Unexpected Role for Connexin37 in Circulating FVIII and Coagulation

The gap junction protein connexin37 (Cx37) plays an important role in cell-cell communication in the vasculature. Cx37 is expressed in endothelial cells, platelets and megakaryocytes. We have recently shown that Cx37 limits thrombus propensity by permitting intercellular signaling between aggregating platelets. Here, we have performed high throughput phage display to identify potential binding partners for the regulatory intracellular C-terminus of Cx37 (Cx37CT). We retrieved 2 consensus binding motifs for Cx37CT: WHK...[K,R]XP... and FH-K...[K,R]XXP.... Sequence alignment against the NCBI protein database indicated 66% homology of one the selected peptides with FVIII B-domain. We performed cross-linking reactions using BS3 and confirmed that an 11-mer peptide of the FVIII B-domain sequence linked to recombinant Cx37CT. In vitro binding of this peptide to Cx37CT was also confirmed by surface plasmon resonance. The dissociation constant of FVIII B-domain peptides to Cx37CT was ~20 uM. Other peptide sequences, designed upstream or downstream of the FVIII B-domain sequence, showed very low or no affinity for Cx37CT. Finally, in vivo studies revealed that thrombin generation in platelet-poor plasma from Cx37-/- mice (endogenous thrombin potential: 634±11 nM min, mean±SEM) was increased compared to Cx37+/+ mice (427±12, P<0.001). Moreover, partial activated thromboplastin time (aPTT) was shorter in Cx37-/- (39.7±1.5 s) than in Cx37+/+ mice (45.9±1.8, P=0.03), whereas prothrombin time was comparable. The shorter aPTT in Cx37-/- mice correlated with higher circulating FVIII activity (46.0±0.7 vs. 53.5±2.7 s for Cx37+/+, P=0.03). Overall, our data show for the first time a functional interaction between FVIII and Cx37. This interaction may be relevant for the control of FVIII secretion and, thereby, in the regulation of levels of FVIII circulating in blood. In addition, these results may open new perspectives to improve the efficiency of recombinant FVIII manufacturing.

Pharmacogenetics-based population pharmacokinetic analysis of etravirine in HIV-1 infected individuals.

OBJECTIVES: Etravirine (ETV) is metabolized by cytochrome P450 (CYP) 3A, 2C9, and 2C19. Metabolites are glucuronidated by uridine diphosphate glucuronosyltransferases (UGT). To identify the potential impact of genetic and non-genetic factors involved in ETV metabolism, we carried out a two-step pharmacogenetics-based population pharmacokinetic study in HIV-1 infected individuals. MATERIALS AND METHODS: The study population included 144 individuals contributing 289 ETV plasma concentrations and four individuals contributing 23 ETV plasma concentrations collected in a rich sampling design. Genetic variants [n=125 single-nucleotide polymorphisms (SNPs)] in 34 genes with a predicted role in ETV metabolism were selected. A first step population pharmacokinetic model included non-genetic and known genetic factors (seven SNPs in CYP2C, one SNP in CYP3A5) as covariates. Post-hoc individual ETV clearance (CL) was used in a second (discovery) step, in which the effect of the remaining 98 SNPs in CYP3A, P450 cytochrome oxidoreductase (POR), nuclear receptor genes, and UGTs was investigated. RESULTS: A one-compartment model with zero-order absorption best characterized ETV pharmacokinetics. The average ETV CL was 41 (l/h) (CV 51.1%), the volume of distribution was 1325 l, and the mean absorption time was 1.2 h. The administration of darunavir/ritonavir or tenofovir was the only non-genetic covariate influencing ETV CL significantly, resulting in a 40% [95% confidence interval (CI): 13-69%] and a 42% (95% CI: 17-68%) increase in ETV CL, respectively. Carriers of rs4244285 (CYP2C19*2) had 23% (8-38%) lower ETV CL. Co-administered antiretroviral agents and genetic factors explained 16% of the variance in ETV concentrations. None of the SNPs in the discovery step influenced ETV CL. CONCLUSION: ETV concentrations are highly variable, and co-administered antiretroviral agents and genetic factors explained only a modest part of the interindividual variability in ETV elimination. Opposing effects of interacting drugs effectively abrogate genetic influences on ETV CL, and vice-versa.

