Agonist-induced internalization and recycling of the glucagon-like peptide-1 receptor in transfected fibroblasts and in insulinomas.

Glucagon-like peptide-1 (GLP-1) is the most potent stimulator of glucose-induced insulin secretion and its pancreatic beta-cell receptor is a member of a new subfamily of G-protein-coupled receptors which includes the receptors for vasoactive intestinal polypeptide, secretin and glucagon. Here we studied agonist-induced GLP-1 receptor internalization in receptor-transfected Chinese hamster lung fibroblasts using three different approaches. First, iodinated GLP-1 bound at 4 degrees C to transfected cells was internalized with a t 1/2 of 2-3 min following warming up of the cells to 37 degrees C. Secondly, exposure to GLP-1 induced a shift in the distribution of the receptors from plasma membrane-enriched to endosomes-enriched membrane fractions, as assessed by Western blot detection of the receptors using specific antibodies. Thirdly, continuous exposure of GLP-1 receptor-expressing cells to iodinated GLP-1 led to a linear accumulation of peptide degradation products in the medium following a lag time of 20-30 min, indicating a continuous cycling of the receptor between the plasma membrane and endosomal compartments. Potassium depletion and hypertonicity inhibited transferrin endocytosis, a process known to occur via coated pit formation, as well as GLP-1 receptor endocytosis. In contrast to GLP-1, the antagonist exendin-(9-39) did not lead to receptor endocytosis. Surface re-expression following one round of GLP-1 receptor endocytosis occurred with a half-time of about 15 min. The difference in internalization and surface re-expression rates led to a progressive redistribution of the receptor in intracellular compartments upon continuous exposure to GLP-1. Finally, endogenous GLP-1 receptors expressed by insulinoma cells were also found to be internalized upon agonist binding. Together our data demonstrate that the GLP-1 receptor is internalized upon agonist binding by a route similar to that taken by single transmembrane segment receptors. The characterization of the pathway and kinetics of GLP-1-induced receptor endocytosis will be helpful towards understanding the role of internalization and recycling in the control of signal transduction by this receptor.

The anti-lymphoma activity of APO866, an inhibitor of nicotinamide adenine dinucleotide biosynthesis, is potentialized when used in combination with anti-CD20 antibody.

Abstract APO866 is an inhibitor of nicotinamide adenine dinucleotide (NAD) biosynthesis that exhibits potent anti-lymphoma activity. Rituximab (RTX), an anti-CD20 antibody, kills lymphoma cells by direct apoptosis and antibody- and complement-dependent cell-mediated cytotoxicities, and has clinical efficacy in non-Hodgkin cell lymphomas. In the present study, we evaluated whether RTX could potentiate APO866-induced human B-lymphoma cell death and shed light on death-mediated mechanisms associated with this drug combination. We found that RTX significantly increases APO866-induced death in lymphoma cells from patients and lines. Mechanisms include enhancement of autophagy-mediated cell death, activation of caspase 3 and exacerbation of mitochondrial depolarization, but not increase of reactive oxygen species (ROS) production, when compared with those induced by each drug alone. In vivo, combined administration of APO866 with RTX in a laboratory model of human aggressive lymphoma significantly decreased tumor burden and prolonged survival over single-agent treatment. Our study demonstrates that the combination of RTX and APO866 optimizes B-cell lymphoma apoptosis and therapeutic efficacy over both compounds administered separately.

New generation vaccine induces effective melanoma-specific CD8+ T cells in the circulation but not in the tumor site.

