Chez les mammifères, le développement du système immunitaire intestinal est initié in utero par un programme déterminé génétiquement. Cependant, le développement complet de ce système immunitaire et sa maturation finale n'ont lieu qu'après la naissance, sous l'influence des très nombreuses bactéries qui colonisent alors l'intestin et qui composent le microbiote intestinal. L'utilisation de modèles in vivo originaux de souris axéniques, sans germe, et gnotoxéniques, colonisées avec des bactéries individuelles ou des groupes de bactéries plus ou moins complexes, nous a permis d'identifier le rôle particulier de la bactérie segmentée filamenteuse (ou SFB) dans la maturation des réponses immunes intestinales adaptatives, IgA et lymphocytaires T. Sa capacité singulière à adhérer à l'épithélium des plaques de Peyer suggère un rôle important de ces formations lymphoïdes dans l'initiation des réponses immunes induites par la SFB. Nos résultats renforcent l'idée du rôle majeur des plaques de Peyer dans l'initiation des réponses IgA intestinales par le microbiote. De façon inattendue, en l'absence de ces structures, la SFB induit dans le chorion des souris, la néogenèse de structures lymphoïdes tertiaires capables d'initier des réponses IgA associées à une forte réponse IL-17 spécifique de la SFB. Ces résultats suggèrent que les puissantes propriétés immunostimulantes de la SFB sont soutenues par de multiples voies d'induction des réponses immunes qui pourraient être liées à son adhésion à la muqueuse intestinale

Au cours des dernières années, Salmonella Enteritidis est devenus les sérotypes les plus souvent isolés chez les patients canadiens, les cas étant liés à la consommation de viande de poulet et d’œufs crus. Les vaccins tués commercialement disponibles pour la volaille, stimulent mal l'immunité mucosale, tandis que l'utilisation de vaccins vivants reste controversée. Par conséquent, un vaccin sous-unitaire par voie orale peut être une solution. Cinq protéines bactériennes ont été choisies comme candidates potentielles et identifiées, soit Glyceraldehyde-3-phosphate dehydrogenase, Enolase, Lipoamide dehydrogenase, DNA protection during starvation protein et Elongation factor-Tu. Notre objectif a été de produire et de purifier ces protéines et de démontrer leur immunogénicité. Les gènes des protéines ont été amplifiés et clonés dans le vecteur pQE-30 pour expression dans Escherichia coli M15. La purification a été effectuée par FPLC. Des poules pondeuses SPF ont été séparées en 6 groupes et injectées par voie intramusculaire à different âges avec une des 5 protéines, ou le PBS chez le groupe témoin. Les œufs ont été ramassés pendant l'expérience et du sang a été prélevé à 36 semaines d'âge. Les anticorps IgY ont été extraits à partir du jaune d'oeuf et du sérum, et les IgA à partir du blanc d'oeuf. Des immunodots, westernblots et ELISA ont évalué l'immunogénicité des protéines et les niveaux d'anticorps induits . Nous avons constaté que ces cinq protéines pourraient stimuler la production d'anticorps spécifiques in vivo. GAPDH, Enolase et DPS ont induit des titres d'anticorps plus élevés que LpdA et EF-Tu. / Over the past years, Salmonella Enteritidis (SE) has become the most prevalent serovars isolated in Canadian patients. Most cases in humans are associated with consumption of chicken meat, raw egg and related products. For controlling Salmonella transmission and infection in poultry, available commercially killed vaccines poorly stimulate mucosal immunity, while the use of live vaccines remains controversial. Therefore an oral subunit vaccine may be a solution. Five bacterial proteins were chosen as potential candidates and identified as Glyceraldehyde-3-phosphate dehydrogenase, Enolase, Lipoamide dehydrogenase, DNA protection during starvation protein and Elongation factor-Tu. Our objectives were to produce and purify these proteins and study their immunogenicity. The proteins genes were amplified and cloned into pQE-30 vector, then transformed into Escherichia coli M15 for expression. Purification was performed using FPLC. SPF laying hens were separated into 6 groups and injected intramuscularly 3 times at 16, 20 and 28 weeks of age. Five groups were injected with a single protein respectively while the sixth group was injected with PBS as control. Eggs were collected during the duration of the experiment and blood was collected when hens were sacrificed at 36 weeks of age. IgY was extracted from egg yolk and serum and IgA from egg white. Immunodot, westernblot and ELISA were used to evaluate the immunogenicity of proteins and antibody levels they induced. We found that these five proteins could stimulate production of specific antibody in vivo. GAPDH, Enolase and DPS induced higher antibody titer than LpdA and Ef-Tu.