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No description available.

No description available.

Les lymphocytes T (LT) doivent analyser efficacement un nombre important de cellules présentatrices d'antigène (CPA) afin de détecter un antigène spécifique et d'initier une réponse immunitaire adaptée. La reconnaissance par le récepteur des cellules T (TCR) d'un peptide spécifique associé au complexe majeur d'histocompatibilité (pMHC) doit donc être extrêmement sensible et rapide. Alors que les voies de signalisation en aval du TCR sont largement étudiées, la cinétique de la discrimination des ligands par le TCR et de l'activation initiale du LT reste mal connue. Des surfaces solides recouvertes de ligands du TCR nous ont permis d'étudier les événements cellulaires initiés par la détection d'un antigène. L'utilisation de la microscopie a fluorescence par réflexion totale interne (TIRFM) et la microscopie interférentielle (IRM) nous a permis de montrer que l'interaction initiale entre surface et LT est attribuable à des microvillosités mobiles. La stimulation du TCR active en quelques secondes un mouvement de rétraction de ces microvillosités. Ces mouvements actifs sont régulés par l'activation de la PLC- γ1 et sont dépendants des myosines (IIA, Ic et Ig), de la cofiline et de l'ezrine. Après cette phase d'analyse de l'environnement extracellulaire, la stimulation du TCR entraîne l'étalement rapide et actif de la cellule. L'amplitude et la vitesse d'étalement reflètent l'efficacité activatrice du ligand reconnu par le TCR. En moins de 5 minutes différents pMHC, ayant des propriétés biophysiques proches, sont donc discriminés par les LT et capables d'induire une première réponse cellulaire spécifique. / T lymphocytes need to efficiently probe a large number of antigen-presenting cell (APC) in order to detect an agonist antigen and to initiate an effective immune response. Therefore, engagement of the T-cell receptor (TCR) by peptide-bound major histocompatibility complex (pMHC) is a highly sensitive and rapid process. While signaling pathways downstream the TCR are extensively studied, little is known about antigen discrimination kinetic and initial T-cell response.Model planar surfaces coated with TCR ligand allowed us to study cellular events initiated by antigen detection. Using TIRFM (total internal reflexion microscopy) and IRM (interference reflexion microscopy) we showed that T-cell initial contacts with the surface were mediated by mobile microvilli. TCR engagement triggers a pulling motion of T cell surface microvilli within seconds. Active microvilli movements are dependent on PLC-γ1 activation and on the presence of myosins (IIA, Ic and Ig) as well as cofilin and ezrin. After this extracellular environment probing period, TCR stimulation triggered a rapid and active cell spreading. Cell spreading amplitude and speed reflect the ligand activating efficacity. In less than 5 minutes pMHC with different biophysical properties were discriminated by T-cells and triggered a first specific cellular response.

Abstract The purpose of this study was to develop sensitive and specific laboratory diagnostic methods for the demonstration of pneumococcal surface antigens or pneumococcus-specific antibodies in clinical samples. The work took account of epidemiological aspects of both pneumococcal disease and nasopharyngeal carriage of pneumococcus. We first compared the sensitivity of pneumococcal culture and antigen detection methods in nasopharyngeal samples in a developing country setting and then investigated the possibility of improving the sensitivity of the antigen detection by introducing an enrichment step in the procedure. — Further investigations were designed to determine the validity of pneumolysin-specific immune complex bound antibody assay as a tool for diagnosing pneumococcal ALRI in a developing country setting. Finally, we developed an enzyme immunoassay for the detection of pneumococcal capsular polysaccharide antigens, using type-specific antibodies produced in-house in rabbits through immunisation with an in-house-produced pneumococcal whole cell vaccine. The method was tested in nasopharyngeal and middle ear fluid samples. The first results indicated that antigen detection might be more sensitive than culture in demonstrating pneumococci in URT, particularly in children with prior antimicrobial therapy. Antigen detection is a feasible method for studies on pneumococci in developing countries. For type-specific demonstration of S. pneumoniae, detection of pneumococcal antigen after an enrichment step proved a sensitive method that can be applied for epidemiologic study purposes, e.g., in vaccine trials, in areas without ready access to a good microbiology laboratory. Determination of IC-bound pneumolysin IgG antibodies appears to be a useful method for species-specific diagnosis of pneumococcal infections. The results indicating pneumococcal aetiology in ALRI patients in this study compare well with the best results obtained by the use of lung aspirates. Increasing the number of serial samples improves the sensitivity of the assay, but even two samples provide more positive findings than other methods currently in routine use. Criteria of positivity need to be confirmed in subsequent larger studies with both healthy controls and patients with confirmed pneumococcal disease. It is also important to control the findings in patients with pneumonia of non-pneumococcal origin. The novel enzyme immunoassay was shown to work well with enrichment culture samples, with an almost 100% sensitivity compared with the culture. Middle ear fluid samples were too diluted for the enzyme immunoassay method used, and only 74% sensitivity compared with culture was achieved. Provided that adequate samples can be obtained, the method will be a useful complement to the current laboratory methods used to diagnose pneumococcal disease. With the existence of a broad spectrum of microbiological and immunological methods, it is imperative to seek international consensus for standard methods to demonstrate pneumococcus. Otherwise it is very difficult to compare results from different clinical studies. A WHO Working Group recently proposed a standard method for detecting upper respiratory carriage of pneumococcus, but a lot of work remains to be done in other areas of research on pneumococcal infections.

