aux jours -2, 18 et 28. Les hépatocytes ont été isolés pour évaluer l’activité et l’expression du CYP3A6, CYP1A2 et NADPH et aussi le ARNm de ces protéines. In vitro, nous avons étudié l’effet de différentes concentrations de CS-disaccharides sulfatés, 4S, 6S, et 4,6S de CS, sur l’activité et l’expression du CYP1A2 et du CYP3A6. Pour documenter la présence de la réaction inflammatoire, nous avons mesure les mucoprotéines, dans le sérum des lapins avec une réaction inflammatoire. Aussi nous avons mesuré la présence de l’oxide nitrique (NO) chez les hépatocytes de lapins contrôles et chez les hépatocytes des lapins avec une réaction inflammatoire. La translocation nucléaire du NF-κB a été etudiée par fluorescence chez les hépatocytes. Par comparaison aux lapins témoins, l’administration du CS pendant 20 et 30 jours n’affecte pas l’activité du CYP3A6 et du CYP1A2. La RIA a augmenté les mucoprotéines à 95,1±5,7 vs 8,4±1,6 mg/dl dans les lapins témoins (p<0,05). La RIA a diminué l’activité du CYP3A6 de 62% et l’activité du CYP 1A2 de 54%. Le CS n’empêché pas la diminution du CYP1A2 produite par la RIA. Par ailleurs, le CS n’affecte pas l’activité ni l’expression de la NADPH. La translocation nucléaire de NF-κB a été empêche par l’administration chronique de CS aux lapins avec RIA; en plus, la concentration de l’oxide nitrique n’a pas démontré une augmentation en présence de CS; par contre, CS n’empêche pas l’augmentation des séromucoïdes. Au contraire, CS affecte la diminution du CYP3A6 en fonction de temps et secondaire à la RIA. Dans ce group, CS a rétabli le niveau des protéines du CYP3A6 observé dans le group de lapins témoins. Pourtant cette croissance été independante de mRNA qui garde un niveau trés bas. Le plus remarcable a été la manière dont CS a augmenté la protéine du CYP3A6, sans avoir rétabli l’activité de cet isoforme. Finalement, in vitro, CS et ses trois disaccharides sulfatés (4S, 6S et 4,6S) n’affectent ni l’activité ni l’expression de CYP1A2, CYP3A6 et de la NADPH. En conclusion, l’administration chronique de CS n’affecte pas l’activité ni l’expression du CYP1A2, ou la diminution du CYP1A2 produite par la réaction inflammatoire. Le CS n’affecte pas l’activité ni l’expression du NADPH. Cependant, CS empêche la diminution du CYP3A6 en fonction de temps et secondaire à la RIA.

Detection and quantification of staphylococcus aureus enterotoxin B in food product using isotopic dilution techniques and mass spectrometry

L’entérotoxine B staphylococcique (SEB) est une toxine entérique hautement résistante à la chaleur et est responsable de plus de 50 % des cas d’intoxication d’origine alimentaire par une entérotoxine. L’objectif principal de ce projet de maîtrise est de développer et valider une méthode basée sur des nouvelles stratégies analytiques permettant la détection et la quantification de SEB dans les matrices alimentaires. Une carte de peptides tryptiques a été produite et 3 peptides tryptiques spécifiques ont été sélectionnés pour servir de peptides témoins à partir des 9 fragments protéolytiques identifiés (couverture de 35 % de la séquence). L’anhydride acétique et la forme deutérée furent utilisés afin de synthétiser des peptides standards marqués avec un isotope léger et lourd. La combinaison de mélanges des deux isotopes à des concentrations molaires différentes fut utilisée afin d’établir la linéarité et les résultats ont démontré que les mesures faites par dilution isotopique combinée au CL-SM/SM respectaient les critères généralement reconnus d’épreuves biologiques avec des valeurs de pente près de 1, des valeurs de R2 supérieure à 0,98 et des coefficients de variation (CV%) inférieurs à 8 %. La précision et l’exactitude de la méthode ont été évaluées à l’aide d’échantillons d’homogénat de viande de poulet dans lesquels SEB a été introduite. SEB a été enrichie à 0,2, 1 et 2 pmol/g. Les résultats analytiques révèlent que la méthode procure une plage d’exactitude de 84,9 à 91,1 %. Dans l’ensemble, les résultats présentés dans ce mémoire démontrent que les méthodes protéomiques peuvent être utilisées efficacement pour détecter et quantifier SEB dans les matrices alimentaires. Mots clés : spectrométrie de masse; marquage isotopique; protéomique quantitative; entérotoxines