Gastric evacuation and feeding in the gilthead sea bream reared under semi-intensive conditions

Gastric evacuation rates of the gilthead sea bream, Sparus aurata, fed with commercial pelleted food and polychaetes (Nereis diversicolor) were determined under experimental conditions. The estimated gastric evacuation rate for pelleted food was 7.97% h(-1), with a total time of digestion of approximately 9 h. The respective values for the natural food were 6.24% h(-1), with a total digestion time of approximately 12 h. The daily consumption of fish reared in earth ponds in a semi-intensive aquaculture facility was estimated through 24h cycles performed between April and August. The daily consumption varied from 18.58 to 31.98 mg g(-1). There was a constant increase in the average daily consumption per individual of 1.8-4.6 g (dry weight). During these cycles, samples of stomachs were taken and the contents preserved for further observation. The feeding behaviour of the reared fish was compared with a fish sample caught in the Ria Formosa lagoon. No common species were found between samples. A total of 38 prey were identified, which suggests that the gilthead sea bream is a non-specific predator. Despite the high abundance of natural prey in the ponds, the dependence of sea bream on pelleted food was high.

Endemic zoonoses: a one health approach

Endemic zoonotic diseases remain a serious but poorly recognised problem in affected communities in developing countries. Despite the overall burden of zoonoses on human and animal health, information about their impacts in endemic settings is lacking and most of these diseases are continuously being neglected. The non-specific clinical presentation of these diseases has been identified as a major challenge in their identification (even with good laboratory diagnosis), and control. The signs and symptoms in animals and humans respectively, are easily confused with other non-zoonotic diseases, leading to widespread misdiagnosis in areas where diagnostic capacity is limited. The communities that are mostly affected by these diseases live in close proximity with their animals which they depend on for livelihood, which further complicates the understanding of the epidemiology of zoonoses. This thesis reviewed the pattern of reporting of zoonotic pathogens that cause febrile illness in malaria endemic countries, and evaluates the recognition of animal associations among other risk factors in the transmission and management of zoonoses. The findings of the review chapter were further investigated through a laboratory study of risk factors for bovine leptospirosis, and exposure patterns of livestock coxiellosis in the subsequent chapters. A review was undertaken on 840 articles that were part of a bigger review of zoonotic pathogens that cause human fever. The review process involves three main steps: filtering and reference classification, identification of abstracts that describe risk factors, and data extraction and summary analysis of data. Abstracts of the 840 references were transferred into a Microsoft excel spread sheet, where several subsets of abstracts were generated using excel filters and text searches to classify the content of each abstract. Data was then extracted and summarised to describe geographical patterns of the pathogens reported, and determine the frequency animal related risk factors were considered among studies that investigated risk factors for zoonotic pathogen transmission. Subsequently, a seroprevalence study of bovine leptospirosis in northern Tanzania was undertaken in the second chapter of this thesis. The study involved screening of serum samples, which were obtained from an abattoir survey and cross-sectional study (Bacterial Zoonoses Project), for antibodies against Leptospira serovar Hardjo. The data were analysed using generalised linear mixed models (GLMMs), to identify risk factors for cattle infection. The final chapter was the analysis of Q fever data, which were also obtained from the Bacterial Zoonoses Project, to determine exposure patterns across livestock species using generalized linear mixed models (GLMMs). Leptospira spp. (10.8%, 90/840) and Rickettsia spp. (10.7%, 86/840) were identified as the most frequently reported zoonotic pathogens that cause febrile illness, while Rabies virus (0.4%, 3/840) and Francisella spp. (0.1%, 1/840) were least reported, across malaria endemic countries. The majority of the pathogens were reported in Asia, and the frequency of reporting seems to be higher in areas where outbreaks are mostly reported. It was also observed that animal related risk factors are not often considered among other risk factors for zoonotic pathogens that cause human fever in malaria endemic countries. The seroprevalence study indicated that Leptospira serovar Hardjo is widespread in cattle population in northern Tanzania, and animal husbandry systems and age are the two most important risk factors that influence seroprevalence. Cattle in the pastoral systems and adult cattle were significantly more likely to be seropositive compared to non-pastoral and young animals respectively, while there was no significant effect of cattle breed or sex. Exposure patterns of Coxiella burnetii appear different for each livestock species. While most risk factors were identified for goats (such as animal husbandry systems, age and sex) and sheep (animal husbandry systems and sex), there were none for cattle. In addition, there was no evidence of a significant influence of mixed livestock-keeping on animal coxiellosis. Zoonotic agents that cause human fever are common in developing countries. The role of animals in the transmission of zoonotic pathogens that cause febrile illness is not fully recognised and appreciated. Since Leptospira spp. and C. burnetii are among the most frequently reported pathogens that cause human fever across malaria endemic countries, and are also prevalent in livestock population, control and preventive measures that recognise animals as source of infection would be very important especially in livestock-keeping communities where people live in close proximity with their animals.

