Imatinib plasma concentrations variability in hemato-oncologic patients

Abstract Imatinib (Glivec~ has transformed the treatment and prognosis of chronic myeloid leukaemia (CML) and of gastrointestinal stromal tumor (GIST). However, the treatment must be taken indefinitely and is not devoid of inconvenience and toxicity. Moreover, resistance or escape from disease control occurs. Considering the large interindividual differences in the function of the enzymatic and transport systems involved in imatinib disposition, exposure to this drug can be expected to vary widely among patients. Among those known systems is a cytochrome P450 (CYI'3A4) that metabolizes imatinib, the multidrug transporter P-glycoprotein (P-gp; product of the MDR1 gene) that expels imatinib out of cells, and al-acid glycoprotein (AGP), a circulating protein binding imatinib in the plasma. The aim of this observational study was to explore the influence of these covariates on imatinib pharmacokinetics (PK), to assess the interindividual variability of the PK parameters of the drug, and to evaluate whether imatinib use would benefit from a therapeutic drug monitoring (TDM) program. A total of 321 plasma concentrations were measured in 59 patients receiving imatinib, using a validated chromatographic method developed for this study (HPLC-LTV). The results were analyzed by non-linear mixed effect modeling (NONMEM). A one-compartment pharmacokinetic model with first-order absorption appropriately described the data, and a large interindividual variability was observed. The MDK> polymorphism 3435C>T and the CYP3A4 activity appeared to modulate the disposition of imatinib, albeit not significantly. A hyperbolic relationship between plasma AGP levels and oral clearance, as well as volume of distribution, was observed. A mechanistic approach was built up, postulating that only the unbound imatinib concentration was able to undergo first-order elimination. This approach allowed determining an average free clearance (CL,~ of 13101/h and a volume of distribution (Vd) of 301 1. By comparison, the total clearance determined was 141/h (i.e. 233 ml/min). Free clearance was affected by body weight and pathology diagnosis. The estimated variability of imatinib disposition (17% for CLu and 66% for Vd) decreased globally about one half with the model incorporating the AGP impact. Moreover, some associations were observed between PK parameters of the free imatinib concentration and its efficacy and toxicity. Finally, the functional influence of P-gp activity has been demonstrated in vitro in cell cultures. These elements are arguments to further investigate the possible usefulness of a TDM program for imatinib. It may help in individualizing the dosing regimen before overt disease progression or development of treatment toxicity, thus improving both the long-term therapeutic effectiveness and tolerability of this drug. Résumé L'imatinib (Glivec ®) a révolutionné le traitement et le pronostic de la leucémie myéloïde chronique (LMC) et des tumeurs stromales d'origine digestive (GIST). Il s'agit toutefois d'un traitement non dénué d'inconvénients et de toxicité, et qui doit être pris indéfiniment. Par ailleurs, une résistance, ou des échappements au traitement, sont également rencontrés. Le devenir de ce médicament dans l'organisme dépend de systèmes enzymatiques et de transport connus pour présenter de grandes différences interindividuelles, et l'on peut s'attendre à ce que l'exposition à ce médicament varie largement d'un patient à l'autre. Parmi ces systèmes, on note un cytochrome P450 (le CYP3A4) métabolisant l'imatinib, la P-glycoprotéine (P-gp ;codée par le gène MDR1), un transporteur d'efflux expulsant le médicament hors des cellules, et l'atglycoprotéine acide (AAG), une protéine circulante sur laquelle se fixe l'imatinib dans le plasma. L'objectif de la présente étude clinique a été de déterminer l'influence de ces covariats sur la pharmacocinétique (PK) de l'imatinib, d'établir la variabilité interindividuelle des paramètres PK du médicament, et d'évaluer dans quelle mesure l'imatinib pouvait bénéficier d'un programme de suivi thérapeutique (TDM). En utilisant une méthode chromatographique développée et validée à cet effet (HPLC-UV), un total de 321 concentrations plasmatiques a été dosé chez 59 patients recevant de l'imatinib. Les résultats ont été analysés par modélisation non linéaire à effets mixtes (NONMEM). Un modèle pharmacocinétique à un compartiment avec absorption de premier ordre a permis de décrire les données, et une grande variabilité interindividuelle a été observée. Le polymorphisme du gène MDK1 3435C>T et l'activité du CYP3A4 ont montré une influence, toutefois non significative, sur le devenir de l'imatinib. Une relation hyperbolique entre les taux plasmatiques d'AAG et la clairance, comme le volume de distribution, a été observée. Une approche mécanistique a donc été élaborée, postulant que seule la concentration libre subissait une élimination du premier ordre. Cette approche a permis de déterminer une clairance libre moyenne (CLlibre) de 13101/h et un volume de distribution (Vd) de 301 l. Par comparaison, la clairance totale était de 141/h (c.à.d. 233 ml/min). La CLlibre est affectée par le poids corporel et le type de pathologie. La variabilité interindividuelle estimée pour le devenir de l'imatinib (17% sur CLlibre et 66% sur Vd) diminuait globalement de moitié avec le modèle incorporant l'impact de l'AAG. De plus, une certaine association entre les paramètres PK de la concentration d'imatinib libre et l'efficacité et la toxicité a été observée. Finalement, l'influence fonctionnelle de l'activité de la P-gp a été démontrée in nitro dans des cultures cellulaires. Ces divers éléments constituent des arguments pour étudier davantage l'utilité potentielle d'un programme de TDM appliqué à l'imatinib. Un tel suivi pourrait aider à l'individualisation des régimes posologiques avant la progression manifeste de la maladie ou l'apparition de toxicité, améliorant tant l'efficacité que la tolérabilité de ce médicament. Résumé large public L'imatinib (un médicament commercialisé sous le nom de Glivec ®) a révolutionné le traitement et le pronostic de deux types de cancers, l'un d'origine sanguine (leucémie) et l'autre d'origine digestive. Il s'agit toutefois d'un traitement non dénué d'inconvénients et de toxicité, et qui doit être pris indéfiniment. De plus, des résistances ou des échappements au traitement sont également rencontrés. Le devenir de ce médicament dans le corps humain (dont l'étude relève de la discipline appelée pharmacocinétique) dépend de systèmes connus pour présenter de grandes différences entre les individus, et l'on peut s'attendre à ce que l'exposition à ce médicament varie largement d'un patient à l'autre. Parmi ces systèmes, l'un est responsable de la dégradation du médicament dans le foie (métabolisme), l'autre de l'expulsion du médicament hors des cellules cibles, alors que le dernier consiste en une protéine (dénommée AAG) qui transporte l'imatinib dans le sang. L'objectif de notre étude a été de déterminer l'influence de ces différents systèmes sur le comportement pharmacocinétique de l'imatinib chez les patients, et d'étudier dans quelle mesure le devenir de ce médicament dans l'organisme variait d'un patient à l'autre. Enfin, cette étude avait pour but d'évaluer à quel point la surveillance des concentrations d'imatinib présentes dans le sang pourrait améliorer le traitement des patients cancéreux. Une telle surveillance permet en fait de connaître l'exposition effective de l'organisme au médicament (concept abrégé par le terme anglais TDM, pour Therapeutic Drag Monitoring. Ce projet de recherche a d'abord nécessité la mise au point d'une méthode d'analyse pour la mesure des quantités (ou concentrations) d'imatinib présentes dans le sang. Cela nous a permis d'effectuer régulièrement des mesures chez 59 patients. Il nous a ainsi été possible de décrire le devenir du médicament dans le corps à l'aide de modèles mathématiques. Nous avons notamment pu déterminer chez ces patients la vitesse à laquelle l'imatinib est éliminé du sang et l'étendue de sa distribution dans l'organisme. Nous avons également observé chez les patients que les concentrations sanguines d'imatinib étaient très variables d'un individu à l'autre pour une même dose de médicament ingérée. Nous avons pu aussi mettre en évidence que les concentrations de la protéine AAG, sur laquelle l'imatinib se lie dans le sang, avait une grande influence sur la vitesse à laquelle le médicament est éliminé de l'organisme. Ensuite, en tenant compte des concentrations sanguines d'imatinib et de cette protéine, nous avons également pu calculer les quantités de médicament non liées à cette protéine (= libres), qui sont seules susceptibles d'avoir une activité anticancéreuse. Enfin, il a été possible d'établir qu'il existait une certaine relation entre ces concentrations, l'effet thérapeutique et la toxicité du traitement. Tous ces éléments constituent des arguments pour approfondir encore l'étude de l'utilité d'un programme de TDM appliqué à l'imatinib. Comme chaque patient est différent, un tel suivi pourrait aider à l'ajustement des doses du médicament avant la progression manifeste de la maladie ou l'apparition de toxicité, améliorant ainsi tant son efficacité que son innocuité.

