Antifungal activity against planktonic and biofilm Candida albicans in an experimental model of foreign-body infection.

OBJECTIVES: The treatment of Candida implant-associated infections remains challenging. We investigated the antifungal activity against planktonic and biofilm Candida albicans in a foreign-body infection model. METHODS: Teflon cages were subcutaneously implanted in guinea pigs, infected with C. albicans (ATCC 90028). Animals were treated intraperitoneally 12 h after infection for 4 days once daily with saline, fluconazole (16 mg/kg), amphotericin B (2.5 mg/kg), caspofungin (2.5 mg/kg) or anidulafungin (20 mg/kg). Planktonic Candida was quantified, the clearance rate and cure rate determined. RESULTS: In untreated animals, planktonic Candida was cleared from cage fluid in 25% (infected with 4.5 × 10(3) CFU/cage), 8% (infected with 4.8 × 10(4) CFU/cage) and 0% (infected with 6.2 × 10(5) CFU/cage). Candida biofilm persisted on all explanted cages. Compared to untreated controls, caspofungin reduced the number of planktonic C. albicans to 0.22 and 0.0 CFU/ml, respectively, and anidulafungin to 0.11 and 0.13 CFU/ml, respectively. Fluconazole cured 2/12 cages (17%), amphotericin B and anidulafungin 1/12 cages (8%) and caspofungin 3/12 cages (25%). CONCLUSION: Echinocandins showed superior activity against planktonic C. albicans. Caspofungin showed the highest cure rate of C. albicans biofilm. However, no antifungal exceeded 25% cure rate, demonstrating the difficulty of eradicating Candida biofilms from implants.

Islet Brain 1 Protects Insulin Producing Cells against Lipotoxicity.

Chronic intake of saturated free fatty acids is associated with diabetes and may contribute to the impairment of functional beta cell mass. Mitogen activated protein kinase 8 interacting protein 1 also called islet brain 1 (IB1) is a candidate gene for diabetes that is required for beta cell survival and glucose-induced insulin secretion (GSIS). In this study we investigated whether IB1 expression is required for preserving beta cell survival and function in response to palmitate. Chronic exposure of MIN6 and isolated rat islets cells to palmitate led to reduction of the IB1 mRNA and protein content. Diminution of IB1 mRNA and protein level relied on the inducible cAMP early repressor activity and proteasome-mediated degradation, respectively. Suppression of IB1 level mimicked the harmful effects of palmitate on the beta cell survival and GSIS. Conversely, ectopic expression of IB1 counteracted the deleterious effects of palmitate on the beta cell survival and insulin secretion. These findings highlight the importance in preserving the IB1 content for protecting beta cell against lipotoxicity in diabetes.

L-Lactate protects neurons against excitotoxicity: implication of an ATP-mediated signaling cascade.

Converging experimental data indicate a neuroprotective action of L-Lactate. Using Digital Holographic Microscopy, we observe that transient application of glutamate (100 μM; 2 min) elicits a NMDA-dependent death in 65% of mouse cortical neurons in culture. In the presence of L-Lactate (or Pyruvate), the percentage of neuronal death decreases to 32%. UK5099, a blocker of the Mitochondrial Pyruvate Carrier, fully prevents L-Lactate-mediated neuroprotection. In addition, L-Lactate-induced neuroprotection is not only inhibited by probenicid and carbenoxolone, two blockers of ATP channel pannexins, but also abolished by apyrase, an enzyme degrading ATP, suggesting that ATP produced by the Lactate/Pyruvate pathway is released to act on purinergic receptors in an autocrine/paracrine manner. Finally, pharmacological approaches support the involvement of the P2Y receptors associated to the PI3-kinase pathway, leading to activation of KATP channels. This set of results indicates that L-Lactate acts as a signalling molecule for neuroprotection against excitotoxicity through coordinated cellular pathways involving ATP production, release and activation of a P2Y/KATP cascade.