Patologías lumbares: determinación de los factores ocupacionales y carga prestacional en una entidad promotora de salud en Bogotá

Resumen: Introducción El dolor lumbar es un trastorno músculo esquelético que afecta la parte baja de la espalda, considerado como un problema de salud pública y catalogado como un desastre en el sitio de trabajo, se encuentra en las 10 primeras causas de enfermedad profesional reportadas por las entidades prestadoras de servicios de salud, generando ausentismo y discapacidad laboral en los países industrializados, con costos que oscilan de los 20 mil a los 98 millones de dólares en los Estados Unidos. Objetivo Determinar la prevalencia de patologías lumbares calificadas y sus factores ocupacionales asociados en una entidad promotora de salud de Bogotá Colombia durante 2013 al 2014. Metodología Se realizó un estudio de corte transversal con datos secundarios pertenecientes a 318 pacientes de una entidad promotora de salud en la ciudad de Bogotá que fueron diagnosticados con patologías lumbares (lumbalgia-lumbago, discopatía lumbar, trastorno de disco intervertebral, espondilolistesis, espondilólisis, hernia discal), y remitidos a medicina laboral o solicitaron calificación de origen en primera oportunidad, en el periodo comprendido entre el año 2013 al 2014. Las variables incluidas fueron sociodemográficas, ocupacionales y diagnósticos médicos, específicamente patologías lumbares. Se realizó distribuciones de frecuencias, medidas de tendencia central y dispersión, análisis de asociación mediante la prueba Chi cuadrado de Pearson y un análisis multivariado a través del modelo de regresión binaria logística y el análisis de concordancia usando el índice de Kappa. Para las pruebas se utilizó un nivel de significación de 0,05. Se digitó y depuró en SPSS versión 23. Resultado El total de usuarios diagnosticados con patologías lumbares fue de 318 de los cuales el 57,2% fueron de sexo masculino con edad promedio de 43 años (D.E 7,9 años). Se encontró asociación significativa entre lumbalgia y movimientos de columna lumbar y levantamiento de carga (p<0,05); discopatía lumbar y movimientos de columna lumbar y factores multicausales (p<0,05); trastorno de disco intervertebral y factores multicausales (p<0.05), hernia de disco y levantamiento de cargas (p<0,05). Respecto a espondilolistesis y espondilólisis no se encontró asociación con ningún factor de riesgo, pero si se encontró asociación significativa entre origen y movimientos de columna lumbar (p= 0.010), con postura mantenida (p= 0.014), con causas multifactoriales (p= 0.000). El grado de concordancia entre la entidad promotora de salud y la administradora de riesgos laborales arrojó un valor en el índice de kappa de 0.432 (p= 0.000) correspondiendo a un grado de acuerdo moderado; para la concordancia entre la entidad promotora de salud y la junta de calificación el índice de kappa fue de 0.680 (p= 0.000) grado de acuerdo alto. Conclusión Las patologías lumbares tienen un alta prevalencia en la población trabajadora como en la no trabajadora, encontrándose un gran número de factores condicionantes a estas enfermedades generando altos costos en días perdidos laborales y en días de incapacidad: Por lo tanto, es importante determinar si estas son catalogadas de origen común o de origen laboral, para establecer programas de vigilancia epidemiológica y programas preventivos.

Efectividad del tratamiento de lumbalgia con acupuntura

Se realizó un estudio observacional retrospectivo longitudinal en una Institución prestadora de Servicios de Salud de la ciudad de Bogotá, con el objetivo de evaluar la efectividad en el manejo del dolor de la terapia con acupuntura en el tratamiento de lumbalgia. Se tomaron 150 historias clínicas de pacientes con lumbalgia atendidos de enero de 2014 a mayo de 2016, las cuales fueron sometidas a los criterios de inclusión definidos por los autores, arrojando 48 historias sometidas a la prueba de Friedman con el fin de identificar el impacto sobre el dolor del tratamiento con acupuntura en los pacientes seleccionados. Adicionalmente, bajo un muestreo aleatorio simple de distribución normal sobre las 48 historias clínicas evaluadas, se seleccionaron 25 casos a los cuales se les aplicó una encuesta no estructurada, con el fin de obtener información sobre el estado de la patología después de finalizar el tratamiento e identificar las posibles causas de deserción. Con este estudio se concluye que la terapia con acupuntura es efectiva en el manejo del dolor de pacientes con lumbalgia, y que es necesario realizar más estudios que puedan sustentar la inclusión de la terapéutica en el manejo de esta patología.

Trastornos musculoesqueléticos en trabajadores de la industria automotriz: revisión de la literatura 2000 a 2016.

Los trastornos musculoesqueléticos (TME) tienen alta relación con la industria automotriz afectando a los trabajadores en quienes se puede encontrar varias patologías como Síndrome Del Túnel Del Carpo, Epicondilitis, Síndrome del Manguito Rotador, discopatía lumbar y lumbalgias, entre otros. Entre los factores de riesgo asociados a estos trastornos están los movimientos repetitivos, posturas inadecuadas, vibración, uso manual de herramientas, tareas físicas demandantes y el mal levantamiento de pesos. Todas estas patologías son causa de ausentismo laboral en todo el mundo, lo que conlleva a un aumento en el costo económico por incapacidades, ayudas diagnósticas y tratamientos. Se realizó una revisión de la literatura científica de artículos publicados del año 2000 a 2016 con relación a los trastornos musculoesqueléticos en la industria automotriz en las bases de datos de Pubmed, Ebsco Host, ScienceDirect y Embase. La evidencia encontrada sugiere que la patología lumbar es la que presenta mayor prevalencia en la industria, con 65% en la población Europea, 42% en Asia, Norteamérica en un 20% y en América Latina en un 46%. A pesar que en la industria automotriz predominan como fuerza laboral los hombres, se reportó que las mujeres eran las que tenían mayores factores de riesgo para desarrollar un TME y dentro de estos las posturas inadecuadas, movimientos repetitivos, sobrecarga laboral y levantamiento de pesos, sumado al tiempo de exposición que fue un común denominador en cada uno de los estudios analizados. Las conclusiones fueron que la prevalencia de TME en esta industria es elevada y esto amerita la implementación de programas de prevención más enfocados en este tema. Además no se encontró en la literatura la existencia de un método eficiente para análisis postural y de sobrecarga física, lo que habla de una necesidad urgente de realizar más investigaciones enfocadas en este tipo de población.

