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

Objective evaluation of shoulder function using body-fixed sensors: a new way to detect early treatment failures?

Background: Variable definitions of outcome (Constant score, Simple Shoulder Test [SST]) have been used to assess outcome after shoulder treatment, although none has been accepted as the universal standard. Physicians lack an objective method to reliably assess the activity of their patients in dynamic conditions. Our purpose was to clinically validate the shoulder kinematic scores given by a portable movement analysis device, using the activities of daily living described in the SST as a reference. The secondary objective was to determine whether this device could be used to document the effectiveness of shoulder treatments (for glenohumeral osteoarthritis and rotator cuff disease) and detect early failures.Methods: A clinical trial including 34 patients and a control group of 31 subjects over an observation period of 1 year was set up. Evaluations were made at baseline and 3, 6, and 12 months after surgery by 2 independent observers. Miniature sensors (3-dimensional gyroscopes and accelerometers) allowed kinematic scores to be computed. They were compared with the regular outcome scores: SST; Disabilities of the Arm, Shoulder and Hand; American Shoulder and Elbow Surgeons; and Constant.Results: Good to excellent correlations (0.61-0.80) were found between kinematics and clinical scores. Significant differences were found at each follow-up in comparison with the baseline status for all the kinematic scores (P < .015). The kinematic scores were able to point out abnormal patient outcomes at the first postoperative follow-up.Conclusion: Kinematic scores add information to the regular outcome tools. They offer an effective way to measure the functional performance of patients with shoulder pathology and have the potential to detect early treatment failures.Level of evidence: Level II, Development of Diagnostic Criteria, Diagnostic Study. (C) 2011 Journal of Shoulder and Elbow Surgery Board of Trustees.

Involvement of the kallikrein-kinin system in the antihypertensive effect of the angiotensin converting enzyme inhibitors.

1. Studies were performed in normal subjects and in rats to assess the effect of angiotensin converting enzyme (ACE) inhibition on the kallikrein-kinin system. As ACE is identical to kininase II, one of the enzymes physiologically involved in bradykinin degradation, bradykinin may be expected to accumulate during ACE inhibition. 2. A competitive antagonist of bradykinin was used to explore in unanaesthetized rats the contribution of circulating bradykinin to blood pressure control under ACE inhibition. 3. No evidence was found for a role of this vasodilating peptide in the blood pressure lowering effect of acute ACE inhibition. 4. The plasma activity of carboxypeptidase N (= kininase I), another pathway of bradykinin degradation, remained intact during a 1 week course of treatment with an ACE inhibitor in normal subjects. This therefore indicates that bradykinin formed during ACE inhibition can still be metabolized.

Multi-Atlas based Segmentation of Head and Neck CT Images using Active Contour

This paper presents the segmentation of bilateral parotid glands in the Head and Neck (H&N) CT images using an active contour based atlas registration. We compare segmentation results from three atlas selection strategies: (i) selection of "single-most-similar" atlas for each image to be segmented, (ii) fusion of segmentation results from multiple atlases using STAPLE, and (iii) fusion of segmentation results using majority voting. Among these three approaches, fusion using majority voting provided the best results. Finally, we present a detailed evaluation on a dataset of eight images (provided as a part of H&N auto segmentation challenge conducted in conjunction with MICCAI-2010 conference) using majority voting strategy.

Dosimetry of 90Y-ibritumomab tiuxetan as consolidation of first remission in advanced-stage follicular lymphoma: results from the international phase 3 first-line indolent trial.

The objective of this analysis was to assess the radiation exposure associated with (90)Y-ibritumomab tiuxetan when used as consolidation therapy in adults with low or minimal tumor burden after first-line therapy of advanced follicular lymphoma (FL). METHODS: The patients who were enrolled in the phase 3 first-line indolent trial were 18 y or older, with CD20(+) grade 1 or 2 stage III or IV FL, and a partial response, complete response, or unconfirmed complete response to first-line chemotherapy. The patients were allocated randomly to receive a single infusion of unlabeled rituximab 250 mg/m(2) on day -7 and consolidation on day 0 with a single dose of (90)Y-ibritumomab tiuxetan, 14.8 MBq/kg, immediately after unlabeled rituximab, 250 mg/m(2), or no further treatment. On day -7, a subset of patients received an injection of 185 MBq of (111)In-ibritumomab tiuxetan immediately after unlabeled rituximab, 250 mg/m(2), for central dosimetry analysis. Correlations were assessed between organ radiation absorbed dose and toxicity, body weight, body mass index, and progression-free survival. RESULTS: Central dosimetry evaluations were available from 57 of 70 patients. Median radiation absorbed doses were 100 cGy (range, 28-327 cGy) for the red marrow and 72 cGy (range, 46-106 cGy) for the whole body. Radiation absorbed doses did not differ significantly between patients who had a partial response or complete response to initial therapy. Progression-free survival correlated significantly with the whole-body (r = 0.4401; P = 0.0006) and bone marrow (r = 0.2976; P = 0.0246) radiation dose. Body weight was significantly negatively correlated with whole-body radiation dose (r = -0.4971; P < 0.0001). Neither the whole-body radiation dose nor the bone marrow radiation dose correlated with hematologic toxicity. CONCLUSION: In patients with low or minimal residual tumor burden after first-line chemotherapy of advanced FL, whole-body and bone marrow exposure after (90)Y-ibritumomab tiuxetan consolidation showed a significant positive correlation with progression-free survival, whereas dosimetric data could not predict hematologic toxicity.