Although increasing evidence suggests that CTL are important to fight the development of some cancers, the frequency of detectable tumor-specific T cells is low in cancer patients, and these cells have generally poor functional capacities, compared with virus-specific CD8(+) T cells. The generation with a vaccine of potent CTL responses against tumor Ags therefore remains a major challenge. In the present study, ex vivo analyses of Melan-A-specific CD8(+) T cells following vaccination with Melan-A peptide and CpG oligodeoxynucleotides revealed the successful induction in the circulation of effective melanoma-specific T cells, i.e., with phenotypic and functional characteristics similar to those of CTL specific for immunodominant viral Ags. Nonetheless, the eventual impact on tumor development in vaccinated melanoma donors remained limited. The comprehensive study of vaccinated patient metastasis shows that vaccine-driven tumor-infiltrating lymphocytes, although activated, still differed in functional capacities compared with blood counterparts. This coincided with a significant increase of FoxP3(+) regulatory T cell activity within the tumor. The consistent induction of effective tumor-specific CD8(+) T cells in the circulation with a vaccine represents a major achievement; however, clinical benefit may not be achieved unless the tumor environment can be altered to enable CD8(+) T cell efficacy.

Effects of carbamazepine coadministration on plasma concentrations of the enantiomers of mianserin and of its metabolites.

Concentrations of the enantiomers of unconjugated and of total (unconjugated plus conjugated) mianserin, desmethylmianserin and 8-hydroxymianserin were measured in 12 patients before and after the introduction of carbamazepine. The dose of mianserin was 60 mg/d, carbamazepine was coadministered at 400 mg/d for 4 weeks, and blood samples were taken at weekly intervals after the introduction of carbamazepine. Each week, carbamazepine significantly decreased plasma concentrations of unconjugated and total (S)-mianserin (the more potent enantiomer) and of unconjugated and total (R)-mianserin. On average, plasma concentrations of unconjugated and total (S)-mianserin and of unconjugated and total (R)-mianserin were 55%, 56%, 66%, and 55%, respectively, of the corresponding values before introduction of carbamazepine. These results strongly suggest the involvement of CYP3A4, the major CYP enzyme induced by carbamazepine, in the metabolism of both enantiomers of mianserin. A strong decrease in the concentrations of (S)-8-hydroxymianserin was also measured (on average, the concentrations were 69% of the corresponding values before carbamazepine introduction). Conversely, plasma concentrations of unconjugated and of total (S)-desmethylmianserin, (R)-desmethylmianserin, and (R)-8-hydroxymianserin were only slightly modified by carbamazepine. From a clinical point of view, as a therapeutic window for (S)-mianserin has been recently suggested, the dose of racemic mianserin for a patient whose (S)-mianserin concentrations have been stabilized within this therapeutic window would need to be approximately doubled if carbamazepine, at 400 mg/d, is introduced as a comedication.

Cancers du sein : comment associer l'hormonothérapie et la radiothérapie en situation adjuvante ? [How to combine hormonotherapy and radiation treatment in adjuvant breast cancer ?]

L'hormonoradiothérapie concomitante est utilisée depuis plusieurs années en pratique clinique quotidienne dans les cancers localement évolués de la prostate. Le transfert de ce concept en pathologie mammaire a été très peu rapporté dans la littérature, mais semble pourtant licite devant l'hormonodépendance fréquente des cancers du sein et la synergie potentielle de ces deux armes thérapeutiques. En situation adjuvante, deux stratégies sont actuellement utilisées : la prescription d'un inhibiteur de l'aromatase d'emblée ou après un délai plus ou moins long de tamoxifène. En pratique, ces molécules peuvent donc interagir avec la radiothérapie adjuvante. Les études rétrospectives récemment publiées n'ont pas mis en évidence de différence significative sur l'incidence des évènements, notamment locorégionaux, de l'association concomitante ou séquentielle du tamoxifène à la radiothérapie. La toxicité de l'association reste discutable en termes de fibroses sous-cutanée et pulmonaire. Il semble que le tamoxifène aggraverait les séquelles postradiques uniquement chez les patientes prédisposées à souffrir d'effets tardifs de la radiothérapie et identifiées par un test prédictif biologique. La prudence reste donc encore de mise du moins pour ces patientes. Cet article détaille les avantages et les risques de l'utilisation concomitante de la radiothérapie et de l'hormonothérapie adjuvantes des cancers localisés du sein. Combined radiation and hormone therapies have become common clinical practice in recent years for locally-advanced prostate cancers. The use of such concomitant therapy in the treatment of breast disease has been infrequently reported in the literature, but seems justified given the common hormonal dependence of breast cancer and the potential synergistic effect of these two treatment modalities. As adjuvant therapy, two strategies are used in daily clinical practice: upfront aromatase inhibitors or sequentially after a variable delay of tamoxifen. These molecules may, thus, interact with radiotherapy. Retrospectives studies recently published did not show any differences in terms of locoregional recurrences between concurrent or sequential radiohormonotherapy. Lung and skin fibroses due to concurrent treatment are still under debate. Nevertheless, late side effects appeared to be increased by such a treatment, particularly in hypersensitive patients identified at risk by the lymphocyte predictive test. Concurrent radiohormonotherapy should, thus, be delivered cautiously at least for these patients. This article details the potent advantages and risks of concurrent use of adjuvant hormonotherapy and radiotherapy in localized breast cancers.