Melanin-based colorations signal strategies to cope with poor and rich environments

One hypothesis for the maintenance of genetic variation states that alternative genotypes are adapted to different environmental conditions (i.e., genotype-by-environment interaction GxE) that vary in space and time. Although GxE has been demonstrated for morphological traits, little evidence has been given whether these GxE are associated with traits used as signal in mate choice. In three wild bird species, we investigated whether the degree of melanin-based coloration, a heritable trait, covaries with nestling growth rate in rich and poor environments. Variation in the degree of reddish-brown phaeomelanism is pronounced in the barn owl (Tyto alba) and tawny owl (Strix aluco), and variation in black eumelanism in the barn owl and Alpine swift (Apus melba). Melanin-based coloration has been shown to be a criterion in mate choice in the barn owl. We cross-fostered hatchlings to test whether nestlings sired by parents displaying melanin-based colorations to different extent exhibit alternative growth trajectories when raised by foster parents in poor (experimentally enlarged broods) and rich (experimentally reduced broods) environments. With respect to phaeomelanism, barn owl and tawny owl offspring sired by redder parents grew more rapidly in body mass only in experimentally reduced broods. With respect to eumelanism, Alpine swift offspring of darker fathers grew their wings more rapidly only in experimentally enlarged broods, a difference that was not detected in reduced broods. These interactions between parental melanism and offspring growth rate indicate that individuals display substantial plasticity in response to the rearing environment which is associated with the degree of melanism: at least with respect to nestling growth, phaeomelanic and eumelanic individuals are best adapted to rich and poor environments, respectively. It now remains to be investigated why eumelanism and phaeomelanism have a different signaling function and what the lifelong consequences of these melanism-dependent allocation strategies are. This is important to fully appraise the role played by environmental heterogeneity in maintaining variation in the degree of melanin-based coloration.

Polyphenol-rich foods in the Mediterranean diet are associated with better cognitive function in elderly subjects at high cardiovascular risk

Brain oxidative processes play a major role in age-related cognitive decline, thus consumption of antioxidant-rich foods might help preserve cognition. Our aim was to assess whether consumption of antioxidant-rich foods in the Mediterranean diet relates to cognitive function in the elderly. In asymptomatic subjects at high cardiovascular risk (n = 447; 52% women; age 5580 y) enrolled in the PREDIMED study, a primary prevention dietary-intervention trial, we assessed food intake and cardiovascular risk profile, determined apolipoprotein E genotype, and used neuropsychological tests to evaluate cognitive function.We also measured urinary polyphenols as an objective biomarker of intake. Associations between energy-adjusted food consumption, urinary polyphenols, and cognitive scores were assessed by multiple linear regression models adjusted for potential confounders. Consumption of some foods was independently related to better cognitive function. The specific associations [regression coefficients (95% confidence intervals)] were: total olive oil with immediate verbal memory [0.755 (0.1511.358)]; virgin olive oil and coffee with delayed verbal memory [0.163 (0.0100.316) and 0.294 (0.0550.534), respectively];walnuts with working memory [1.191 (0.0612.322)]; and wine with Mini-Mental State Examination scores [0.252 (0.0060.496)]. Urinary polyphenols were associated with better scores in immediate verbal memory [1.208 (0.2362.180)]. Increased consumption of antioxidant-rich foods in general and of polyphenols in particular is associated with better cognitive performance in elderly subjects at high cardiovascular risk. The results reinforce the notion that Mediterranean diet components might counteract age-related cognitive decline.

Prediction of free imatinib concentrations based on total plasma concentrations in patients with gastrointestinal stromal tumours.

AIM: Total imatinib concentrations are currently measured for the therapeutic drug monitoring of imatinib, whereas only free drug equilibrates with cells for pharmacological action. Due to technical and cost limitations, routine measurement of free concentrations is generally not performed. In this study, free and total imatinib concentrations were measured to establish a model allowing the confident prediction of imatinib free concentrations based on total concentrations and plasma proteins measurements. METHODS: One hundred and fifty total and free plasma concentrations of imatinib were measured in 49 patients with gastrointestinal stromal tumours. A population pharmacokinetic model was built up to characterize mean total and free concentrations with inter-patient and intrapatient variability, while taking into account α1 -acid glycoprotein (AGP) and human serum albumin (HSA) concentrations, in addition to other demographic and environmental covariates. RESULTS: A one compartment model with first order absorption was used to characterize total and free imatinib concentrations. Only AGP influenced imatinib total clearance. Imatinib free concentrations were best predicted using a non-linear binding model to AGP, with a dissociation constant Kd of 319 ng ml(-1) , assuming a 1:1 molar binding ratio. The addition of HSA in the equation did not improve the prediction of imatinib unbound concentrations. CONCLUSION: Although free concentration monitoring is probably more appropriate than total concentrations, it requires an additional ultrafiltration step and sensitive analytical technology, not always available in clinical laboratories. The model proposed might represent a convenient approach to estimate imatinib free concentrations. However, therapeutic ranges for free imatinib concentrations remain to be established before it enters into routine practice.