Exposure to Leishmania braziliensis triggers neutrophil activation and apoptosis.

BACKGROUND: Neutrophils are the first line of defense against invading pathogens and are rapidly recruited to the sites of Leishmania inoculation. During Leishmania braziliensis infection, depletion of inflammatory cells significantly increases the parasite load whereas co-inoculation of neutrophils plus L. braziliensis had an opposite effect. Moreover, the co-culture of infected macrophages and neutrophils also induced parasite killing leading us to ask how neutrophils alone respond to an L. braziliensis exposure. Herein we focused on understanding the interaction between neutrophils and L. braziliensis, exploring cell activation and apoptotic fate. METHODS AND FINDINGS: Inoculation of serum-opsonized L. braziliensis promastigotes in mice induced neutrophil accumulation in vivo, peaking at 24 h. In vitro, exposure of thyoglycollate-elicited inflammatory or bone marrow neutrophils to L. braziliensis modulated the expression of surface molecules such as CD18 and CD62L, and induced the oxidative burst. Using mCherry-expressing L. braziliensis, we determined that such effects were mainly observed in infected and not in bystander cells. Neutrophil activation following contact with L. braziliensis was also confirmed by the release of TNF-α and neutrophil elastase. Lastly, neutrophils infected with L. braziliensis but not with L. major displayed markers of early apoptosis. CONCLUSIONS: We show that L. braziliensis induces neutrophil recruitment in vivo and that neutrophils exposed to the parasite in vitro respond through activation and release of inflammatory mediators. This outcome may impact on parasite elimination, particularly at the early stages of infection.

A phase IIa randomised clinical study of GNbAC1, a humanised monoclonal antibody against the envelope protein of multiple sclerosis-associated endogenous retrovirus in multiple sclerosis patients.

BACKGROUND: GNbAC1 is an immunoglobulin (IgG4) humanised monoclonal antibody against multiple sclerosis-associated retrovirus (MSRV)-Env, a protein of endogenous retroviral origin, expressed in multiple sclerosis (MS) lesions, which is pro-inflammatory and inhibits oligodendrocyte precursor cell differentiation. OBJECTIVE: This is a randomised, double-blind placebo-controlled dose-escalation study followed by a six-month open-label phase to test GNbAC1 in MS patients. The primary objective was to assess GNbAC1 safety in MS patients, and the other objectives were pharmacokinetic and pharmacodynamic assessments. METHODS: Ten MS patients were randomised into two cohorts to receive a single intravenous infusion of GNbAC1/placebo at doses of 2 or 6 mg/kg. Then all patients received five infusions of GNbAC1 at 2 or 6 mg/kg at four-week intervals in an open-label setting. Safety, brain magnetic resonance imaging (MRI), pharmacokinetics, immunogenicity, cytokines and MSRV RNA expression were studied. RESULTS: All patients completed the study. GNbAC1 was well tolerated in all patients. GNbAC1 pharmacokinetics is dose-linear with mean elimination half-life of 27-37 d. Anti-GNbAC1 antibodies were not detected. Cytokine analysis did not indicate an adverse effect. MSRV-transcripts showed a decline after the start of treatment. Nine patients had stable brain lesions at MRI. CONCLUSION: The safety, pharmacokinetic profile, and pharmacodynamic responses to GNbAC1 are favourable in MS patients over a six-month treatment period.

Vitamin D time profile based on the contribution of non-genetic and genetic factors in HIV-infected individuals of European ancestry.

BACKGROUND: Vitamin D deficiency is prevalent in HIV-infected individuals and vitamin D supplementation is proposed according to standard care. This study aimed at characterizing the kinetics of 25(OH)D in a cohort of HIV-infected individuals of European ancestry to better define the influence of genetic and non-genetic factors on 25(OH)D levels. These data were used for the optimization of vitamin D supplementation in order to reach therapeutic targets. METHODS: 1,397 25(OH)D plasma levels and relevant clinical information were collected in 664 participants during medical routine follow-up visits. They were genotyped for 7 SNPs in 4 genes known to be associated with 25(OH)D levels. 25(OH)D concentrations were analysed using a population pharmacokinetic approach. The percentage of individuals with 25(OH)D concentrations within the recommended range of 20-40 ng/ml during 12 months of follow-up and several dosage regimens were evaluated by simulation. RESULTS: A one-compartment model with linear absorption and elimination was used to describe 25(OH)D pharmacokinetics, while integrating endogenous baseline plasma concentrations. Covariate analyses confirmed the effect of seasonality, body mass index, smoking habits, the analytical method, darunavir/ritonavir and the genetic variant in GC (rs2282679) on 25(OH)D concentrations. 11% of the inter-individual variability in 25(OH)D levels was explained by seasonality and other non-genetic covariates, and 1% by genetics. The optimal supplementation for severe vitamin D deficient patients was 300,000 IU two times per year. CONCLUSIONS: This analysis allowed identifying factors associated with 25(OH)D plasma levels in HIV-infected individuals. Improvement of dosage regimen and timing of vitamin D supplementation is proposed based on those results.