Evaluation of errors in alpine skiing video analysis

INTRODUCTION: Video records are widely used to analyze performance in alpine skiing at professional or amateur level. Parts of these analyses require the labeling of some movements (i.e. determining when specific events occur). If differences among coaches and differences for the same coach between different dates are expected, they have never been quantified. Moreover, knowing these differences is essential to determine which parameters reliable should be used. This study aimed to quantify the precision and the repeatability for alpine skiing coaches of various levels, as it is done in other fields (Koo et al, 2005). METHODS: A software similar to commercialized products was designed to allow video analyses. 15 coaches divided into 3 groups (5 amateur coaches (G1), 5 professional instructors (G2) and 5 semi-professional coaches (G3)) were enrolled. They were asked to label 15 timing parameters (TP) according to the Swiss ski manual (Terribilini et al, 2001) for each curve. TP included phases (initiation, steering I-II), body and ski movements (e.g. rotation, weighting, extension, balance). Three video sequences sampled at 25 Hz were used and one curve per video was labeled. The first video was used to familiarize the analyzer to the software. The two other videos, corresponding to slalom and giant slalom, were considered for the analysis. G1 realized twice the analysis (A1 and A2) at different dates and TP were randomized between both analyses. Reference TP were considered as the median of G2 and G3 at A1. The precision was defined as the RMS difference between individual TP and reference TP, whereas the repeatability was calculated as the RMS difference between individual TP at A1 and at A2. RESULTS AND DISCUSSION: For G1, G2 and G3, a precision of +/-5.6 frames, +/-3.0 and +/-2.0 frames, was respectively obtained. These results showed that G2 was more precise than G1, and G3 more precise than G2, were in accordance with group levels. The repeatability for G1 was +/-3.1 frames. Furthermore, differences among TP precision were observed, considering G2 and G3, with largest differences of +/-5.9 frames for "body counter rotation movement in steering phase II", and of 0.8 frame for "ski unweighting in initiation phase". CONCLUSION: This study quantified coach ability to label video in term of precision and repeatability. The best precision was obtained for G3 and was of +/-0.08s, which corresponds to +/-6.5% of the curve cycle. Regarding the repeatability, we obtained a result of +/-0.12s for G1, corresponding to +/-12% of the curve cycle. The repeatability of G2 and G3 are expected to be lower than the precision of G1 and the corresponding repeatability will be assessed soon. In conclusion, our results indicate that the labeling of video records is reliable for some TP, whereas caution is required for others. REFERENCES Koo S, Gold MD, Andriacchi TP. (2005). Osteoarthritis, 13, 782-789. Terribilini M, et al. (2001). Swiss Ski manual, 29-46. IASS, Lucerne.

N-Glycosylation and conserved cysteine residues in RAMP3 play a critical role for the functional expression of CRLR/RAMP3 adrenomedullin receptor.

The calcitonin receptor-like receptor (CRLR) and receptor activity modifying protein-3 (RAMP3) can assemble into a CRLR/RAMP3 heterodimeric receptor that exhibits the characteristics of a high affinity adrenomedullin receptor. RAMP3 participates in adrenomedullin (AM) binding via its extracellular N-terminus characterized by the presence of six highly conserved cysteine residues and four N-glycosylation consensus sites. Here, we assessed the usage of these conserved residues in cotranslational modifications of RAMP3 and addressed their role in functional expression of the CRLR/RAMP3 receptor. Using a Xenopus oocyte expression system, we show that (i) RAMP3 is assembled with CRLR as a multiple N-glycosylated species in which two, three, or four consensus sites are used; (ii) elimination of all N-glycans in RAMP3 results in a significant inhibition of receptor [(125)I]AM binding and an increase in the EC(50) value for AM; (iii) several lines of indirect evidence indicate that each of the six cysteines is involved in disulfide bond formation; (iv) when all cysteines are mutated to serines, RAMP3 is N-glycosylated at all four consensus sites, suggesting that disulfide bond formation inhibits N-gylcosylation; and (v) elimination of all cysteines abolishes adrenomedullin binding and leads to a complete loss of receptor function. Our data demonstrate that cotranslational modifications of RAMP3 play a critical role in the function of the CRLR/RAMP3 adrenomedullin receptor.