A SHORTENED RNA-FISH PROTOCOL FOR ANALYZING ARTWORKS’ MICROCOLONIZERS

Microorganisms are involved in the deterioration of Cultural Heritage. Thus, there is a need to enhance the techniques used for their detection and identification. RNA Fluorescent In Situ Hybridization (RNA-FISH) has been successfully applied for phylogenetic identification of the viable components of the microbial communities colonizing artworks both in situ and ex situ. Until recently, it was time-consuming, taking not less than 6 h for the analysis. We have developed an RNA-FISH in suspension protocol that allowed ex situ analysis of microorganisms involved in artworks’ biodeterioration in 5 h. In this work, three modified protocols, involving microwave heating, were evaluated for further shortening two of the four main critical steps in RNA-FISH: hybridization and washing. The original and modified protocols were applied in cellular suspensions of bacteria and yeast isolates. The results obtained were evaluated and compared in terms of detectability and specificity of the signals detected by epifluorescence microscopy. One of the methods tested showed good and specific FISH signals for all the microorganisms selected and did not produce signals evidencing non-specific or fixation-induced fluorescence. This 3 h protocol allows a remarkable reduction of the time usually required for performing RNA-FISH analysis in Cultural Heritage samples. Thus, a rapid alternative for analyzing yeast and bacteria cells colonizing artworks’ surfaces by RNA-FISH is presented in this work.

Il contratto di compravendita immobiliare da un punto di vista pragmatico. Modello per un'analisi testuale contrastiva sviluppato su corpora da Italia, Germania e Austria

Oggetto del presente studio è un'analisi del genere testuale del contratto di compravendita immobiliare negli ordinamenti di Italia, Germania e Austria in un'ottica sincronica e pragmatica. Il testo è considerato come un atto comunicativo legato a convenzioni prestabilite e volto ad assolvere a specifiche funzioni sociali. L'obbiettivo principale del lavoro è lo sviluppo di un modello di analisi testuale che possa evidenziare l'interazione tra la funzione primaria e l'assetto macro- e microstrutturale di questo genere testuale, ovvero tra il piano giuridico e quello linguistico-testuale. L'analisi svolta permette inoltre di confrontare tre sistemi giuridici rispetto alla modalità di attuare questo negozio, nonché le lingue italiana e tedesca ed altresì due varietà di quest'ultima. Il corpus è composto da 40 atti autentici e 9 atti da formulari, compresi in un arco temporale che va dal 2000 al 2018. L'analisi parte con la definizione delle coordinate intra-ed extratestuali che determinano questo genere testuale e da una sua classificazione all'interno dei testi dell'ambito giuridico. Su questa base, i contratti dei corpora di Italia, Germania e Austria vengono analizzati separatamente rispetto alla loro macrostruttura, comprendendo in ciò tre piani macrostrutturali, ovvero quello giuridico da un lato e quelli funzionale e tematico dall'altro. L'interazione tra la funzione giuridica e l'assetto linguistico-testuale del contratto di compravendita immobiliare emerge in particolare a livello di quello funzionale, ossia relativo alla sequenza delle funzioni linguistiche realizzate sulla base dei contenuti giuridici. I risultati evinti dall'analisi dei tre corpora sono, infine, messi a confronto e integrati con una classificazione delle forme verbali che caratterizzano determinati macro-ambiti d'uso/funzionali all'interno di questo genere testuale, ovvero la realizzazione di specifiche funzioni linguistiche e giuridiche. Il metodo proposto offre nuovi spunti per ricerche future, tanto nell'ambito della linguistica contrastiva applicata a testi specialistici, che della traduzione e linguistica giuridica.

Evaluation of alpha-synuclein RT-QuIC for an early and differential diagnosis of Lewy body disease