Impact of T cell receptor affinity on the molecular mechanisms modulating CD8 T cell signalling and function against NY-ESO-1 tumour antigen

It is well established that cytotoxic T lymphocytes play a pivotal role in the protection against intracellular pathogens and tumour cells. Such protective immune responses rely on the specific T cell receptor (TCR)-mediated recognition by CD8 T cells of small antigenic peptides presented in the context of class-I Major Histocompatibility Complex molecules (pMHCs) on the surface of infected or malignant cells. The strength (affinity/avidity) of this interaction is a major correlate of protection. Although tumour-reactive CD8 T cells can be observed in cancer patients, anti-tumour immune responses are often ineffective in controlling or eradicating the disease due to the relative low TCR affinity of these cells. To overcome this limitation, tumour-specific CD8 T cells can be genetically modified to express TCRs of improved binding strength against a defined tumour antigen before adoptive cell transfer into cancer patients. We previously generated a panel of TCRs specific for the cancer-testis antigen NY-ESO-l,57.165 with progressively increased affinities for the pMHC complex, thus providing us with a unique tool to investigate the causal link between the surface expression of such TCRs and T cell activation and function. We recently demonstrated that anti-tumour CD8 T cell reactivity could only be improved within physiological affinity limits, beyond which drastic functional declines were observed, suggesting the presence of multiple regulatory mechanisms limiting T cell activation and function in a TCR affinity-dependent manner. The overarching goal of this thesis was (i) to assess the precise impact of TCR affinity on T cell activation and signalling at the molecular level and (ii) to gain further insights on the mechanisms that regulate and delimitate maximal/optimized CD8 T cell activation and signalling. Specifically, by combining several technical approaches we characterized the activation status of proximal (i.e. CD3Ç, Lek, and ZAP-70) and distal (i.e. ERK1/2) signalling molecules along the TCR affinity gradient. Moreover, we assessed the extent of TCR downmodulation, a critical step for initial T cell activation. CD8 T cells engineered with the optimal TCR affinity variants showed increased activation levels of both proximal and distal signalling molecules when compared to the wild-type T cells. Our analyses also highlighted the "paradoxical" status of tumour-reactive CD8 T cells bearing very high TCR affinities, which retained strong proximal signalling capacity and TCR downmodulation, but were unable to propagate signalling distally (i.e. pERKl/2), resulting in impaired cell-mediated functions. Importantly, these very high affinity T cells displayed maximal levels of SHP-1 and SHP-2 phosphatases, two negative regulatory molecules, and this correlated with a partial pERKl/2 signalling recovery upon pharmacological SHP-l/SHP-2 inhibition. These findings revealed the putative presence of inhibitory regulators of the TCR signalling cascade acting very rapidly following tumour-specific stimulation. Moreover, the very high affinity T cells were only able to transiently express enhanced proximal signalling molecules, suggesting the presence of an additional level of regulation that operates through the activation of negative feedback loops over time, limiting the duration of the TCR-mediated signalling. Overall, the determination of TCR-pMHC binding parameters eliciting optimal CD8 T cell activation, signalling, and effector function while guaranteeing high antigen specificity, together with the identification of critical regulatory mechanisms acting proximally in the TCR signalling cascade, will directly contribute to optimize and support the development of future TCR-based adoptive T cell strategies for the treatment of malignant diseases. -- Les lymphocytes T CD8 cytotoxiques jouent un rôle prédominant dans la protection contre les pathogènes intracellulaires et les cellules tumorales. Ces réponses immunitaires dépendent de la spécificité avec