Clinical experience to date with nilotinib in gastrointestinal stromal tumors.

Nilotinib, a novel tyrosine kinase inhibitor (TKI) that inhibits BCR-ABL, the stem cell factor receptor (KIT), and platelet-derived growth factor receptor-alpha (PDGFRα), is approved for the treatment of patients with newly diagnosed Philadelphia chromosome-positive chronic myelogenous leukemia (CML) and those with CML that is imatinib-resistant or -intolerant. Due to its potent inhibition of KIT and PDGFRα--the two tyrosine kinases that are the central oncogenic mechanisms of gastrointestinal stromal tumors (GIST)--nilotinib also has been investigated for potential efficacy and safety in patients with GIST who have progressed on other approved treatments. Initial results have been encouraging, as nilotinib has demonstrated clinical efficacy and safety in a phase I trial as either a single agent or in combination with imatinib, as well as in heavily pretreated patients with GIST in a compassionate use program. In addition, the phase III trial of nilotinib versus best supportive care (with or without a TKI at the investigator's discretion) indicated that nilotinib may have efficacy in some third-line patients. Furthermore, the Evaluating Nilotinib Efficacy and Safety in Clinical Trials (ENEST g1 trial), a phase III randomized, open-label study comparing the safety and efficacy of imatinib versus nilotinib in the first-line treatment of patients with GIST, is currently under way. Other studies with nilotinib either have been initiated or are in development. Based on published and accruing clinical data, nilotinib shows potential as a new drug in the clinician's armamentarium for the management of GIST.