Antibiotic susceptibility and molecular epidemiology of Panton-Valentine leukocidin-positive meticillin-resistant Staphylococcus aureus: An international survey

The antibiotic susceptibility and molecular epidemiology of Panton-Valentine leukocidin (PVL)-positive meticillin-resistant Staphylococcus aureus (MRSA) isolates reported from 17 countries in the Americas, Europe and, Australia-Asia were analysed. Among a total of 3236 non-duplicate isolates, the lowest susceptibility was observed to erythromycin in all regions. Susceptibility to ciprofloxacin showed large variation (25%, 75% and 84% in the Americas, Europe and Australia-Asia, respectively). Two vancomycin-intermediate PVL-positive MRSA isolates were reported, one from Hong Kong and the other from The Netherlands. Resistance to trimethoprim/sulfamethoxazole and linezolid was <1%. Among 1798 MRSA isolates from 13 countries that were tested for the requested 10 non-β-lactam antibiotics, 49.4% were multisusceptible. However, multiresistant isolates (resistant to at least three classes of non-β-lactam antibiotics) were reported from all regions. Sequence type 30 (ST30) was reported worldwide, whereas ST80 and ST93 were exclusive to Europe and Australia, respectively. USA300 and related clones (ST8) are progressively replacing the ST80 clone in several European countries. Eight major clusters were discriminated by multilocus variable-number tandem repeat assay (MLVA), showing a certain geographic specificity. PVL-positive MRSA isolates frequently remain multisusceptible to non-β-lactam agents, but multiresistance is already prevalent in all regions. Surveillance of MRSA susceptibility patterns should be monitored to provide clinicians with the most current information regarding changes in resistance patterns.

"Live High-Train Low and High" Hypoxic Training Improves Team-Sport Performance.

PURPOSE: This study aims to investigate physical performance and hematological changes in 32 elite male team-sport players after 14 d of "live high-train low" (LHTL) training in normobaric hypoxia (≥14 h·d at 2800-3000 m) combined with repeated-sprint training (six sessions of four sets of 5 × 5-s sprints with 25 s of passive recovery) either in normobaric hypoxia at 3000 m (LHTL + RSH, namely, LHTLH; n = 11) or in normoxia (LHTL + RSN, namely, LHTL; n = 12) compared with controlled "live low-train low" (LLTL; n = 9) training. METHODS: Before (Pre), immediately after (Post-1), and 3 wk after (Post-2) the intervention, hemoglobin mass (Hbmass) was measured in duplicate [optimized carbon monoxide (CO) rebreathing method], and vertical jump, repeated-sprint (8 × 20 m-20 s recovery), and Yo-Yo Intermittent Recovery level 2 (YYIR2) performances were tested. RESULTS: Both hypoxic groups similarly increased their Hbmass at Post-1 and Post-2 in reference to Pre (LHTLH: +4.0%, P < 0.001 and +2.7%, P < 0.01; LHTL: +3.0% and +3.0%, both P < 0.001), whereas no change occurred in LLTL. Compared with Pre, YYIR2 performance increased by ∼21% at Post-1 (P < 0.01) and by ∼45% at Post-2 (P < 0.001), with no difference between the two intervention groups (vs no change in LLTL). From Pre to Post-1, cumulated sprint time decreased in LHTLH (-3.6%, P < 0.001) and LHTL (-1.9%, P < 0.01), but not in LLTL (-0.7%), and remained significantly reduced at Post-2 (-3.5%, P < 0.001) in LHTLH only. Vertical jump performance did not change. CONCLUSIONS: "Live high-train low and high" hypoxic training interspersed with repeated sprints in hypoxia for 14 d (in season) increases the Hbmass, YYIR2 performance, and repeated-sprint ability of elite field team-sport players, with benefits lasting for at least 3 wk postintervention.

Genetics-Based Population Pharmacokinetics and Pharmacodynamics of Risperidone in a Psychiatric Cohort.