laquelle les récepteurs T (TCR) des lymphocytes CD8 reconnaissent les peptides antigéniques présentés par les molécules du complexe Majeur de Histocompatibilité de classe I (pCMH) à la surface des cellules infectées ou malignes. La force (ou affinité/avidité) de l'interaction du TCR-pCMH est un corrélat majeur de protection. Les réponses immunitaires sont cependant souvent inefficaces et ne permettent pas de contrôler ou d'éliminer les cellules tumorales chez les patients atteint du cancer, et ce à cause de la relative faible reconnaissance des TCRs exprimés par les lymphocytes T CD8 envers les antigènes tumoraux. Afin de surmonter cette limitation, les cellules T anti-tumorales peuvent être génétiquement modifiées en les dotant de TCRs préalablement optimisés afin d'augmenter leur reconnaissance ou affinité contre les antigènes tumoraux, avant leur ré¬infusion dans le patient. Nous avons récemment généré des cellules T CD8 exprimant un panel de TCRs spécifiques pour l'antigène tumoral NY-ESO-l157.16J avec des affinités croissantes, permettant ainsi d'investiguer la causalité directe entre l'affinité du TCR-pCMH et la fonction des cellules T CD8. Nous avons démontré que la réactivité anti-tumorale pouvait être améliorée en augmentant l'affinité du TCR dans une intervalle physiologique, mais au delà duquel nous observons un important déclin fonctionnel. Ces résultats suggèrent la présence de mécanismes de régulation limitant l'activation des cellules T de manière dépendante de l'affinité du TCR. Le but de cette thèse a été (i) de définir l'impact précis de l'affinité du TCR sur l'activation et la signalisation des cellules T CD8 au niveau moléculaire et (ii) d'acquérir de nouvelles connaissances sur les mécanismes qui régulent et délimitent l'activation et la signalisation maximale des cellules T CD8 optimisées. Spécifiquement, en combinant plusieurs approches technologiques, nous avons caractérisé l'état d'activation de différentes protéines de la voie de signalisation proximale (CD3Ç, Lek et ZAP-70) et distale (ERK1/2) le long du gradient d'affinité du TCR, ainsi que l'internalisation du TCR, une étape clef dans l'activation initiale des cellules T. Les lymphocytes T CD8 exprimant des TCRs d'affinité optimale ont montré des niveaux d'activation augmentés des molécules proximales et distales par rapport aux cellules de type sauvage (wild-type). Nos analyses ont également mis en évidence un paradoxe chez les cellules T CD8 équipées avec des TCRs de très haute affinité. En effet, ces cellules anti-tumorales sont capables d'activer leurs circuits biochimiques au niveau proximal et d'internaliser efficacement leur TCR, mais ne parviennent pas à propager les signaux biochimiques dépendants du TCR jusqu'au niveau distal (via phospho-ERKl/2), avec pour conséquence une limitation de leur capacité fonctionnelle. Finalement, nous avons démontré que SHP-1 et SHP-2, deux phosphatases avec des propriétés régulatrices négatives, étaient majoritairement exprimées dans les cellules T CD8 de très hautes affinités. Une récupération partielle des niveaux d'activation de ERK1/2 a pu être observée après l'inhibition pharmacologique de ces phosphatases. Ces découvertes révèlent la présence de régulateurs moléculaires qui inhibent le complexe de signalisation du TCR très rapidement après la stimulation anti-tumorale. De plus, les cellules T de très hautes affinités ne sont capables d'activer les molécules de la cascade de signalisation proximale que de manière transitoire, suggérant ainsi un second niveau de régulation via l'activation de mécanismes de rétroaction prenant place progressivement au cours du temps et limitant la durée de la signalisation dépendante du TCR. En résumé, la détermination des paramètres impliqués dans l'interaction du TCR-pCMH permettant l'activation de voies de signalisation et des fonctions effectrices optimales ainsi que l'identification des mécanismes de régulation au niveau proximal de la cascade de signalisation du TCR contribuent directement à l'optimisation et au développement de stratégies anti-tumorales basées sur l'ingénierie des TCRs pour le traitement des maladies malignes.