Therapeutic drug monitoring and metabolites profil analysis of antiviral drugs

The widespread use of combination antiretroviral therapy (ARVs) has considerably improved the prognosis of patients infected with HIV. Conversely, considerable advances have been recently realized for the therapy of hepatitis C infection with the recent advent of potent new anti-HCV drugs that allow an increasing rate HCV infection cure. Despite their overall efficacy, a significant number of patients do not achieve or maintain adequate clinical response, defined as an undetectable viral load for HIV, and a sustained virological response (or cure) in HCV infection. Treatment failure therefore still remains an important issue besides drugs toxicities and viral resistance which is not uncommon in a significant percentage of patients who do not reach adequate virological suppression. The reasons of variability in drug response are multifactorial and apart from viral genetics, other factors such as environmental factors, drug- drug interactions, and imperfect compliance may have profound impact on antiviral drugs' clinical response. The possibility of measuring plasma concentration of antiviral drugs enables to guide antiviral drug therapy and ensure optimal drug exposure. The overall objective of this research was to widen up the current knowledge on pharmacokinetic and pharmacogenetic factors that influence the clinical response and toxicity of current and newly approved antiretroviral and anti-HCV drugs. To that endeavour, analytical methods using liquid chromatography coupled with tandem mass spectrometry have been developed and validated for the precise and accurate measurement of new antiretroviral and anti-HCV drugs . These assays have been applied for the TDM of ARVs and anti-HCV in patients infected with either HIV or HCV respectively, and co-infected with HIV- HCV. A pharmacokinetic population model was developed to characterize inter and intra-patient variability of rilpivirine, the latest marketed Non Nucleoside Reverse transcriptase (NNRTI) Inhibitor of HIVand to identify genetic and non genetic covariates influencing rilpivirine exposure. None of the factors investigated so far showed however any influence of RPV clearance. Importantly, we have found that the standard daily dosage regimen (25 mg QD) proposed for rilpivirine results in concentrations below the proposed therapeutic target in about 40% of patients. In these conditions, virologie escape is a potential risk that remains to be further investigated, notably via the TDM approach that can be a useful tool to identify patients who are at risk for being exposed to less than optimal levels of rilpivirine in plasma. Besides the last generation NNRTI rilpivirine, we have studied efavirenz, the major NNRTI clinically used so far. Namely for efavirenz, we aimed at identifying a potential new marker of toxicity that may be incriminated for the neuropsychological sides effects and hence discontinuation of efavirenz therapy. To that endeavour, a comprehensive analysis of phase I and phase II metabolites profiles has been performed in plasma, CSF and in urine from patients under efavirenz therapy. We have found that phase II metabolites of EFV constitute the major species circulating in blood, sometimes exceeding the levels of the parent drug efavirenz. Moreover we have identified a new metabolite of efavirenz in humans, namely the 8-OH-EFV- sulfate which is present at high concentrations in all body compartments from patients under efavirenz therapy. These investigations may open the way to possible alternate phenotypic markers of efavirenz toxicity. Finally, the specific influence of P-glycoprotein on the cellular disposition of a series ARVs (NNRTIs and Pis] has been studies in in vitro cell systems using the siRNA silencing approach. -- Depuis l'introduction de la thérapie antirétrovirale (ARVs) la morbidité et la mortalité liées au VIH ont considérablement diminué. En parallèle le traitement contre le virus de l'hépatite C (VHC) a connu récemment d'énormes progrès avec l'arrivée de nouveaux médicaments puissants, ce qui a permis une augmentation considérable de la guérison de l'infection par le VHC. En dépit de l'efficacité de ces traitements antiviraux, les échecs thérapeutiques ainsi que les effets secondaires des traitements restent un problème important. Une réponse imparfaite ou la toxicité du traitement est certainement multifactorielle. Le suivi thérapeutique des médicaments [Therapeutic Drug Monitoring TDM) à travers la mesure des concentrations plasmatiques constitue une approche importante pour guider le traitement médicamenteux et de s'assurer que les patients sont exposés à des concentrations optimales des médicaments dans le sang, et puissent tirer tout le bénéfice potentiel du traitement. L'objectif global de cette thèse était d'étudier les facteurs pharmacocinétiques et pharmacogénétiques qui influencent l'exposition des médicaments antiviraux (ARVs et anti- VHC) récemment approuvés. A cet effet, des méthodes de quantification des concentrations plasmatiques des médicaments antirétroviraux, anti-VHC ainsi que pour certains métabolites ont été développées et validées en utilisant la Chromatographie liquide couplée à la spectrométrie de masse tandem. Ces méthodes ont été utilisées pour le TDM des ARVs et pour les agents anti-VHC chez les patients infectés par le VIH, et le VHC, respectivement, mais aussi chez les patients co-infectés par le VIH-VHC. Un modèle de pharmacocinétique de population a été développé pour caractériser la variabilité inter-et intra-patient du médicament rilpivirine, un inhibiteur non nucléosidique de la transcriptase de VIH