Synucleinopathies are a group of neurodegenerative diseases characterized by tissue deposition of insoluble aggregates of the protein α-synuclein. Currently, the clinical diagnosis of these diseases, including Parkinson’s disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA), is very challenging, especially at an early disease stage, due to the heterogeneous and often non-specific clinical manifestations. Therefore, identifying specific biomarkers to aid the diagnosis and improve the clinical management of patients with these disorders represents a primary goal in the field. Pursuing this aim, we applied the α-Syn Real-Time Quaking-Induced Conversion (RT-QuIC), an ultrasensitive technique able to detect minute amounts of amyloidogenic proteins, to a large cohort of 953 CSF samples from clinically well-characterized (“clinical” group), or neuropathologically verified (“NP” group) patients with parkinsonism or dementia. Of significance, we also studied patients with prodromal synucleinopathies (“prodromal” group), such as pure autonomic failure (PAF) (n = 28), isolated REM sleep behavior disorder (iRBD) (n = 18), and mild cognitive impairment due to probable Lewy body (LB) disease (MCI-LB) (n = 81). Our findings show that α-syn RT-QuIC can accurately detect α-Syn seeding activity across the whole spectrum of LB-related disorders (LBD), exhibiting a mean sensitivity of 95.2% in the “clinical” and “NP” group, while ranging between 89.3% (PAF) and 100% (RBD) in the “prodromal group”. Moreover, we observed 95.1% sensitivity and 96.6% specificity in the distinction between MCI-LB patients and cognitively unimpaired controls, demonstrating the solid diagnostic potential of α-Syn RT-QuIC in the early phase of the disease. Finally, 13.3% of MCI-AD patients also had a positive test, suggesting an underlying LB co-pathology. This work demonstrated that α-Syn RT-QuIC is an efficient assay for accurate and early diagnosis of LBD, which should be implemented for clinical management and recruitment for clinical trials in memory clinics.

Leishmania-Specific Surface Antigens Show Sub-Genus Sequence Variation and Immune Recognition

Background: A family of hydrophilic acylated surface (HASP) proteins, containing extensive and variant amino acid repeats, is expressed at the plasma membrane in infective extracellular (metacyclic) and intracellular (amastigote) stages of Old World Leishmania species. While HASPs are antigenic in the host and can induce protective immune responses, the biological functions of these Leishmania-specific proteins remain unresolved. Previous genome analysis has suggested that parasites of the sub-genus Leishmania (Viannia) have lost HASP genes from their genomes. Methods/Principal Findings: We have used molecular and cellular methods to analyse HASP expression in New World Leishmania mexicana complex species and show that, unlike in L. major, these proteins are expressed predominantly following differentiation into amastigotes within macrophages. Further genome analysis has revealed that the L. (Viannia) species, L. (V.) braziliensis, does express HASP-like proteins of low amino acid similarity but with similar biochemical characteristics, from genes present on a region of chromosome 23 that is syntenic with the HASP/SHERP locus in Old World Leishmania species and the L. (L.) mexicana complex. A related gene is also present in Leptomonas seymouri and this may represent the ancestral copy of these Leishmania-genus specific sequences. The L. braziliensis HASP-like proteins (named the orthologous (o) HASPs) are predominantly expressed on the plasma membrane in amastigotes and are recognised by immune sera taken from 4 out of 6 leishmaniasis patients tested in an endemic region of Brazil. Analysis of the repetitive domains of the oHASPs has shown considerable genetic variation in parasite isolates taken from the same patients, suggesting that antigenic change may play a role in immune recognition of this protein family. Conclusions/Significance: These findings confirm that antigenic hydrophilic acylated proteins are expressed from genes in the same chromosomal region in species across the genus Leishmania. These proteins are surface-exposed on amastigotes (although L. (L.) major parasites also express HASPB on the metacyclic plasma membrane). The central repetitive domains of the HASPs are highly variant in their amino acid sequences, both within and between species, consistent with a role in immune recognition in the host.

Prolonged antiretroviral therapy initiated at seroconversion is associated with a polyfunctional HIV-1-specific T-cell profile comparable to that of long-term non-progressors (LTNPs)

Background: Early initiation of antiretroviral therapy (ART) may dramatically curtail cumulative immunological damage allowing maximal levels of immune preservation/reconstitution and induce an immunovirological status similar to that of HIV-1 LTNPs with low viral reservoirs and polyfunctional HIV-1 specific T cell responses.Methods: We performed a cross-sectional study of an HIV-1 seroconverter cohort on long-term ART (LTTS) and compared it to one of LTNPs. Inclusion criteria for 20 LTTS were: (a) ?4 years ART; (b) long-term aviremia and (c) absence of treatment failure and for 15 LTNPs: (a) ?7 years of documented HIV-1 infection; (b) <1000 HIV-1 RNA copies/mL and ?500 CD4+ T-cells/mm3 in >90% of measurements; (d) absence of AIDS-defining conditions; (e) ART-naı¨ve except for temporary ART for prevention of MTCT. In both cohorts, we analysed residual viral replication and reservoirs in peripheral blood, as measured by cellassociated HIV-1 RNA and DNA in PBMCs, respectively and used polychromatic flow cytometry to analyse HIV-1-specific CD4+ and CD8+ T-cell functional profile in terms of cytokine production using IFN-c, IL-2, TNF-a production.Results: Cell-associated DNA [47.7 (4.8-583.2) in LTTS and 19.7 (0.5-295.5) in LTNPS, p=0.10], and RNA [3.9 (0-36) and 5.8 (0-10.3), respectively] were shown to be similarly low in both cohorts. We identified 103 CD8 T cell epitope-specific responses, all subjects responding to ?1 epitope. Mean responding number of responding epitopes per patient was 2 and 4 in LTTS and LTNPS, respectively. Mean% of cytokine-secreting CD8 T cells was 0.37% and 0.50% (p=0.06), of these 43% and 39% (p=0.12) were secreting simultaneously IFN-c, IL-2 and TNF-a. Respective values for CD4 T cells were 0.28% and 0.33% (p=0.28) of which 33% and 30% (0.32) were secreting these 3 cytokines simultaneously.Conclusions: Long-term aviremia after very early ART initiation is associated with low levels of reservoirs saturation ad residual replication. Although less broad CD8 T cell responses were found in LTTS, HIV-1 specific CD4 and CD8 T cell responses showed similar magnitude and functional profile in the 2 cohorts. Our results indicate that prolonged ART initiated at the time of HIV-1 seroconversion is associated with immuno-virological features which resemble those of LTNPs. (BHIVA Research Award Winner 2008: Anna Garcia-Diaz.)