Antibody levels against GLURP R2, MSP1 block 2 hybrid and AS202.11 and the risk of malaria in children living in hyperendemic (Burkina Faso) and hypo-endemic (Ghana) areas.

Differences in parasite transmission intensity influence the process of acquisition of host immunity to Plasmodium falciparum malaria and ultimately, the rate of malaria related morbidity and mortality. Potential vaccines being designed to complement current intervention efforts therefore need to be evaluated against different malaria endemicity backgrounds. The associations between antibody responses to the chimeric merozoite surface protein 1 block 2 hybrid (MSP1 hybrid), glutamate-rich protein region 2 (GLURP R2) and the peptide AS202.11, and the risk of malaria were assessed in children living in malaria hyperendemic (Burkina Faso, n = 354) and hypo-endemic (Ghana, n = 209) areas. Using the same reagent lots and standardized protocols for both study sites, immunoglobulin (Ig) M, IgG and IgG sub-class levels to each antigen were measured by ELISA in plasma from the children (aged 6-72 months). Associations between antibody levels and risk of malaria were assessed using Cox regression models adjusting for covariates. There was a significant association between GLURP R2 IgG3 and reduced risk of malaria after adjusting age of children in both the Burkinabe (hazard ratio 0.82; 95 % CI 0.74-0.91, p < 0.0001) and the Ghanaian (HR 0.48; 95 % CI 0.25-0.91, p = 0.02) cohorts. MSP1 hybrid IgM was associated (HR 0.85; 95 % CI 0.73-0.98, p = 0.02) with reduced risk of malaria in Burkina Faso cohort while IgG against AS202.11 in the Ghanaian children was associated with increased risk of malaria (HR 1.29; 95 % CI 1.01-1.65, p = 0.04). These findings support further development of GLURP R2 and MSP1 block 2 hybrid, perhaps as a fusion vaccine antigen targeting malaria blood stage that can be deployed in areas of varying transmission intensity.

The skill paradox: Explaining and reducing employment discrimination against skilled immigrants

Using a social identity theory approach, we theorized that recruiters might be particularly biased against skilled immigrant applicants. We refer to this phenomenon as a skill paradox, according to which immigrants are more likely to be targets of employment discrimination the more skilled they are. Furthermore, building on the common ingroup identity model, we proposed that this paradox can be resolved through human resource management (HRM) strategies that promote inclusive hiring practices (e.g., by emphasizing fit with a diverse clientele). The results from a laboratory experiment were consistent with our predictions: Local recruiters preferred skilled local applicants over skilled immigrant applicants, but only when these applicants were qualified for a specific job. This bias against qualified and skilled immigrant applicants was attenuated when fit with a diverse clientele was emphasized, but not when fit with a homogeneous clientele was emphasized or when the hiring strategy was not explained. We discuss the implications of our findings for research on employment discrimination against skilled immigrants, including the role of inclusiveness for reducing discriminatory biases.

The Impact of Supervisor's Advice, Codes of Conduct, Accountability and Moral Attentiveness of Individuals on Discriminatory Behaviour Against Immigrants.

Past studies on the personnel selection demonstrated that a supervisor's advice to discriminate can lead to compliant behaviours. This study had the aim to extend past findings by examining what can overcome the powerful influence of the hierarchy. 50 Swiss managers participated to an in-basket exercise. The main task was to evaluate Swiss candidates (in-group) and foreigners (out-groups: Spanish and Kosovo Albanians) and to select two applicants for a job interview. Main results were the effect of codes of conduct to prevent discrimination against out-group applicants in the presence of a supervisor's advice to prefer in-group members. But, when participants were accountable to an audience, this beneficial effect disappears because participants followed the supervisor's advice. The second aim was to assess if the difference in responses between participants was related to their difference in moral attentiveness. Results showed some significant relationships but not always in the direction expected.