BACKGROUND: High interindividual variability in plasma concentrations of risperidone and its active metabolite, 9-hydroxyrisperidone, may lead to suboptimal drug concentration. OBJECTIVE: Using a population pharmacokinetic approach, we aimed to characterize the genetic and non-genetic sources of variability affecting risperidone and 9-hydroxyrisperidone pharmacokinetics, and relate them to common side effects. METHODS: Overall, 150 psychiatric patients (178 observations) treated with risperidone were genotyped for common polymorphisms in NR1/2, POR, PPARα, ABCB1, CYP2D6 and CYP3A genes. Plasma risperidone and 9-hydroxyrisperidone were measured, and clinical data and common clinical chemistry parameters were collected. Drug and metabolite concentrations were analyzed using non-linear mixed effect modeling (NONMEM(®)). Correlations between trough concentrations of the active moiety (risperidone plus 9-hydroxyrisperidone) and common side effects were assessed using logistic regression and linear mixed modeling. RESULTS: The cytochrome P450 (CYP) 2D6 phenotype explained 52 % of interindividual variability in risperidone pharmacokinetics. The area under the concentration-time curve (AUC) of the active moiety was found to be 28 % higher in CYP2D6 poor metabolizers compared with intermediate, extensive and ultrarapid metabolizers. No other genetic markers were found to significantly affect risperidone concentrations. 9-hydroxyrisperidone elimination was decreased by 26 % with doubling of age. A correlation between trough predicted concentration of the active moiety and neurologic symptoms was found (p = 0.03), suggesting that a concentration >40 ng/mL should be targeted only in cases of insufficient, or absence of, response. CONCLUSIONS: Genetic polymorphisms of CYP2D6 play an important role in risperidone, 9-hydroxyrisperidone and active moiety plasma concentration variability, which were associated with common side effects. These results highlight the importance of a personalized dosage adjustment during risperidone treatment.

A new approach for the separation of spermatozoa from other cell types in forensically relevant samples.

Due to imbalance in genetic material contribution, gynecological samples collected following a sexual assault are challenging to process in order to resolve the male contributor's DNA profile. We set up a new and fast procedure for the recovery and separation of cells from cotton swabs, or other supports. Using spermatozoa specific CD52 antibody coupled to magnetic beads along with magnetic columns, this procedure was first developed and optimized by flow cytometry. It allows the recovery of two enriched cell fractions: a sperm fraction, mostly enriched with the alleged offender's spermatozoa, and a non-sperm fraction, mostly enriched with cells from the victim. Processing fresh as well as six months old mock samples, made of buccal swabs loaded with sperm dilutions, resulted in full single NGM SElect DNA profiles of the sperm donors, respectively the epithelial cells donors, for the sperm and the non-sperm fractions. Untreated duplicate samples processed in parallel only provided the autosomal DNA profiles of the epithelial cells donors. This new procedure can be rapidly tested and adopted by forensic laboratories worldwide as it uses material already commercially available. Moreover it can be easily automated with existing platform, and could therefore provide a mean to rapidly reduce existing backlogs.

Influence of PTPN2 and pre-existing immunity on the generation of effector and memory CD8 T cells