et d'identifier les variables génétiques et non génétiques influençant l'exposition au médicament. Aucun des facteurs étudiés n'a montré d'influence notable sur la clairance de la rilpivirine. Toutefois, la concentration résiduelle extrapolée selon le modèle de pharmacocinétique de population qui a été développé, a montré qu'une grande proportion des patients présente des concentrations minimales inférieures à la cible thérapeutique proposée. Dans ce contexte, la relation entre les concentrations minimales et l'échappement virologique nécessite une surveillance étroite des taux sanguins des patients recevant de la rilpivirine. A cet effet, le suivi thérapeutique est un outil important pour l'identification des patients à risque soient sous-exposés à lai rilpivirine. Pour identifier de nouveaux marqueurs de la toxicité qui pourraient induire l'arrêt du traitement, le profil des métabolites de phase I et de phase II a été étudié dans différentes matrices [plasma, LCR et urine) provenant de patients recevant de l'efavirenz. Les métabolites de phase II, qui n'avaient à ce jour jamais été investigués, constituent les principales espèces présentes dans les matrices étudiées. Au cours de ces investigations, un nouveau métabolite 8- OH-EFV-sulfate a été identifié chez l'homme, et ce dernier est. présent à des concentrations importantes. L'influence de certains facteurs pharmacogénétique des patients sur le profil des métabolites a été étudiée et ouvre la voie à de possibles nouveaux marqueurs phénotypiques alternatifs qui pourraient possiblement mieux prédire la toxicité associée au traitement par l'efavirenz. Finalement, nous nous sommes intéressés à étudier dans un modèle in vitro certains facteurs, comme la P-glycoprotéine, qui influencent la disposition cellulaire de certains médicaments antirétroviraux, en utilisant l'approche par la technologie du siRNA permettant de bloquer sélectivement l'expression du gène de cette protéine d'efflux des médicaments. -- Depuis l'introduction de la thérapie antiretrovirale (ARVs] la morbidité et la mortalité liées au VIH ont considérablement diminué. En parallèle le traitement contre le virus de l'hépatite C (VHC) a connu récemment d'énormes progrès avec l'arrivée de nouveaux médicaments puissants, ce qui a permis une augmentation considérable de la guérison de l'infection par le VHC. En dépit de l'efficacité de ces traitements antiviraux, les échecs thérapeutiques ainsi que les effets secondaires des traitements restent un problème important. Il a pu être démontré que la concentration de médicament présente dans l'organisme est corrélée avec l'efficacité clinique pour la plupart des médicaments agissant contre le VIH et contre le VHC. Les médicaments antiviraux sont généralement donnés à une posologie fixe et standardisée, à tous les patients, il existe cependant une importante variabilité entre les concentrations sanguines mesurées chez les individus. Cette variabilité peut être expliquée par plusieurs facteurs démographiques, environnementaux ou génétiques. Dans ce contexte, le suivi des concentrations sanguines (ou Therapeutic Drug Monitoring, TDM) permet de contrôler que les patients soient exposés à des concentrations suffisantes (pour bloquer la réplication du virus dans l'organisme) et éviter des concentrations excessives, ce qui peut entraîner l'apparition d'intolérence au traitement. Le but de ce travail de thèse est d'améliorer la compréhension des facteurs pharmacologiques et génétiques qui peuvent influencer l'efficacité et/ou la toxicité des médicaments antiviraux, dans le but d'améliorer le suivi des patients. A cet effet, des méthodes de dosage très sensibles et ont été mises au point pour permettre de quantifier les médicaments antiviraux dans le sang et dans d'autres liquides biologiques. Ces méthodes de dosage sont maintenant utilisées d'une part dans le cadre de la prise en charge des patients en routine et d'autre part pour diverses études cliniques chez les patients infectés soit par le HIV, le HCV ou bien coinfectés par les deux virus. Une partie de ce travail a été consacrée à l'investigation des différents facteurs démographiques, génétiques et environnementaux qui pourraient l'influencer la réponse clinique à la rilpivirine, un nouveau médicament contre le VIH. Toutefois, parmi tous les facteurs étudiés à ce jour, aucun n'a permis d'expliquer la variabilité de l'exposition à la rilpivirine chez les patients. On a pu cependant observer qu'à la posologie standard recommandée, un pourcentage relativement élevé de patients pourrait présenter des concentrations inférieures à la concentration sanguine minimale actuellement proposée. Il est donc utile de surveiller étroitement les concentrations de rilpivirine chez les patients pour identifier sans délai ceux qui risquent d'être sous-exposés. Dans l'organisme, le médicament subit diverses transformations (métabolisme) par des enzymes, notamment dans le foie, il est transporté dans les cellules et tissus par des protéines qui modulent sa concentration au site de son action pharmacologique. A cet effet, différents composés (métabolites) produits dans l'organisme après l'administration d'efavirenz, un autre médicament anti-VIH, ont été étudiés. En conclusion, nous nous sommes intéressés à la fois aux facteurs pharmacologiques et génétiques des traitements antiviraux, une approche qui s'inscrit dans l'optique d'une stratégie globale de prise en charge du patient. Dans ce contexte, le suivi des concentrations sanguines de médicaments constitue une des facettes du domaine émergent de la Médecine Personnalisée qui vise à maximiser le bénéfice thérapeutique et le profil de tolérance des médicaments antiviraux

Peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) but not PPARalpha serves as a plasma free fatty acid sensor in liver.

Peroxisome proliferator-activated receptor alpha (PPARalpha) is an important transcription factor in liver that can be activated physiologically by fasting or pharmacologically by using high-affinity synthetic agonists. Here we initially set out to elucidate the similarities in gene induction between Wy14643 and fasting. Numerous genes were commonly regulated in liver between the two treatments, including many classical PPARalpha target genes, such as Aldh3a2 and Cpt2. Remarkably, several genes induced by Wy14643 were upregulated by fasting independently of PPARalpha, including Lpin2 and St3gal5, suggesting involvement of another transcription factor. Using chromatin immunoprecipitation, Lpin2 and St3gal5 were shown to be direct targets of PPARbeta/delta during fasting, whereas Aldh3a2 and Cpt2 were exclusive targets of PPARalpha. Binding of PPARbeta/delta to the Lpin2 and St3gal5 genes followed the plasma free fatty acid (FFA) concentration, consistent with activation of PPARbeta/delta by plasma FFAs. Subsequent experiments using transgenic and knockout mice for Angptl4, a potent stimulant of adipose tissue lipolysis, confirmed the stimulatory effect of plasma FFAs on Lpin2 and St3gal5 expression levels via PPARbeta/delta. In contrast, the data did not support activation of PPARalpha by plasma FFAs. The results identify Lpin2 and St3gal5 as novel PPARbeta/delta target genes and show that upregulation of gene expression by PPARbeta/delta is sensitive to plasma FFA levels. In contrast, this is not the case for PPARalpha, revealing a novel mechanism for functional differentiation between PPARs.

CD62L(high) Treg cells with superior immunosuppressive properties accumulate within the CNS during remissions of EAE.

The role of regulatory T cell populations within the CNS in the regulation of CNS-autoimmunity is controversial. We show that during recovery from relapsing remitting experimental autoimmune encephalomyelitis, regulatory T cells accumulate within the CNS that express high levels of CD62L. These CD62L(high) Treg cells express increased amounts of CTLA-4, ICOS and TGF-β and are more potent than CD62L(low) Treg cells in suppressing proliferation and inducing apoptosis in effector T cells. CD62L(high) Treg cells thus represent a population of Treg cells that display superior immunosuppressive properties and accumulate in the CNS during recovery from CNS-autoimmunity.

Mechanism, regulation, and ecological role of bacterial cyanide biosynthesis.

A few bacterial species are known to produce and excrete hydrogen cyanide (HCN), a potent inhibitor of cytochrome c oxidase and several other metalloenzymes. In the producer strains, HCN does not appear to have a role in primary metabolism and is generally considered a secondary metabolite. HCN synthase of proteobacteria (especially fluorescent pseudomonads) is a membrane-bound flavoenzyme that oxidizes glycine, producing HCN and CO2. The hcnABC structural genes of Pseudomonas fluorescens and P. aeruginosa have sequence similarities with genes encoding various amino acid dehydrogenases/oxidases, in particular with nopaline oxidase of Agrobacterium tumefaciens. Induction of the hcn genes of P. fluorescens by oxygen limitation requires the FNR-like transcriptional regulator ANR, an ANR recognition sequence in the -40 region of the hcn promoter, and nonlimiting amounts of iron. In addition, expression of the hcn genes depends on a regulatory cascade initiated by the GacS/GacA (global control) two-component system. This regulation, which is typical of secondary metabolism, manifests itself during the transition from exponential to stationary growth phase. Cyanide produced by P. fluorescens strain CHA0 has an ecological role in that this metabolite accounts for part of the biocontrol capacity of strain CHA0, which suppresses fungal diseases on plant roots. Cyanide can also be a ligand of hydrogenases in some anaerobic bacteria that have not been described as cyanogenic. However, in this case, as well as in other situations, the physiological function of cyanide is unknown.