Increased plasma levels of N-terminal brain natriuretic peptide (NT-proBNP) in type 2 diabetic patients with vascular complications.

AIMS: The plasma levels of either brain natriuretic peptide (BNP) or the N-terminal fragment of the prohormone (NT-proBNP) have recently gained extreme importance as markers of myocardial dysfunction. Patients with type 2 diabetes are at high risk of developing cardiovascular complications. This study was aimed to assess whether plasma NT-proBNP levels are at similar levels in type 2 diabetics with or without overt cardiovascular diseases. METHODS: We assayed plasma NT-proBNP in 54 type 2 diabetics, 27 of whom had no overt macro- and/or microvascular complications, while the remaining ones had either or both. The same assay was carried out in 38 healthy control subjects age and sex matched as a group with the diabetics. RESULTS: Plasma NT-proBNP was higher in diabetics (median 121 pg/ml, interquartile range 50-240 pg/ml, ) than in those without complications (37 pg/ml, 21-54 pg/ml, P<0.01). Compared with the controls (55 pg/ml, 40-79 pg/ml), only diabetics with vascular complications had significantly increased plasma NT-proBNP levels (P<0.001). In the diabetics, coronary heart disease and nephropathy (defined according to urinary excretion of albumin) were each independently associated with elevated values of plasma NT-proBNP. CONCLUSIONS: In type 2 diabetes mellitus, patients with macro- and/or micro-vascular complications exhibit an elevation of plasma NT-proBNP levels compared to corresponding patients with no evidence of vascular disease. The excessive secretion of this peptide is independently associated with coronary artery disease and overt nephropathy. The measurement of circulating NT-proBNP concentration may therefore be useful to screen for the presence of macro- and/or microvascular disease.

Plasma PCSK9 concentrations during an oral fat load and after short term high-fat, high-fat high-protein and high-fructose diets.

BACKGROUND: PCSK9 (Proprotein Convertase Subtilisin Kexin type 9) is a circulating protein that promotes hypercholesterolemia by decreasing hepatic LDL receptor protein. Under non interventional conditions, its expression is driven by sterol response element binding protein 2 (SREBP2) and follows a diurnal rhythm synchronous with cholesterol synthesis. Plasma PCSK9 is associated to LDL-C and to a lesser extent plasma triglycerides and insulin resistance. We aimed to verify the effect on plasma PCSK9 concentrations of dietary interventions that affect these parameters. METHODS: We performed nutritional interventions in young healthy male volunteers and offspring of type 2 diabetic (OffT2D) patients that are more prone to develop insulin resistance, including: i) acute post-prandial hyperlipidemic challenge (n=10), ii) 4 days of high-fat (HF) or high-fat/high-protein (HFHP) (n=10), iii) 7 (HFruc1, n=16) or 6 (HFruc2, n=9) days of hypercaloric high-fructose diets. An acute oral fat load was also performed in two patients bearing the R104C-V114A loss-of-function (LOF) PCSK9 mutation. Plasma PCSK9 concentrations were measured by ELISA. For the HFruc1 study, intrahepatocellular (IHCL) and intramyocellular lipids were measured by 1H magnetic resonance spectroscopy. Hepatic and whole-body insulin sensitivity was assessed with a two-step hyperinsulinemic-euglycemic clamp (0.3 and 1.0 mU.kg-1.min-1). FINDINGS: HF and HFHP short-term diets, as well as an acute hyperlipidemic oral load, did not significantly change PCSK9 concentrations. In addition, post-prandial plasma triglyceride excursion was not altered in two carriers of PCSK9 LOF mutation compared with non carriers. In contrast, hypercaloric 7-day HFruc1 diet increased plasma PCSK9 concentrations by 28% (p=0.05) in healthy volunteers and by 34% (p=0.001) in OffT2D patients. In another independent study, 6-day HFruc2 diet increased plasma PCSK9 levels by 93% (p<0.0001) in young healthy male volunteers. Spearman's correlations revealed that plasma PCSK9 concentrations upon 7-day HFruc1 diet were positively associated with plasma triglycerides (r=0.54, p=0.01) and IHCL (r=0.56, p=0.001), and inversely correlated with hepatic (r=0.54, p=0.014) and whole-body (r=-0.59, p=0.0065) insulin sensitivity. CONCLUSIONS: Plasma PCSK9 concentrations vary minimally in response to a short term high-fat diet and they are not accompanied with changes in cholesterolemia upon high-fructose diet. Short-term high-fructose intake increased plasma PCSK9 levels, independent on cholesterol synthesis, suggesting a regulation independent of SREBP-2. Upon this diet, PCSK9 is associated with insulin resistance, hepatic steatosis and plasma triglycerides.