Notre système immunitaire joue un rôle important pour la protection envers les maladies infectieuses. Au cours d'une réponse à une infection primaire, des cellules B et des cellules T spécifiques, dirigées contre le pathogène en question, sont générées et certaines d'entre elles deviennent des cellules dites mémoires. Leur fonction est de nous protéger contre une nouvelle infection avec le même pathogène, une infection secondaire. Dans certaines situations, comme c'est par exemple le cas avec la grippe, les pathogènes ne sont pas toujours complètement identiques et les cellules mémoires ne sont pas à même d'assurer leur rôle protecteur et d'empêcher une réinfection. Pourtant, on ne sait à l'heure actuelle que très peu comment une immunité acquise, mais non protectrice, influence le développement d'une réponse immunitaire ultérieure. Dans la première partie de cette thèse, nous avons étudié comment les cellules T mémoires cytotoxiques altèrent la réponse de cellules T cytotoxiques nouvellement induites. Au cours d'une réaction immunitaire dirigée contre une infection primaire, un vaste répertoire de lymphocytes T est créé, constitué de cellules T possédant divers degrés d'affinité pour le pathogène. Lors d'une infection secondaire, seules les cellules T ayant une forte affinité pour le pathogène participent à la réponse. Nous avons pu démontrer que ce phénomène de restriction du répertoire des cellules T est principalement causé par les cellules T mémoires qui sont à même de reconnaître un antigène pathogénique présent dans les deux infections. Dans un deuxième projet, nous avons étudié comment l'absence de PTPN2 influence la réponse des cellules T. Chez l'homme, une mutation dans le gène de PTPN2 est associée à des maladies auto-immunes et résulte en une activité réduite de cette phosphatase dans les lymphocytes T. Nous avons montré que la baisse d'activité de la phosphatase PTNP2 conduit à une meilleure expansion des cellules T ayant une qualité comparable à des cellules T auto-antigène spécifiques. De plus, nous avons observé que la survie de ces cellules T effectues ayant une phosphatase diminuée est nettement améliorée. Cela peut conduire à une réponse immunitaire plus efficace ou, éventuellement, à une pathologie auto-immune plus grave. En outre, nos résultats montrent qu'en manipulant l'activité de cette phosphatase, il est possible d'augmenter l'efficacité du transfert des cellules T dans un hôte receveur. Un tel transfert de cellules T est pratiqué chez des patients atteints de tumeurs. Nos travaux suggèrent que la manipulation de la phosphatase PTPN2 pourrait donc représenter une approche thérapeutique novatrice et prometteuse. -- Notre système immunitaire joue un rôle important pour la protection contre les maladies. Les cellules T CD8+ ont une importance primordiale pour le contrôle d'infections primaires causées par des virus ou bactéries, mais également contre certaines tumeurs. Par conséquent, mieux comprendre les exigences nécessaires à l'induction de bonnes réponses des cellules T CD8 pourrait nous permettre de construire des vaccins contre les pathogènes contre lesquels nous n'avons pour l'instant pas de vaccins mais aussi d'améliorer les réactions immunitaires dirigées anti-tumorales. Dans la première partie de cette thèse, nous avons étudié l'influence qu'une immunité préexistante a sur la réponse des cellules T CD8. Nous sommes souvent exposés à des pathogènes qui sont similaires mais pas identiques à ceux que nous avons rencontrés auparavant. De telles infections hétérologues ne sont pas l'objet de beaucoup d'études et certains exemples indiquent même qu'une immunité préexistante partielle peut mener à une aggravation de la maladie. Nous avons étudié le répertoire des lymphocytes T CD8 qui sont générés lors d'une rencontre avec un nouvel antigène, et ce en comparant infection primaire et secondaire. En utilisant le modèle expérimental d'infections à Listeria monocytogenes, nous avons pu montrer que lors d'une infection primaire, un répertoire diversifié comprenant des cellules T CD8 de forte et faible affinité est constitué. Au contraire, dans le cas d'une infection secondaire, le répertoire des cellules T est fortement limité et seulement les lymphocytes T de forte affinité sont impliqués dans la réponse immunitaire. Nous avons pu démontrer que ces Rangements sont provoqués par des cellules T CD8 mémoires capables de reconnaître un antigène présent dans les deux infections. Cette augmentation du seuil d'activation des cellules effectrices est majoritairement causée par les lymphocytes T CD8 mémoires non transférables. Ces observations indiquent que les vaccins visant à induire des cellules T anti-tumorales de faible affinité seraient inefficaces si le vaccin contient des épitopes contre lesquels il existe une mémoire immunologique. Les réponses immunitaires conduites par les cellules T contre les antigènes tumoraux dépendent des cellules T CD8 de faible réactivité contre les antigènes tumoraux puisque les cellules à forte réactivité sont éliminées par les mécanismes de tolérance. Nous basant sur l'existence dans la littérature de preuves indiquant que PTPN2 influence la réponse des cellules T de faible affinité, nous