Cross-neutralization of four paramyxoviruses by a human monoclonal antibody.

Broadly neutralizing antibodies reactive against most and even all variants of the same viral species have been described for influenza and HIV-1 (ref. 1). However, whether a neutralizing antibody could have the breadth of range to target different viral species was unknown. Human respiratory syncytial virus (HRSV) and human metapneumovirus (HMPV) are common pathogens that cause severe disease in premature newborns, hospitalized children and immune-compromised patients, and play a role in asthma exacerbations. Although antisera generated against either HRSV or HMPV are not cross-neutralizing, we speculated that, because of the repeated exposure to these viruses, cross-neutralizing antibodies may be selected in some individuals. Here we describe a human monoclonal antibody (MPE8) that potently cross-neutralizes HRSV and HMPV as well as two animal paramyxoviruses: bovine RSV (BRSV) and pneumonia virus of mice (PVM). In its germline configuration, MPE8 is HRSV-specific and its breadth is achieved by somatic mutations in the light chain variable region. MPE8 did not result in the selection of viral escape mutants that evaded antibody targeting and showed potent prophylactic efficacy in animal models of HRSV and HMPV infection, as well as prophylactic and therapeutic efficacy in the more relevant model of lethal PVM infection. The core epitope of MPE8 was mapped on two highly conserved anti-parallel β-strands on the pre-fusion viral F protein, which are rearranged in the post-fusion F protein conformation. Twenty-six out of the thirty HRSV-specific neutralizing antibodies isolated were also found to be specific for the pre-fusion F protein. Taken together, these results indicate that MPE8 might be used for the prophylaxis and therapy of severe HRSV and HMPV infections and identify the pre-fusion F protein as a candidate HRSV vaccine.

Circulating matrix γ-carboxyglutamate protein (MGP) species are refractory to vitamin K treatment in a new case of Keutel syndrome.

Summary.  Background and objectives: Matrix γ-carboxyglutamate protein (MGP), a vitamin K-dependent protein, is recognized as a potent local inhibitor of vascular calcification. Studying patients with Keutel syndrome (KS), a rare autosomal recessive disorder resulting from MGP mutations, provides an opportunity to investigate the functions of MGP. The purpose of this study was (i) to investigate the phenotype and the underlying MGP mutation of a newly identified KS patient, and (ii) to investigate MGP species and the effect of vitamin K supplements in KS patients. Methods: The phenotype of a newly identified KS patient was characterized with specific attention to signs of vascular calcification. Genetic analysis of the MGP gene was performed. Circulating MGP species were quantified and the effect of vitamin K supplements on MGP carboxylation was studied. Finally, we performed immunohistochemical staining of tissues of the first KS patient originally described focusing on MGP species. Results: We describe a novel homozygous MGP mutation (c.61+1G>A) in a newly identified KS patient. No signs of arterial calcification were found, in contrast to findings in MGP knockout mice. This patient is the first in whom circulating MGP species have been characterized, showing a high level of phosphorylated MGP and a low level of carboxylated MGP. Contrary to expectations, vitamin K supplements did not improve the circulating carboxylated MGP levels. Phosphorylated MGP was also found to be present in the first KS patient originally described. Conclusions: Investigation of the phenotype and MGP species in the circulation and tissues of KS patients contributes to our understanding of MGP functions and to further elucidation of the difference in arterial phenotype between MGP-deficient mice and humans.