Clinical Prognostic Factors Predicting Outcomes Of Patients With Recurrent Gbm And Nomograms

BACKGROUND: Prognostic models and nomograms were recently developed to predict survival of patients with newly diagnosed glioblastoma multiforme (GBM).1 To improve predictions, models should be updated with the most recent patient and disease information. Nomograms predicting patient outcome at the time of disease progression are required. METHODS: Baseline information from 299 patients with recurrent GBM recruited in 8 phase I or II trials of the EORTC Brain Tumor Group was used to evaluate clinical parameters as prognosticators of patient outcome. Univariate (log rank) and multivariate (Cox models) analyses were made to assess the ability of patients' characteristics (age, sex, performance status [WHO PS], and MRC neurological deficit scale), disease history (prior treatments, time since last treatment or initial diagnosis, and administration of steroids or antiepileptics) and disease characteristics (tumor size and number of lesions) to predict progression free survival (PFS) and overall survival (OS). Bootstrap technique was used for models internal validation. Nomograms were computed to provide individual patients predictions. RESULTS: Poor PS and more than 1 lesion had a significant prognostic impact for both PFS and OS. Antiepileptic drug use was significantly associated with worse PFS. Larger tumors (split by the median of the largest tumor diameter >42.5 mm) and steroid use had shorter OS. Age, sex, neurologic deficit, prior therapies, and time since last therapy or initial diagnosis did not show independent prognostic value for PFS or OS. CONCLUSIONS: This analysis confirms that PS but not age is a major prognostic factor for PFS and OS. Multiple or large tumors and the need to administer steroids significantly increase the risk of progression and death. Nomograms at the recurrence could be used to obtain accurate predictions for the design of new targeted therapy trials or retrospective analyses. (1. T. Gorlia et al., Nomograms for predicting survival of patients with newly diagnosed glioblastoma. Lancet Oncol 9 (1): 29-38, 2008.)

Endogenous angiotensin II induces atherosclerotic plaque vulnerability and elicits a Th1 response in ApoE-/- mice.

Rupture of vulnerable plaques is the main cause of acute cardiovascular events. However, mechanisms responsible for transforming a stable into a vulnerable plaque remain elusive. Angiotensin II, a key regulator of blood pressure homeostasis, has a potential role in atherosclerosis. To study the contribution of angiotensin II in plaque vulnerability, we generated hypertensive hypercholesterolemic ApoE-/- mice with either normal or endogenously increased angiotensin II production (renovascular hypertension models). Hypertensive high angiotensin II ApoE-/- mice developed unstable plaques, whereas in hypertensive normal angiotensin II ApoE-/- mice plaques showed a stable phenotype. Vulnerable plaques from high angiotensin II ApoE-/- mice had thinner fibrous cap (P<0.01), larger lipid core (P<0.01), and increased macrophage content (P<0.01) than even more hypertensive but normal angiotensin II ApoE-/- mice. Moreover, in mice with high angiotensin II, a skewed T helper type 1-like phenotype was observed. Splenocytes from high angiotensin II ApoE-/- mice produced significantly higher amounts of interferon (IFN)-gamma than those from ApoE-/- mice with normal angiotensin II; secretion of IL4 and IL10 was not different. In addition, we provide evidence for a direct stimulating effect of angiotensin II on lymphocyte IFN-gamma production. These findings suggest a new mechanism in plaque vulnerability demonstrating that angiotensin II, within the context of hypertension and hypercholesterolemia, independently from its hemodynamic effect behaves as a local modulator promoting the induction of vulnerable plaques probably via a T helper switch.