Persistence of EBV Antigen-Specific CD8 T Cell Clonotypes during Homeostatic Immune Reconstitution in Cancer Patients.

Persistent viruses are kept in check by specific lymphocytes. The clonal T cell receptor (TCR) repertoire against Epstein-Barr virus (EBV), once established following primary infection, exhibits a robust stability over time. However, the determinants contributing to this long-term persistence are still poorly characterized. Taking advantage of an in vivo clinical setting where lymphocyte homeostasis was transiently perturbed, we studied EBV antigen-specific CD8 T cells before and after non-myeloablative lympho-depleting chemotherapy of melanoma patients. Despite more advanced T cell differentiation, patients T cells showed clonal composition comparable to healthy individuals, sharing a preference for TRBV20 and TRBV29 gene segment usage and several co-dominant public TCR clonotypes. Moreover, our data revealed the presence of relatively few dominant EBV antigen-specific T cell clonotypes, which mostly persisted following transient lympho-depletion (TLD) and lymphocyte recovery, likely related to absence of EBV reactivation and de novo T cell priming in these patients. Interestingly, persisting clonotypes frequently co-expressed memory/homing-associated genes (CD27, IL7R, EOMES, CD62L/SELL and CCR5) supporting the notion that they are particularly important for long-lasting CD8 T cell responses. Nevertheless, the clonal composition of EBV-specific CD8 T cells was preserved over time with the presence of the same dominant clonotypes after non-myeloablative chemotherapy. The observed clonotype persistence demonstrates high robustness of CD8 T cell homeostasis and reconstitution.

Gliotransmission in the hippocampus, mechanisms and interaction with synaptic functions