La tuberculose (TB) est une maladie infectieuse causée par M. tuberculosis. En 2009, la mortalité était élevée avec 1,7 millions de décès enregistrés dans le monde. La co-infection par le VIH, notamment en Afrique, et les tuberculoses à bacilles multi-résistants aux antibiotiques, rendent la maîtrise de la pandémie encore plus complexe. Les tests actuels de diagnostic présentent des lacunes notamment en terme de sensibilité pour la microscopie qui est le test le plus utilisé, ou en terme de praticité, en ce qui concerne la culture qui est le test de référence. Le développement de nouveaux tests pour le diagnostic de la TB active représente un enjeu majeur de nos sociétés modernes. Les tests immunologiques, en particulier les tests sérologiques actuellement disponibles, présentent une alternative aux tests bactériologiques couramment utilisés mais leurs performances restent faibles. Pour cela, il est indispensable de cibler de nouveaux biomarqueurs pour développer un test efficace, sensible, rapide et peu onéreux. C'est dans de cadre que s'inscrit ce travail de thèse.Nous rapportons dans ce mémoire, la recherche de marqueurs innovants dans le cadre de la mise au point d'un test de diagnostic de la tuberculose. Nous avons ciblé plusieurs marqueurs protéiques (OmpATb, LipY, Rv0183, Rv1984c et Rv3452) et un marqueur glycolipide, le tréhalose-6,6'-dimycolate (TDM). Parmi ces candidats, plusieurs présentent de réelles performances sur le plan diagnostique. Parallèlement à cette étude, nous avons mis au point une technique de séparation des différentes formes de TDM. La séparation des différentes formes de TDM devrait permettre de déterminer de façon précise les fonctions de ces composants majeurs de la paroi de M. tuberculosis et d'évaluer leur potentiel en terme de diagnostic.Mots clés : tuberculose, sérodiagnostic, détection antigène, détection anticorps, enzymes lipolytiques, glycolipide. / Tuberculosis (TB) is an infectious disease caused by M. tuberculosis. In 2009, mortality was high with 1,7 million of recorded deaths in the world. The co-infection by the HIV, in particular in Africa, and tuberculosis with multi-resistant bacilli to antibiotics, make the control of pandemia more complex. The current tests of diagnosis present gaps, in particular, in term of sensitivity for microscopy, which is currently the used test, or in term of praticity, for the culture which is considered as the gold standard.The development of new tests for the diagnosis of TB active represents a major challenge of our modern societies. The immunological tests, in particular the serologic tests available, can present an alternative to the currently bacteriological tests. However, their performances are not sufficient. That's why, it is essential to target new biomarkers which permit to establish an effective, sensitive, fast and cheap test. It is in this context that my study takes place.We report in this memory, the research of innovating markers for the development of a diagnostic test of tuberculosis. We targeted several protenic markers (OmpATb, LipY, Rv0183, Rv1984c and for Rv3452) and a glycolipid marker, the trehalose-6,6'-dimycolate (TDM). Among these candidates, several shown good performances for active TB diagnosis. In parallel to this study, a technique of separation of the various forms of TDM was developed. The separation of the various forms of TDM will permit to study their role on the biological activities and evaluate their potential in term of diagnosis.Key words: tuberculosis, serodiagnostic, antigen detection, antibody detection, lipolytic enzymes, glycolipid.

Background: Helicobacter pylori (H. pylori) infection is a significant risk factor for peptic ulcer (PU) and gastric cancer (GC). Apart from the virulent CagA genotype of H. pylori environmental and dietary factors influence disease outcomes. There have been no studies addressing these factors in Western India. Hence, we conducted a case control study enrolling PU, GC patients and controls at Pune, India. Methods: Risk factors for PU and H. pylori infection were assessed in participant interview. H. pylori status was assessed from stool by monoclonal antigen detection. To understand treatment effect, we followed 100 H. pylori positive patients. Results: We enrolled 190 PU patients, 125 Controls and 35 GU patients. Prevalence of H. pylori was 61% among symptomatic patients and 45% among controls. H. pylori infection (OR: 1.70, 95% CI: 1.03-2.89), meat (OR: 1.10, 95% CI: 1.02-1.75), fish (OR: 1.05, 95% CI: 1.02-1.89) consumption, and family history of ulcer (OR: 1.20, 95% CI: 1.08-1.60) were risk factors for PU. Consumption of snacks with alcohol (OR: 0.32, 95% CI: 0.13-0.78) and history of anti-parasite treatment (OR: 0.51, 95% CI: 0.30-0.86) were protective factors against PU. Lower socioeconomic status (SES) (OR: 1.10, 95% CI: 1.02-1.39), meat consumption (OR: 2.35, 95% CI: 1.30-4.23), smoking (OR: 2.23, 95% CI: 1.24-4.02), eating restaurant food thrice per week (OR: 3.77, 95% CI: 1.39-10.23) and drinking non-filtered or non-boiled water (OR: 1.05, 95% CI: 1.01-1.23) were risk factors for H. pylori infection. Consumption of chili peppers (OR: 0.20, 95% CI: 0.10-0.37) and concurrent parasite infestation (OR: 0.44, 95% CI: 0.24-0.80) were protective against H. pylori infection. H. pylori infection was eradicated only in 53% (40/75) of treated patients. Conclusion: This study indicates that H. pylori infection is associated PU. Consumption of meat, fish and family history of PU are risk factors for PU. Lower SES, consumption of restaurant food, meat, non filtered water and smoking are risk factors for H. pylori infection. Consumption of chili peppers and concurrent parasite infestation are protective against H. pylori infectionwhile history of anti parasite treatment protects against PU. H. pylori were eradicated only in 53% of patients.