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Introduction: HPV infection is the most common sexually transmitted disease in humans, which can cause benign diseases (warty lesion) or malignant as the anogenital cancer, oral cancer and, with the highest incidence, cervical cancer. Overall, the main HPV detection method is based on cytological evaluation of Papanicolaou; however, false-negative results and unsatisfactory sampling complicate the disease diagnosis and prevention. In this context, the investigation of new diagnosis platforms and prophylaxys has great interest. Objectives: The aim of this study was to conduct an epidemiological study of HPV infection in the women population of southeastern Brazil and then propose new diagnostic and vaccine approaches. Methodology: Genital samples of 5,223 women were evaluated by a new molecular tool based on PCR (Nested Multiplex PCR - NMPCR), using a cocktail of primers for simultaneous detection of 38 different HPV types, in single tube. Another molecular approach was proposed by Phage Display technique to select the IgA binding peptides from cervical samples of patients with HPV infection. In silico (Linear and 3D bioinformatics) and in vitro (ELISA, Slot blot and Bioelectrode) analysis were performed to validate the selected phages and synthetic peptides in cervical (n=91) and salivary specimens (n=66). The sensitivity and specificity parameters of HPV diagnosis were determined by ROC curve. To test the vaccine potential of synthetic peptides, in silico (3D bioinformatic), in vivo (female BALB/c mice immunization) or in vitro analyzes (MTT, ELISA, splenocytes culture, CBA and Neutralization assay) were carried out. Results: Among the 5,223 women evaluated, there was a prevalence of 58.9% of HPV infection, especially the types 53, 52 and 06, showing the relevance to propose new alternatives for HPV detection and prevention. In molecular approach of Phage Display, thirty-two distinct phage clones were selected against IgA from positive HPV cervical samples. Phage C.B1 showed higher reactivity against HPV samples compared to the negative control group by ELISA. This peptide is a putative epitope of HPV major capsid protein (L1) and has distinguished efficiently infection from HPV controls in cervical and saliva samples (p <0.0001), with high sensitivity (above 95%) and specificity (above 71%) in both fluids. The C.B1 peptide was also successfully incorporated (p<0.05) onto a graphite bioelectrode for HPV direct diagnosis in saliva. In the vaccine approach, three peptides (PEP1, PEP3 and PEP4) showed no toxicity in murine macrophages. Immunizations showed potent induction of serum IgG antibodies production by PEP3, and increase in IgA titer in the vaginal mucosa under PEP1 and PEP3 stimuli. The evaluation of cellular immune response indicated that the PEP1 and, particularly, the PEP3 chimeric peptide were able to polarize the immune response towards to Th1 profile and sensitize the cells to a pro-inflammatory \"status\", suitable for antiviral activity. The HPV-16 PsVs neutralization assay showed that PEP3 was able to reduce the infection by 49%. Conclusions: Overall, the studies presented here showed a high prevalence of HPV among Brazilian women by viral types still little explored in the literature. We selected mimotopes by Phage Display tool capable of detecting HPV in both saliva samples as in the cervical secretion, providing a low cost, simple and non-invasive diagnostics for population screening. In addition, the mimotopes selected have attractive applicability in vaccine formulations, but further studies are still needed. / Introdução: A infecção por HPV é a doença sexualmente transmissível mais comum em humanos, que pode causar doenças benignas (lesões verrucosas) ou malignas como o câncer anogenital, câncer oral e, com maior incidência, o câncer do colo uterino. Globalmente, o principal método de detecção do HPV baseia-se na avaliação citopatológica de Papanicolaou, entretanto, resultados falsonegativos e obtenção de amostras insatisfatórias dificultam o diagnóstico e a prevenção da doença. Neste contexto, a busca por novas plataformas diagnósticas e profiláticas para o HPV é de grande interesse. Objetivos: O objetivo do presente estudo foi realizar um levantamento epidemiológico da infecção por HPV na populacão feminina da região Sudeste do Brasil e, a partir de então, propor novas abordagens diagnósticas e vacinais. Metodologia: Amostras genitais de 5.223 mulheres foram avaliadas por uma nova ferramenta molecular baseada em PCR (Nested Multiplex PCR - NMPCR), utilizando-se um coquetel de primers para detecção simultânea de 38 diferentes tipos virais de HPV, em um único tubo. Outra abordagem molecular foi proposta através da técnica de Phage Display, a fim de selecionar peptídeos ligantes a anticorpos IgA oriundos de amostras cervicas de pacientes com infecção por HPV. Análises in silico (Bioinformática linear e 3D) e in vitro (ELISA, Slot blot e Bioeletrodo) foram realizadas para a validação dos fagos selecionados e dos peptídeos sintetizados em espécimes cervicais (n=91) e salivares (n=66). Os