SUMMARYAstrocytes represent the largest cell population in the human brain. In addition to a well established role as metabolic support for neuronal activity, in the last years these cells have been found to accomplish other important and, sometimes, unexpected functions. The tight enwrapping of synapses by astrocytic processes and the predominant expression of glutamate uptake carriers in the astrocytic rather than neuronal plasma membranes brought to the definition of a critical involvement of astrocytes in the clearance of glutamate from synaptic junctions. Moreover, several publications showed that astrocytes are able to release chemical transmitters (gliotransmitters) suggesting their active implication in the control of synaptic functions. Among gliotransmitters, the best characterized is glutamate, which has been proposed to be released from astrocytes in a Ca2+ dependent manner via exocytosis of synaptic-like microvesicles.In my thesis I present results leading to substantial advancement of the understanding of the mechanisms by which astrocytes modulate synaptic activity in the hippocampus, notably at excitatory synapses on dentate granule cells. I show that tumor necrosis factor- alpha (TNFa), a molecule that is generally involved in immune system functions, critically controls astrocyte-to-synapse communication (gliotransmission) in the brain. With constitutive levels of TNFa present, activation of purinergic G protein-coupled receptors in astrocytes, called P2Y1 receptors, induces localized intracellular calcium ([Ca2+]j) elevation in astrocytic processes (measured by two-photon microscopy) followed by glutamate release and activation of pre-synaptic NMDA receptors resulting in synaptic potentiation. In preparations lacking TNFa, astrocytes respond with identical [Ca2+]i elevations but fail to induce neuromodulation. I find that TNFa specifically controls the glutamate release step of gliotransmission. Addition of very low (picomolar) TNFa concentrations to preparations lacking the cytokine, promptly reconstitutes both normal exocytosis in cultured astrocytes and gliotransmission in hippocampal slices. These data provide the first demonstration that gliotransmission and its synaptic effects are controlled not only by astrocyte [Ca2+]i elevations but also by permissive/homeostatic factors like TNFa.In addition, I find that higher and presumably pathological TNFa concentrations do not act just permissively but instead become direct and potent triggers of glutamate release from astrocytes, leading to a strong enhancement of excitatory synaptic activity. The TNFa action, like the one observed upon P2Y1R activation, is mediated by pre-synaptic NMDA receptors, but in this case the effect is long-lasting, and not reversible. Moreover, I report that a necessary molecular target for this action of TNFa is TNFR1, one of the two specific receptors for the cytokine, as I found that TNFa was unable to induce synaptic potentiation when applied in slices from TNFR1 knock-out (Tnfrlv") mice. I then created a double transgenic mouse model where TNFR1 is knocked out in all cells but can be re-expressed selectively in astrocytes and I report that activation of the receptors in these cells is sufficient to reestablish TNFa-dependent long-lasting potentiation of synaptic activity in the TNFR1 knock-out mice.I therefore discovered that TNFa is a primary molecule displaying both permissive and instructive roles on gliotransmission controlling synaptic functions. These reports might have profound implications for the understanding of both physiological and pathological processes associated to TNFa production, including inflammatory processes in the brain.RÉSUMÉLes astrocytes sont les cellules les plus abondantes du cerveau humain. Outre leur rôle bien établi dans le support métabolique de l'activité neuronale, d'autres fonctions importantes, et parfois inattendues de ces cellules ont été mises en lumière au cours de ces dernières années. Les astrocytes entourent étroitement les synapses de leurs fins processus qui expriment fortement les transporteurs du glutamate et permettent ainsi aux astrocytes de jouer un rôle critique dans l'élimination du glutamate de la fente synaptique. Néanmoins, les astrocytes semblent être capables de jouer un rôle plus intégratif en modulant l'activité synaptique, notamment par la libération de transmetteurs (gliotransmetteurs). Le gliotransmetteur le plus étudié est le glutamate qui est libéré par l'exocytose régulée de petites vésicules ressemblant aux vésicules synaptiques (SLMVs) via un mécanisme dépendant du calcium.Les résultats présentés dans cette thèse permettent une avancée significative dans la compréhension du mode de communication de ces cellules et de leur implication dans la transmission de l'information synaptique dans l'hippocampe, notamment des synapses excitatrices des cellules granulaires du gyrus dentelé. J'ai pu montrer que le « facteur de nécrose tumorale alpha » (TNFa), une cytokine communément associée au système immunitaire, est aussi fondamentale pour la communication entre astrocyte et synapse. Lorsqu'un niveau constitutif très bas de TNFa est présent, l'activation des récepteurs purinergiques P2Y1 (des récepteurs couplés à protéine G) produit une augmentation locale de calcium (mesurée en microscopie bi-photonique) dans l'astrocyte. Cette dernière déclenche ensuite une libération de glutamate par les astrocytes conduisant à l'activation de récepteurs NMDA présynaptiques et à une augmentation de l'activité synaptique. En revanche, dans la souris TNFa knock-out cette modulation de l'activité synaptique par les astrocytes n'est pas bien qu'ils présentent toujours une excitabilité calcique normale. Nous avons démontré que le TNFa contrôle spécifiquement l'exocytose régulée des SLMVs astrocytaires en permettant la fusion synchrone de ces vésicules et la libération de glutamate à destination des récepteurs neuronaux. Ainsi, nous avons, pour la première fois, prouvé que la modulation de l'activité synaptique par l'astrocyte nécessite, pour fonctionner correctement, des facteurs « permissifs » comme le TNFa, agissant sur le mode de sécrétion du glutamate astrocytaire.J'ai pu, en outre, démontrer que le TNFa, à des concentrations plus élevées (celles que l'on peut observer lors de conditions pathologiques) provoque une très forte augmentation de l'activité synaptique, agissant non plus comme simple facteur permissif mais bien comme déclencheur de la gliotransmission. Le TNFa provoque 1'activation des récepteurs NMD A pré-synaptiques (comme dans le cas des P2Y1R) mais son effet est à long terme et irréversible. J'ai découvert que le TNFa active le récepteur TNFR1, un des deux récepteurs spécifiques pour le TNFa. Ainsi, l'application de cette cytokine sur une tranche de cerveau de souris TNFR1 knock-out ne produit aucune modification de l'activité synaptique. Pour vérifier l'implication des astrocytes dans ce processus, j'ai ensuite mis au point un modèle animal doublement transgénique qui exprime le TNFR1 uniquement dans les astrocytes. Ce dernier m'a permis de prouver que l'activation des récepteurs TNFR1 astrocytaires est suffisante pour induire une augmentation de l'activité synaptique de manière durable.Nous avons donc découvert que le TNFa possède un double rôle, à la fois un rôle permissif et actif, dans le contrôle de la gliotransmission et, par conséquent, dans la modulation de l'activité synaptique. Cette découverte peut potentiellement être d'une extrême importance pour la compréhension des mécanismes physiologiques et pathologiques associés à la production du TNFa, en particulier lors de conditions inflammatoires.RÉSUMÉ GRAND PUBLICLes astrocytes représentent la population la plus nombreuse de cellules dans le cerveau humain. On sait, néanmoins, très peu de choses sur leurs fonctions. Pendant très longtemps, les astrocytes ont uniquement été considérés comme la colle du cerveau, un substrat inerte permettant seulement de lier les cellules neuronales entre elles. Il n'y a que depuis peu que l'on a découvert de nouvelles implications de ces cellules dans le fonctionnement cérébral, comme, entre autres, une fonction de support métabolique de l'activité neuronale et un rôle dans la modulation de la neurotransmission. C'est ce dernier aspect qui fait l'objet de mon projet de thèse.Nous avons découvert que l'activité des synapses (régions qui permettent la communication d'un neurone à un autre) qui peut être potentialisée par la libération du glutamate par les astrocytes, ne peut l'être que dans des conditions astrocytaires très particulières. Nous avons, en particulier, identifié une molécule, le facteur de nécrose tumorale alpha (TNFa) qui joue un rôle critique dans cette libération de glutamate astrocytaire.Le TNFa est surtout connu pour son rôle dans le système immunitaire et le fait qu'il est massivement libéré lors de processus inflammatoires. Nous avons découvert qu'en concentration minime, correspondant à sa concentration basale, le TNFa peut néanmoins exercer un rôle indispensable en permettant la communication entre l'astrocyte et le neurone. Ce mode de fonctionnement est assez probablement représentatif d'un processus physiologique qui permet d'intégrer la communication astrocyte/neurone au fonctionnement général du cerveau. Par ailleurs, nous avons également démontré qu'en quantité plus importante, le TNFa change son mode de fonctionnement et agit comme un stimulateur direct de la libération de glutamate par l'astrocyte et induit une activation persistante de l'activité synaptique. Ce mode de fonctionnement est assez probablement représentatif d'un processus pathologique.Nous sommes également arrivés à ces conclusions grâce à la mise en place d'une nouvelle souche de souris doublement transgéniques dans lesquelles seuls les astrocytes (etnon les neurones ou les autres cellules cérébrales) sont capables d'être activés par le TNFa.