nous sommes intéressés à comprendre comment PTPN2 impacte les réponses des cellules T CD8 en général. Nous avons remarqué que des cellules T CD8 déficientes en PTPN2 exhibent une meilleure capacité à proliférer suite à une faible ou courte stimulation du récepteur des lymphocytes T. La phase effectrice est prolongée et la contraction retardée résultant ainsi à globalement plus de cellules effectrices. Ce phénomène est également accompagné d'une meilleure survie des cellules effectrices de différentiation terminale. Une fois transférées dans un nouvel hôte receveur, les cellules effectrices terminales KLRG1+CD127- déficientes en phosphatase PTPN2 peuvent survivre et se transformer en cellules mémoires CD127+ fonctionnelles. De façon inattendue, nous avons découvert que l'élimination de PTPN2 améliore l'efficacité du transfert et la formation des cellules mémoires ainsi que leur capacité protectrice. Manipuler l'activité de cette phosphatase apparaît donc comme une approche intéressante et prometteuse pour la thérapie cellulaire par transfert adoptif de lymphocytes T. Nos observations montrent que la manipulation d'un facteur intrinsèque, l'absence de PTPN2, peut, dans certaines circonstances, améliorer la réponse des cellules T. Une meilleure connaissance des mécanismes contrôlant la réponse des lymphocytes T CD8 pourrait donc permettre la manipulation de ces derniers et conduire à des réponses immunitaires plus vigoureuses. Si ces réponses sont déclenchées par l'utilisation de vaccins, il est nécessaire de considérer l'historique d'une exposition préalable à des agents pathogènes ou à des vaccins puisque celle-ci peut, comme nous l'avons démontré, influencer le répertoire des cellules T recrutées dans la réponse immunitaire et, par conséquent, modifier l'aptitude de notre système immunitaire à faire face à une infection. -- Our immune system plays an important role in the protection from disease. CD8 T cells are critical for the control of primary infections with most viruses and certain bacteria as well as against some tumors. Therefore, better knowledge of CD8 T cell responses might enable us to generate vaccines against pathogens for which currently no vaccines are available or to improve anti-tumor immune responses. In the first part of this thesis we addressed the issue how previously acquired immunity impacts on the response of CD8 T cells. We are often exposed to pathogens that are related but not identical to the previously encountered ones. Such heterologous infections are not well studied and there are some indications that partial pre-existing immunity may in some cases even lead to an enhancement of disease. We specifically studied the T cell repertoire of CD8 T cells that are responding to a newly encountered antigen in secondary compared to primary infections. Using the experimental model of Listeria monocytogenes infections, we showed that in primary infections a wide repertoire including high and low affinity CD8 T cells is recruited into the immune response. In contrast to this, in secondary infections, the T cell repertoire is severely restricted and only T cells of high affinity are responding. We were able to pinpoint this difference to the presence of memory CD8 T cells that recognize an antigen that is shared between the two subsequent infections. This increase in the activation threshold was most effectively mediated via non-transferable memory CD8 T cells. This would argue that vaccines targeting low affinity tumor-specific T cells would fail if the vaccine contains previously encountered CD8 T cell epitopes. T cell mediated immune responses to tumor antigen rely often on T cells which weakly react to tumor antigen as high affinity T cells are eliminated by tolerance mechanisms. Following indication in the literature that PTPN2 impacts on the response of such weakly antigen-reactive T cells, we investigated how PTPN2 impacts in general the response of CD8 T cells. We observed that CD8 T cells lacking PTPN2 show an enhanced expansion following weak or short-term T cell receptor stimulation. The effector phase is prolonged and contraction delayed thus resulting in overall more effector cells. This is accompanied by a better survival of terminal effector cells. When transferred into new recipients, KLRG1+CD127- terminal effector cells lacking PTPN2 can survive and convert into CD127+ functional memory cells. Surprisingly, we discovered that elimination of PTPN2 enhances the transfer efficacy and formation of memory cells as well as the protective capacity. Targeting PTPN2 might thus be a promising approach for adoptive T cell therapy. Our observations show how the manipulation of an intrinsic factor, the absence of PTPN2, can enhance T cell responses under certain circumstances. A better understanding of underlying mechanisms for the control of CDS T cell responses might enable the manipulation of these and allow for more powerful responses. If these responses are induced through vaccines it is imperative that the previous history of exposure to pathogens or vaccines is considered as it can, as we have shown in this thesis, influence the recruited T cell repertoire and thus possibly the ability to handle the infection.