An improved approach for predicting the distribution of rare and endangered species from occurrence and pseudo-absence data

1. Few examples of habitat-modelling studies of rare and endangered species exist in the literature, although from a conservation perspective predicting their distribution would prove particularly useful. Paucity of data and lack of valid absences are the probable reasons for this shortcoming. Analytic solutions to accommodate the lack of absence include the ecological niche factor analysis (ENFA) and the use of generalized linear models (GLM) with simulated pseudo-absences. 2. In this study we tested a new approach to generating pseudo-absences, based on a preliminary ENFA habitat suitability (HS) map, for the endangered species Eryngium alpinum. This method of generating pseudo-absences was compared with two others: (i) use of a GLM with pseudo-absences generated totally at random, and (ii) use of an ENFA only. 3. The influence of two different spatial resolutions (i.e. grain) was also assessed for tackling the dilemma of quality (grain) vs. quantity (number of occurrences). Each combination of the three above-mentioned methods with the two grains generated a distinct HS map. 4. Four evaluation measures were used for comparing these HS maps: total deviance explained, best kappa, Gini coefficient and minimal predicted area (MPA). The last is a new evaluation criterion proposed in this study. 5. Results showed that (i) GLM models using ENFA-weighted pseudo-absence provide better results, except for the MPA value, and that (ii) quality (spatial resolution and locational accuracy) of the data appears to be more important than quantity (number of occurrences). Furthermore, the proposed MPA value is suggested as a useful measure of model evaluation when used to complement classical statistical measures. 6. Synthesis and applications. We suggest that the use of ENFA-weighted pseudo-absence is a possible way to enhance the quality of GLM-based potential distribution maps and that data quality (i.e. spatial resolution) prevails over quantity (i.e. number of data). Increased accuracy of potential distribution maps could help to define better suitable areas for species protection and reintroduction.

Subjective mental time: the functional architecture of projecting the self to past and future.

Abstract Human experience takes place in the line of mental time (MT) created through 'self-projection' of oneself to different time-points in the past or future. Here we manipulated self-projection in MT not only with respect to one's life events but also with respect to one's faces from different past and future time-points. Behavioural and event-related functional magnetic resonance imaging activity showed three independent effects characterized by (i) similarity between past recollection and future imagination, (ii) facilitation of judgements related to the future as compared with the past, and (iii) facilitation of judgements related to time-points distant from the present. These effects were found with respect to faces and events, and also suggest that brain mechanisms of MT are independent of whether actual life episodes have to be re-experienced or pre-experienced, recruiting a common cerebral network including the anteromedial temporal, posterior parietal, inferior frontal, temporo-parietal and insular cortices. These behavioural and neural data suggest that self-projection in time is a fundamental aspect of MT, relying on neural structures encoding memory, mental imagery and self.

Phenotypic and functional analysis of human fetal liver hematopoietic stem cells in culture.

Steady-state hematopoiesis and hematopoietic transplantation rely on the unique potential of stem cells to undergo both self-renewal and multilineage differentiation. Fetal liver (FL) represents a promising alternative source of hematopoietic stem cells (HSCs), but limited by the total cell number obtained in a typical harvest. We reported that human FL nonobese diabetic/severe combined immunodeficient (NOD/SCID) repopulating cells (SRCs) could be expanded under simple stroma-free culture conditions. Here, we sought to further characterize FL HSC/SRCs phenotypically and functionally before and following culture. Unexpanded or cultured FL cell suspensions were separated into various subpopulations. These were tested for long-term culture potential and for in vivo repopulating function following transplantation into NOD/SCID mice. We found that upon culture of human FL cells, a tight association between classical stem cell phenotypes, such as CD34(+) /CD38(-) and/or side population, and NOD/SCID repopulating function was lost, as observed with other sources. Although SRC activity before and following culture consistently correlated with the presence of a CD34(+) cell population, we provide evidence that, contrary to umbilical cord blood and adult sources, stem cells present in both CD34(+) and CD34(-) FL populations can sustain long-term hematopoietic cultures. Furthermore, upon additional culture, CD34-depleted cell suspensions, devoid of SRCs, regenerated a population of CD34(+) cells possessing SRC function. Our studies suggest that compared to neonatal and adult sources, the phenotypical characteristics of putative human FL HSCs may be less strictly defined, and reinforce the accumulated evidence that human FL represents a unique, valuable alternative and highly proliferative source of HSCs for clinical applications.

Survey of prenatal screening policies in Europe for structural malformations and chromosome anomalies, and their impact on detection and termination rates for neural tube defects and Down's syndrome.