Evaluation of the immune response to infection with metastatic and non-metastatic "Leishmania guyanensis" parasites

ABSTRACT : Les infections par le parasite Leishmania guyanensis se caractérisent par une dissémination depuis le site initial d'infection jusqu'aux tissus naso-pharyngés, responsable de la Leishmaniose à lésions secondaires muco-cutanées (LMC). Les lésions des patients atteints de LMC montrent une massive infiltration de cellules immunitaires, une réponse immunitaire élevée et la présence de parasites (bien qu'en très faible quantité). La LMC engendre une augmentation de l'expression de TNFa ainsi qu'un défaut dans le contrôle de la réponse immunitaire caractérisé par une absence de réponse à l'IL 10. La réponse immunitaire de l'hôte ainsi que la virulence du parasite sont deux facteurs reconnus pour le contrôle de l'infection. Le mécanisme de la pathogenèse de la LMC restent grandement incompris, surtout le mécanisme de dissémination de l'infection du site d'inoculation jusqu'aux sites secondaires d'infection (métastases) ainsi que les détails de la réponse de l'hôte contre le pathogène. Dans un modèle d'infection d' hamsters avec des parasites du Nouveau Monde, la classification des parasites Leishmania se fait en fonction de leur capacité à développer des métastases. Ce modéle d'infection a permis de caractériser différentes souches de parasites selon la classification de l'Organisation Mondiale de la Sante (OMS) tel que la souche de référence W>É-II/BR/78/M5313 qui est reconnue comme hautement métastatique alors que ces clones dérivés de M5313 montrent de grandes variations quand a leur capacité à créer des métastases. Les clones 13 et 21 sont métastatiques (M+) alors que les clones 3 et 17 sont nonmétastatiques (NI-). Les objectifs de cette thèse ont été d'étudier le rôle de la réponse immunitaire innée des macrophages après infection in vitro avec différents clones métastatiques et non-métastatiques du parasite L. guyanensis, ainsi que d'étudier la réponse immunitaire générée suite à une infection in vivo par les clones M+ et M- de L. guyanensis dans un modèle marin. L'analyse de la .réponse immunitaire des macrophages in vitro montrent qu'il y aune augmentation significative de leur statut d'activation après infection par des parasites M+ indiquée par la modulation des marqueurs d'activation de surface CD80, CD86 et CD40, ainsi que une augmentation significative de CXCL 10, CCLS, IL6 et TNFa au niveau transcription de l'ARNm et au niveau de la protéine. Cette phénomène d'activation a été observée chez les deux souches de souris C57BL/6 et BALB/c. L'utilisation d'un inhibiteur d'entrée des parasites (Cytochalsin D) ou d'un inhibiteur des fonctions endosomales (Chloroquine) diminue de manière significative la réponse des macrophages aux parasites M+. L'utilisation de macrophages déficients en TLR, MyD88, et TRIF a démontré que la réponse générée après infection par les parasites M+ était dépendante de la voie de signalisation de TRIF et TLR3. Lors d'infection in vivo par des parasites M5313, au moins 50% des souris BALB/c présentent un phénotype sensible caractérisé par des lésions non-nécrotiques qui ne guérissent pas, persistent plus de 13 semaines après infection et contiennent un nombre considérable de parasites. Ces souris développent une réponse immunitaire de type T helper 2 (Th2) avec un niveau élevé d'IL-4 et d'IL-10. Les autres souris ont un phénotype non-sensible, les souris développant peu ou pas de lésion, avec peu de parasites et une réponse immunitaire diminuée, caractérisée par un niveau faible d'IFNy, d'IL4 et d'IL10. De plus, les souris BALB/c infectées par un parasite L. guyanensis isolé à partir des lésions muco-cutanées d'un patient humain atteint de LMC ont démontrés un phénotype similaire aux souris infectées par la souche M5313 avec 50% des souris développant des lésions persistantes, alors qu'un parasite dérivé des lésions cutanées humains n'a montré qu'une faible sensibilité avec une lésion transitoire qui finit par guérir. Nous avons montré que la sensibilité de ces souris BALB/c dépend de l'IL-4 et de l'IL-10 car les souris IL-10-/sur fond génétique BALB/c ainsi que les souris BALB/c traitée avec de l'anti-IL4 étaient capables de contrôler l'infection par M5313. Les souris C57BL/6 sont résistantes à l'infection par le parasite M5313. Elles développent une lésion transitoire qui guérit 9 semaines après infection. Ces souris résistantes ont un très faible taux de parasites au site d'infection et développent une réponse immunitaire de type Thl avec un niveau élevé d'IFNr et peu d'IL4 et d'IL10. Les infections in vivo de souris déficientes en MyD88, TRIF, TLR3 ou TLR9 (sur fond génétique C57BL/6) ont indiqué que MyD88 et TLR9 étaient impliqués dans la résistance à l'infection par L. guyanensi, et que TRIF et TLR3 avaient un rôle important dans la sensibilité. Ce travail met en évidence le fait que la réponse immunitaire de l'hôte est modulée par le parasite selon leur caractérisation d'être soit M+ ou M-. Nous avons démontré également que plusieurs gènes et voies de signalisations étaient impliqués dans cette réponse favorisant le développement d'une LMC. ABSTRACT : Leishmania guyanensis parasites are able to disseminate from the initial site of cutaneous skin infection to the nasopharyngeal tissues resulting in destructive secondary lesions and the disease Mucocutaneous Leishmaniasis (MCL). The secondary lesions in patients have intense immune cell infiltration, elevated immune responses and the presence (albeit at low levels) of parasites. More specifically, MCL patients produce higher levels of TNFa and display impairment in their ability to control the immune response due to a defect in their ability to respond to IL10. Little is known about the pathogenesis of MCL, especially about the dissemination of the infection from the site of inoculation to secondary sites (metastasis) and the response of the host to the pathogen. The hamster model of L. guyanensis infection has previously characterized the WHO reference strain, L. guyanensis WHI/BR/78/M5313, as being highly metastatic. Clones of parasites derived from this reference strain show a differential ability to metastasize. This thesis studied the differential immune response generated by macrophages in vitro, or by mice in vivo, following infection with L. guyanensis parasites. A significant increase in the activation status of macrophages derived from C57BL/6 or BALB/c mice was observed after in vitro infection with L. guyanensis parasites when compared to non-metastatic parasites. This change in status was evidenced by the increased expression of surface activation markers, together with the chemokines, CXCL 10, CCLS, and cytokines, IL6 and TNFa. Furthermore, in vitro infection of macrophages isolated from mice deficient in either a specific Toll Like Receptor (TLR) or the adaptor molecules MyD88 or TRIF, indicated that the immune response generated following L. guyanensis metastatic parasite infection was reliant on the TRIF dependent TLR3 signalling pathway. In vivo footpad infection of BALB/c mice with the L. guyanensis M5313 parasites showed a reproducible susceptible phenotype, whereby at least 50% of infected mice developed non-healing, nonnecrosing lesions with high parasitemia that persisted over 13 weeks post infection. This phenotype was characterized by a Th2 type cytokine immune response with increased levels of IL4 and IL10 detected in the draining lymph nodes. IL 10 deficient mice on a BALB/c background, or BALB/c mice treated with anti-IL4 were able to control infection with L. guyanensis M5313 parasites, thereby proving that these cytokines were indeed implicated in the susceptibility to infection. Moreover, infection of BALB/c mice with patient isolated L. guyanensis parasites confirmed that MCL derived parasites were able to induce a susceptibility phenotype similar to that of L. guyanensis M5313. C57BL/6 mice, on the other hand, were highly resistant to infection with L. guyanensis M5313 parasites and produced transient footpad swelling that healed by week 9 post infection, together with low degrees of footpad parasitemia and a Thl polarized immune response. Infection of mice deficient in MyD88, TRIF, TLR3, and TLR9 (on a C57BL/6 background), indicated that MyD88 and TLR9 were involved in the resistance of these mice to infection, and that TRIF and TLR3 were involved in the susceptibility. This study has shown that the host response can be differentially modulated depending on the infecting parasite with several genes and pathways being identified that could be involved in promoting the development of MCL.