Nanoparticles in SiH4-Ar plasma: Modelling and comparison with experimental data

Experimental and theoretical investigations for growth of silicon nanoparticles (4 to 14 nm) in radio frequency discharge were carried out. Growth processes were performed with gas mixtures of SiH4 and Ar in a plasma chemical reactor at low pressure. A distinctive feature of presented kinetic model of generation and growth of nanoparticles (compared to our earlier model) is its ability to investigate small"critical" dimensions of clusters, determining the rate of particle production and taking into account the influence of SiH2 and Si2Hm dimer radicals. The experiments in the present study were extended to high pressure (≥20 Pa) and discharge power (≥40 W). Model calculations were compared to experimental measurements, investigating the dimension of silicon nanoparticles as a function of time, discharge power, gas mixture, total pressure, and gas flow.

Determination of the Enantiomers of Mianserin and its Metabolites in Plasma by Capillary Electrophoresis After Liquid-Liquid Extraction and On-Column Sample Preconcentration

Capillary electrophoresis has drawn considerable attention in the past few years, particularly in the field of chiral separations because of its high separation efficiency. However, its routine use in therapeutic drug monitoring is hampered by its low sensitivity due to a short optical path. We have developed a capillary zone electrophoresis (CZE) method using 2mM of hydroxypropyl-β-cyclodextrin as a chiral selector, which allows base-to-base separation of the enantiomers of mianserin (MIA), desmethylmianserin (DMIA), and 8-hydroxymianserin (OHMIA). Through the use of an on-column sample concentration step after liquid-liquid extraction from plasma and through the presence of an internal standard, the quantitation limits were found to be 5 ng/mL for each enantiomer of MIA and DMIA and 15 ng/mL for each enantiomer of OHMIA. To our knowledge, this is the first published CE method that allows its use for therapeutic monitoring of antidepressants due to its sensitivity down to the low nanogram range. The variability of the assays, as assessed by the coefficients of variation (CV) measured at two concentrations for each substance, ranged from 2 to 14% for the intraday (eight replicates) and from 5 to 14% for the interday (eight replicates) experiments. The deviations from the theoretical concentrations, which represent the accuracy of the method, were all within 12.5%. A linear response was obtained for all compounds within the range of concentrations used for the calibration curves (10-150 ng/mL for each enantiomer of MIA and DMIA and 20-300 ng/mL for each enantiomer of OHMIA). Good correlations were calculated between [(R) + (S)]-MIA and DMIA concentrations measured in plasma samples of 20 patients by a nonchiral gas chromatography method and CZE, and between the (R)- and (S)-concentrations of MIA and DMIA measured in plasma samples of 37 patients by a previously described chiral high-performance liquid chromatography method and CZE. Finally, no interference was noted from more than 20 other psychotropic drugs. Thus, this method, which is both sensitive and selective, can be routinely used for therapeutic monitoring of the enantiomers of MIA and its metabolites. It could be very useful due to the demonstrated interindividual variability of the stereoselective metabolism of MIA.

A new concept of integrated cardiopulmonary bypass circuit.