Prevalence of ragweed allergy in rural Geneva - a pilot study.

OBJECTIVE: The prevalence of ragweed allergy is increasing worldwide. Ragweed distribution and abundance is spreading in Europe in a wide area ranging from the Rhone valley in France to Hungary and Ukraine, where the rate of the prevalence can peak at as high as 12%. Low-grade ragweed colonisation was seen in Geneva and Ticino, less than two decades ago. There were fears that allergies to ragweed would increase Switzerland. The intent of this study was to assess the rate of prevalence of sensitisation and allergy to ragweed in the population living in the first rural Swiss setting where ragweed had been identified in 1996, and to evaluate indirectly the efficacy of elimination and containment strategies. MATERIAL AND METHODS: In 2009, 35 adults in a rural village in the Canton of Geneva were recruited. Data were collected by means of questionnaires and skin-prick tests were done on each participant. The study was approved by the local Ethics Committee. RESULTS: Based on questionnaires, 48.6% had rhinitis (95% confidence interval [CI] 32.9-64.4; n = 17/35) and 17.1% asthma (95% CI 8.1-32.6; n = 6/35). Atopy was diagnosed in 26.4% (95% CI 12.9-44.4) of the sample (n = 9/34). Ragweed sensitisation was found in 2.9% (95% CI 0.7-19.7; n = 1/34), mugwort sensitisation in 2.9% (95% CI 0.1-14.9; n = 1/35), alder sensitisation in 17.1% (95% CI 6.6-33.6; n = 6/35), ash sensitisation in 12.5% (95% CI 3.5-29.0; n = 4/32) and grass sensitisation in 22.9% (95% CI 10.4-40.1; n = 8/35). Ragweed (95% CI 0.1-14.9; n = 1/34) and mugwort allergies (95% CI 0.1-14.9; n = 1/35) were both found in 2.9% of the population. CONCLUSION: This study showed a surprisingly low incidence of ragweed sensitisation and allergy, of 2.9% and 2.9%, respectively, 20 years after the first ragweed detection in Geneva. The feared rise in ragweed allergy seems not to have happened in Switzerland, compared with other ragweed colonised countries. These results strongly support early field strategies against ragweed.