Cytomegalovirus (CMV)-specific cellular immune responses.

A large percentage of healthy individuals (50-90%) is chronically infected with Cytomegalovirus (CMV). Over the past few years, several techniques were developed in order to monitor CMV-specific T-cell responses. In addition to the identification of antigen-specific T cells with peptide-loaded MHC complexes, most of the current strategies to identify CMV-specific T cells are centered on the assessment of the functions of memory T cells including their ability to mediate effector function, to proliferate or to secrete cytokines following antigen-specific stimulation. The investigation of these functions has allowed the characterization of the CMV-specific T-cell responses that are present during different phases of the infection. Furthermore, it has also been shown that the combination of virus-specific CD4 and CD8 T-cell responses are critical components of the immune response in the control of virus replication.

Detection of functional antigen-specific T cells from urine of non-muscle invasive bladder cancer patients.

To gain further insights into the role of T lymphocytes in immune responses against bladder tumors, we developed a method that monitors the presence of functional antigen-specific T cells in the urine of non-muscle invasive bladder cancer patients. As relatively few immune cells can usually be recovered from urine, we examined different isolation/amplification protocols and took advantage of patients treated with weekly intravesical instillations of Bacillus Calmette-Guérin, resulting in large amounts of immune cells into urine. Our findings demonstrate that, upon in vitro amplification, antigen-specific T cells can be detected by an interferon γ (IFNγ)-specific ELISPOT assay.