OBJECTIVE: Standard cardiopulmonary bypass (CPB) circuits with their large surface area and volume contribute to postoperative systemic inflammatory reaction and hemodilution. In order to minimize these problems a new approach has been developed resulting in a single disposable, compact arterio-venous loop, which has integral kinetic-assist pumping, oxygenating, air removal, and gross filtration capabilities (CardioVention Inc., Santa Clara, CA, USA). The impact of this system on gas exchange capacity, blood elements and hemolysis is compared to that of a conventional circuit in a model of prolonged perfusion. METHODS: Twelve calves (mean body weight: 72.2+/-3.7 kg) were placed on cardiopulmonary bypass for 6 h with a flow of 5 l/min, and randomly assigned to the CardioVention system (n=6) or a standard CPB circuit (n=6). A standard battery of blood samples was taken before bypass and throughout bypass. Analysis of variance was used for comparison. RESULTS: The hematocrit remained stable throughout the experiment in the CardioVention group, whereas it dropped in the standard group in the early phase of perfusion. When normalized for prebypass values, both profiles differed significantly (P<0.01). Both O2 and CO2 transfers were significantly improved in the CardioVention group (P=0.04 and P<0.001, respectively). There was a slightly higher pressure drop in the CardioVention group but no single value exceeded 112 mmHg. No hemolysis could be detected in either group with all free plasma Hb values below 15 mg/l. Thrombocyte count, when corrected by hematocrit and normalized by prebypass values, exhibited an increased drop in the standard group (P=0.03). CONCLUSION: The CardioVention system with its concept of limited priming volume and exposed foreign surface area, improves gas exchange probably because of the absence of detectable hemodilution, and appears to limit the decrease in the thrombocyte count which may be ascribed to the reduced surface. Despite the volume and surface constraints, no hemolysis could be detected throughout the 6 h full-flow perfusion period.

Consommation d'aliments riches en calcium dans la population adulte de Suisse romande et du Tessin. [Consumption of calcium-rich food in the adult population of French-speaking Switzerland and of Tessin].

Food intake high in calcium content is important in the development of skeleton and the prevention of osteoporosis. From a public health perspective, it is therefore important to know the dietary calcium intake of a population. Two population surveys in the French and Italian parts of Switzerland (MONICA, 1988-1989, and Geneva, 1991), were combined to study nutritional habits related to calcium intake. A random population sample, aged 35 et 65 years, answered to a 24 hour recall questionnaire, either self-administered (MONICA, cantons of Vaud, Fribourg and Tessin, N = 2734) or by phone (canton of Geneva, N = 475). In the 4 cantons, for the previous day, 60% of participants ate dairy products, particularly whole milk and cheese. They drunk 3-4 dl/day of milk. A significant proportion (10%) of french and italian speaking Swiss did not consume any food high in calcium content. In Geneva, the mean daily calcium intake was 656 mg in men and 489 mg in women. In conclusion 1) eating habits related to calcium intake are similar across cantons; 2) women consume dairy products more frequently than men, but in smaller quantities; 3) about 60% of men and 80% of women do not get the daily amount of calcium recommended for the prevention of osteoporosis.

Analytical interference of 4-hydroxy-3-methoxymethamphetamine with the measurement of plasma free normetanephrine by ultra-high pressure liquid chromatography-tandem mass spectrometry.

OBJECTIVES: The diagnosis of pheochromocytoma relies on the measurement of plasma free metanephrines assay whose reliability has been considerably improved by ultra-high pressure liquid chromatography tandem mass spectrometry (UHPLC-MS/MS). Here we report an analytical interference occurring between 4-hydroxy-3-methoxymethamphetamine (HMMA), a metabolite of 3,4-methylenedioxymethamphetamine (MDMA, "Ecstasy"), and normetanephrine (NMN) since they share a common pharmacophore resulting in the same product ion after fragmentation. DESIGN AND METHODS: Synthetic HMMA was spiked into plasma samples containing various concentrations of NMN and the intensity of the interference was determined by UPLC-MS/MS before and after improvement of the analytical method. RESULTS: Using a careful adjustment of chromatographic conditions including the change of the UPLC analytical column, we were able to distinguish both compounds. HMMA interference for NMN determination should be seriously considered since MDMA activates the sympathetic nervous system and if confounded with NMN may lead to false-positive tests when performing a differential diagnostic of pheochromocytoma.

Increased trimipramine plasma levels during fluvoxamine comedication.

A depressive patient, a non-responder to trimipramine (TRI), was comedicated first with citalopram (CIT) and then with fluvoxamine (FLUV). Both the TRI-CIT and TRI-FLUV combination treatments led to a worsening of the depressive state and to the appearance of panic attacks. The addition of FLUV to TRI resulted in a twofold increase of the plasma levels of TRI and to a slight increase of its N-demethylated and 2-hydroxylated metabolites. These results suggest that the interaction between FLUV and TRI occurred at the level of cytochrome P-450IID6 and cytochrome P-450meph in this patient, phenotyped as an extensive metabolizer of both dextromethorphan and mephenytoin. The adverse effects were possibly due to (a) a pharmacokinetic interaction between CIT and FLUV with TRI and/or (b) alterations in serotonergic and/or dopaminergic neurotransmission.