Leishmaniavirus : a possible target against metastatic leishmaniasis

Leishmaniasis is widely spread disease found in bath tropical and temperate regions but limited to the habitat of its sand fly vector. lt affects over 12 million people with 2 million new cases each year. As cutaneous leishmaniasis patients show varying levels of immunity to the disease after recovery, the development of a vaccine has much promise as a prevention strategy. Unfortunately however, existing anti-leishmanial vaccines are plagued by safety issues and have only ever shown limited efficacy .So, despite much effort, no effective vaccine is currently available. Recent studies suggest a correlation between the presence of Leishmania RNA virus (LRV) and the development of mucocutaneous leishmaniasis (MCL), which is characterised by the presence of secondary lesions in nasal and buccal mucosa, causing destructive and disfiguring facial lesions. Moreover, recent research has associated the viral presence to treatment fa ilure in patients. ln the first part of this work, we propose that these viral particles may serve as promising vaccine candidates due to their powerful TLR-3 antigenicity, launching an early cell-mediated attack on stimulated cells and thus eliminating their virulent complications. The second part of this work discusses a preliminary study on the lymphocyte immune response against Leishmania guyanensis infection. The lymphocyte response (and in particular, the raie of CDS+ T cells) is controversial and varies greatly between Leishmania species. Here, we illustrate the importance of a small CDS+ T cell subpopulation, expressing the CDSaa+ receptor. These intraepithelial lymphocytes are mainly present in the skin, vagina and intestinal tissue and are best known for their raie in the early immune response against pathogens. Similarly to traditional CDS+ cells, they secrete the tissue-destructive enzymes, perforin and granzyme, which can result in a hyper-inflammatory cutaneous lesion, raising a possibility for their raie in Leishmania infection. lndeed, our initial results in a murine mode( of Leishmania guyanensis infection suggest a pathogenic raie for CDSaa+ T cells. Further research into species-specific immune responses against the various Leishmania parasites is critical to realising the clinical potential of immunotherapy in the treatment and prevention of this disfiguring disease . -- La Leishmaniose est une maladie infectieuse causée par le parasite Leishmania. Elle est localisée dans les régions où son vecteur se reproduit, c'est-à-dire dans des régions tropicales ou tempérées. Cette pathologie affecte 12 millions des personnes dans le monde et 2 millions de nouveaux cas sont recensés chaque année. D'autres facteurs, tels la déforestation, les conditions d'hygiène ou encore l'accès limité aux médicaments, aggravent la pathologie et renforcent sa propagation. Les patients affectés par la leishmaniose et qui arrivent à en guérir, présentent une protection contre une réinfection. Pour cette raison, le développement d'un vaccin reste la meilleure solution pour combattre ce fléau. Mais, à ce jour, et malgré beaucoup d'efforts, aucun vaccin efficace n'a encore été développé. Un autre facteur responsable de l'aggravation de la pathologie et de la résistance de ces parasites aux drogues est un virus qui peut infecter certaines souches de Leishmania. Ce virus, appelé Leishmania RNA virus, peut induire une réponse inflammatoire exagérée, ce qui a comme résultat l'aggravation de la pathologie, la survie et la dissémination de ce parasite au sein de l'hôte infecté. Vu l'absence d'un vaccin contre ce parasite, Leishmania, nous proposons de développer un vaccin non pas contre le parasite lui- même mais contre l'agent qui provoque l'exacerbation de la pathologie, c'est-à-dire le virus. Dans cette étude, nous décrivons le développement d'un vaccin contre LRV, qui empêche le parasite d'induire des inflammations exagérées dans les souris. En d'autres mots, nous essayons de prévenir toutes les complications générées par cet hyperpathogène qu'est le LRV, en utilisant sa capside comme cible pour le développement d'un vaccin. Dans la deuxième partie de ce manuscrit, nous avons aussi étudié plus en détail la réponse immunitaire, et en particulier la réponse des lymphocytes T COB suite à l'infection du parasite Leishmania guyanensis porteur du LRV.