OBJECTIVE: To 'map' the current (2004) state of prenatal screening in Europe. DESIGN: (i) Survey of country policies and (ii) analysis of data from EUROCAT (European Surveillance of Congenital Anomalies) population-based congenital anomaly registers. SETTING: Europe. POPULATION: Survey of prenatal screening policies in 18 countries and 1.13 million births in 12 countries in 2002-04. METHODS: (i) Questionnaire on national screening policies and termination of pregnancy for fetal anomaly (TOPFA) laws in 2004. (ii) Analysis of data on prenatal detection and termination for Down's syndrome and neural tube defects (NTDs) using the EUROCAT database. MAIN OUTCOME MEASURES: Existence of national prenatal screening policies, legal gestation limit for TOPFA, prenatal detection and termination rates for Down's syndrome and NTD. RESULTS: Ten of the 18 countries had a national country-wide policy for Down's syndrome screening and 14/18 for structural anomaly scanning. Sixty-eight percent of Down's syndrome cases (range 0-95%) were detected prenatally, of which 88% resulted in termination of pregnancy. Eighty-eight percent (range 25-94%) of cases of NTD were prenatally detected, of which 88% resulted in termination. Countries with a first-trimester screening policy had the highest proportion of prenatally diagnosed Down's syndrome cases. Countries with no official national Down's syndrome screening or structural anomaly scan policy had the lowest proportion of prenatally diagnosed Down's syndrome and NTD cases. Six of the 18 countries had a legal gestational age limit for TOPFA, and in two countries, termination of pregnancy was illegal at any gestation. CONCLUSIONS: There are large differences in screening policies between countries in Europe. These, as well as organisational and cultural factors, are associated with wide country variation in prenatal detection rates for Down's syndrome and NTD.

Assessment of panobacumab as adjunctive immunotherapy for the treatment of nosocomial Pseudomonas aeruginosa pneumonia.

The fully human anti-lipopolysaccharide (LPS) immunoglobulin M (IgM) monoclonal antibody panobacumab was developed as an adjunctive immunotherapy for the treatment of O11 serotype Pseudomonas aeruginosa infections. We evaluated the potential clinical efficacy of panobacumab in the treatment of nosocomial pneumonia. We performed a post-hoc analysis of a multicenter phase IIa trial (NCT00851435) designed to prospectively evaluate the safety and pharmacokinetics of panobacumab. Patients treated with panobacumab (n = 17), including 13 patients receiving the full treatment (three doses of 1.2 mg/kg), were compared to 14 patients who did not receive the antibody. Overall, the 17 patients receiving panobacumab were more ill. They were an average of 72 years old [interquartile range (IQR): 64-79] versus an average of 50 years old (IQR: 30-73) (p = 0.024) and had Acute Physiology and Chronic Health Evaluation II (APACHE II) scores of 17 (IQR: 16-22) versus 15 (IQR: 10-19) (p = 0.043). Adjunctive immunotherapy resulted in an improved clinical outcome in the group receiving the full three-course panobacumab treatment, with a resolution rate of 85 % (11/13) versus 64 % (9/14) (p = 0.048). The Kaplan-Meier survival curve showed a statistically significantly shorter time to clinical resolution in this group of patients (8.0 [IQR: 7.0-11.5] versus 18.5 [IQR: 8-30] days in those who did not receive the antibody; p = 0.004). Panobacumab adjunctive immunotherapy may improve clinical outcome in a shorter time if patients receive the full treatment (three doses). These preliminary results suggest that passive immunotherapy targeting LPS may be a complementary strategy for the treatment of nosocomial O11 P. aeruginosa pneumonia.

Isolation and in vitro chondrogenic potential of human foetal spine cells.

Cell therapy for nucleus pulposus (NP) regeneration is an attractive treatment for early disc degeneration as shown by studies using autologous NP cells or stem cells. Another potential source of cells is foetal cells. We investigated the feasibility of isolating foetal cells from human foetal spine tissues and assessed their chondrogenic potential in alginate bead cultures. Histology and immunohistochemistry of foetal tissues showed that the structure and the matrix composition (aggrecan, type I and II collagen) of foetal intervertebral disc (IVD) were similar to adult IVD. Isolated foetal cells were cultured in monolayer in basic media supplemented with 10% Fetal Bovine Serum (FBS) and from each foetal tissue donation, a cell bank of foetal spine cells at passage 2 was established and was composed of around 2000 vials of 5 million cells. Gene expression and immunohistochemistry of foetal spine cells cultured in alginate beads during 28 days showed that cells were able to produce aggrecan and type II collagen and very low level of type I and type X collagen, indicating chondrogenic differentiation. However variability in matrix synthesis was observed between donors. In conclusion, foetal cells could be isolated from human foetal spine tissues and since these cells showed chondrogenic potential, they could be a potential cell source for IVD regeneration.