Prognostic factors for progression-free and overall survival in advanced biliary tract cancer.

BACKGROUND: Biliary tract cancer is an uncommon cancer with a poor outcome. We assembled data from the National Cancer Research Institute (UK) ABC-02 study and 10 international studies to determine prognostic outcome characteristics for patients with advanced disease. METHODS: Multivariable analyses of the final dataset from the ABC-02 study were carried out. All variables were simultaneously included in a Cox proportional hazards model, and backward elimination was used to produce the final model (using a significance level of 10%), in which the selected variables were associated independently with outcome. This score was validated externally by receiver operating curve (ROC) analysis using the independent international dataset. RESULTS: A total of 410 patients were included from the ABC-02 study and 753 from the international dataset. An overall survival (OS) and progression-free survival (PFS) Cox model was derived from the ABC-02 study. White blood cells, haemoglobin, disease status, bilirubin, neutrophils, gender, and performance status were considered prognostic for survival (all with P < 0.10). Patients with metastatic disease {hazard ratio (HR) 1.56 [95% confidence interval (CI) 1.20-2.02]} and Eastern Cooperative Oncology Group performance status (ECOG PS) 2 had worse survival [HR 2.24 (95% CI 1.53-3.28)]. In a dataset restricted to patients who received cisplatin and gemcitabine with ECOG PS 0 and 1, only haemoglobin, disease status, bilirubin, and neutrophils were associated with PFS and OS. ROC analysis suggested the models generated from the ABC-02 study had a limited prognostic value [6-month PFS: area under the curve (AUC) 62% (95% CI 57-68); 1-year OS: AUC 64% (95% CI 58-69)]. CONCLUSION: These data propose a set of prognostic criteria for outcome in advanced biliary tract cancer derived from the ABC-02 study that are validated in an international dataset. Although these findings establish the benchmark for the prognostic evaluation of patients with ABC and confirm the value of longheld clinical observations, the ability of the model to correctly predict prognosis is limited and needs to be improved through identification of additional clinical and molecular markers.

Similar Hemoglobin Mass Response in Hypobaric and Normobaric Hypoxia in Athletes

PURPOSE: To compare hemoglobin mass (Hbmass) changes during an 18-d live high-train low (LHTL) altitude training camp in normobaric hypoxia (NH) and hypobaric hypoxia (HH). METHODS: Twenty-eight well-trained male triathletes were split into three groups (NH: n = 10, HH: n = 11, control [CON]: n = 7) and participated in an 18-d LHTL camp. NH and HH slept at 2250 m, whereas CON slept, and all groups trained at altitudes <1200 m. Hbmass was measured in duplicate with the optimized carbon monoxide rebreathing method before (pre-), immediately after (post-) (hypoxic dose: 316 vs 238 h for HH and NH), and at day 13 in HH (230 h, hypoxic dose matched to 18-d NH). Running (3-km run) and cycling (incremental cycling test) performances were measured pre and post. RESULTS: Hbmass increased similar in HH (+4.4%, P < 0.001 at day 13; +4.5%, P < 0.001 at day 18) and NH (+4.1%, P < 0.001) compared with CON (+1.9%, P = 0.08). There was a wide variability in individual Hbmass responses in HH (-0.1% to +10.6%) and NH (-1.4% to +7.7%). Postrunning time decreased in HH (-3.9%, P < 0.001), NH (-3.3%, P < 0.001), and CON (-2.1%, P = 0.03), whereas cycling performance changed nonsignificantly in HH and NH (+2.4%, P > 0.08) and remained unchanged in CON (+0.2%, P = 0.89). CONCLUSION: HH and NH evoked similar Hbmass increases for the same hypoxic dose and after 18-d LHTL. The wide variability in individual Hbmass responses in HH and NH emphasizes the importance of individual Hbmass evaluation of altitude training.