Nonresponse to clozapine and ultrarapid CYP1A2 activity: clinical data and analysis of CYP1A2 gene.

Clozapine (CLO), an atypical antipsychotic, depends mainly on cytochrome P450 1A2 (CYP1A2) for its metabolic clearance. Four patients treated with CLO, who were smokers, were nonresponders and had low plasma levels while receiving usual doses. Their plasma levels to dose ratios of CLO (median; range, 0.34; 0.22 to 0.40 ng x day/mL x mg) were significantly lower than ratios calculated from another study with 29 patients (0.75; 0.22 to 2.83 ng x day/mL x mg; P < 0.01). These patients were confirmed as being CYP1A2 ultrarapid metabolizers by the caffeine phenotyping test (median systemic caffeine plasma clearance; range, 3.85; 3.33 to 4.17 mL/min/kg) when compared with previous studies (0.3 to 3.33 mL/min/kg). The sequencing of the entire CYP1A2 gene from genomic DNA of these patients suggests that the -164C > A mutation (CYP1A2*1F) in intron 1, which confers a high inducibility of CYP1A2 in smokers, is the most likely explanation for their ultrarapid CYP1A2 activity. A marked (2 patients) or a moderate (2 patients) improvement of the clinical state of the patients occurred after the increase of CLO blood levels above the therapeutic threshold by the increase of CLO doses to very high values (ie, up to 1400 mg/d) or by the introduction of fluvoxamine, a potent CYP1A2 inhibitor, at low dosage (50 to 100 mg/d). Due to the high frequency of smokers among patients with schizophrenia and to the high frequency of the -164C > A polymorphism, CYP1A2 genotyping could have important clinical implications for the treatment of patients with CLO.

Molecular basis of selective PPARgamma modulation for the treatment of Type 2 diabetes.

Peroxisome proliferator-activated receptors (PPARs) (alpha, beta/delta and gamma) are lipid sensors capable of adapting gene expression to integrate various lipid signals. As such, PPARs are also very important pharmaceutical targets, and specific synthetic ligands exist for the different isotypes and are either currently used or hold promises in the treatment of major metabolic disorders. In particular, compounds of the class of the thiazolinediones (TZDs) are PPARgamma agonists and potent insulin-sensitizers. The specific but still broad expression patterns of PPARgamma, as well as its implication in numerous pathways, constitutes also a disadvantage regarding drug administration, since this potentially increases the chance to generate side-effects through the activation of the receptor in tissues or cells not affected by the disease. Actually, numerous side effects associated with the administration of TZDs have been reported. Today, a new generation of PPARgamma modulators is being actively developed to activate the receptor more specifically, in a cell and time-dependent manner, in order to induce a specific subset of target genes only and modulate a restricted number of metabolic pathways. We will discuss here why and how the development of such selective PPARgamma modulators is possible, and summarize the results obtained with the published molecules.

The antiviral adaptor proteins Cardif and Trif are processed and inactivated by caspases

The outcome of a viral infection depends on the interplay between the host's capacity to trigger potent antiviral responses and viral mechanisms that counteract them. Although Toll-like receptor (TLR)-3, which recognizes virally derived double-stranded (ds) RNA, transmits downstream antiviral signaling through the TIR adaptor Trif (TICAM-1), viral RNA-sensing RIG-like helicases (RLHs) use the mitochondrial-bound CARD protein Cardif (IPS-1/MAVS/VISA). The importance of these two antiviral signaling pathways is reflected by the fact that both adaptors are inhibited through specific cleavage triggered by the hepatitis C virus serine protease NS3-4A. Here, we show that inactivation can also occur through cellular caspases activated by various pro-apoptotic signals. Upon caspase-dependent cleavage both adaptors loose their capacity to activate the transcription factors interferon regulatory factors (IRF) and NF-kappaB. Importantly, poliovirus infection triggers a caspase-dependent cleavage of Cardif, suggesting that some viruses may activate caspases not only as a mean to facilitate shedding and replication, but also to impair antiviral responses