parâmetros de sensibilidade e especificidade do diagnóstico do HPV foram determinados pela curva ROC. Para testar o potencial vacinal dos peptídeos sintéticos, análises in silico (Bioinformática 3D), in vivo (imunização em camundongos fêmeas BALB/c) e in vitro (MTT, ELISA, Cultura de esplenócitos, CBA e Ensaio de Neutralização) foram conduzidas. Resultados: Dentre as 5.223 mulheres avaliadas, houve uma prevalência de 58,9% de infecção por HPV, com predominância dos tipos 53, 52 e 06, evidenciando a relevância de se propor novas alternativas de detecção e profilaxia do HPV. Na abordagem molecular de Phage Display trinta e dois clones de fagos distintos foram selecionados contra a IgA de amostras cervicais positivas para o HPV. O fago C.B1 apresentou maior reatividade contra amostras de HPV em comparação ao grupo controle negativo, por ELISA. Este peptídeo é um epítopo putativo da proteína principal do capsídeo do HPV (L1) e tem discriminado eficientemente amostras HPV de controles, em amostras cervicais e salivares (p<0,0001); com alta sensibilidade (acima de 95%) e especificidade (acima de 71%) em ambos fluidos. O peptídeo C.B1 também foi incorporado com sucesso (p<0,05) em um bioeletrodo de grafite para o diagnóstico do HPV direto na saliva. Na abordagem vacinal, três peptídeos (PEP1, PEP3 e PEP4) não apresentaram toxicidade em macrófagos murinos. As imunizações revelaram potente indução na produção de anticorpos séricos IgG pelo PEP3, e aumento nos títulos de IgA na mucosa vaginal sob estímulo dos PEP1 e PEP3. A avaliação da resposta imune celular indicou que o PEP1 e, sobretudo o peptídeo quimérico PEP3, foram capazes de polarizar a resposta imune para o perfil Th1 e sensibilizar as células a um status pró-inflamatório, propício para atuação antiviral. O ensaio de neutralização de PsVs do HPV-16 mostrou que o PEP3 foi capaz de reduzir a infecção em 49%. Conclusões: De maneira geral, os estudos aqui apresentados evidenciaram uma alta prevalência de HPV em mulheres brasileiras por tipos virais ainda pouco explorados na literatura mundial. Nós selecionamos mimotopos pela ferramenta de Phage Display capazes de detectar o HPV tanto em amostras de saliva quanto em secreção cervical, possibilitando um diagnóstico simples, de baixo custo, e não invasivo para rastreios populacionais. Além disso, os mimotopos aqui selecionados possuem atraente aplicabilidade em formulações vacinais, porém estudos adicionais ainda são necessários. / Doutor em Genética e Bioquímica

Le système immunitaire muqueux, et plus particulièrement les réponses intestinales IgA sont essentielles non seulement à la défense contre les agents pathogènes, mais aussi au façonnement de la flore intestinale commensale. Dans les modèles murins de déficit en IgA, on observe une dysbiose intestinale majeure associée à une inflammation muqueuse, réversibles après restauration des IgA. Le but de ce travail est de décrire l'impact de l'absence d'IgA chez l'homme sur la composition du microbiote intestinal ainsi que ses conséquences locales et systémiques. L'étude comparative par analyse métagénomique des selles de 17 sujets déficitaires en IgA et de 34 donneurs sains retrouve l'absence de différence majeure en termes de répartition des phyla dominants, de diversité et de richesse génique bactériennes entre les deux groupes. En revanche, en analysant à l'échelon des espèces, on observe dans le déficit en IgA une surreprésentation d'espèces pro-inflammatoires et une sous-représentation d'espèces anti-inflammatoires. En outre, en l'absence d'IgA, nous observons la présence de réponses IgM qui opsonisent partiellement les genres ciblés par l'IgA, mais semblent maintenir la diversité au sein des Actinobactéries. Les patients présentent un biais phénotypique lymphocytaire T circulant (TH17) associé à des stigmates de translocation bactérienne. Enfin, l'absence d'IgA s'associe à une perturbation du réseau bactérien minimal "obligatoire". Ces résultats suggèrent que le déficit en IgA humain s'accompagne d'une dysbiose modérée associée à une altération de l'architecture du réseau bactérien induisant une hyperactivation du système immunitaire, malgré la présence de réponses IgM. / IgA responses play a key role in gut mucosa, defending host against pathogens but also shaping the commensal flora. In order to get insights into the specific contributions of IgA to host/microbial symbiosis in humans, we explored patients that lack only IgA, using gut microbial metagenomics and systems immunology. Microbiota composition was compared between 34 healthy controls and 17 selective IgA deficiency (sIgAd) patients. Contrary to what was observed in murine models of IgA deficiency, we show that human sIgAd is not associated with massive perturbations of gut microbial ecology, regarding phyla distribution, bacterial diversity and gene richness. A clear gut microbial signature is however associated to sIgAd: we found 19 over-represented MGS mainly described to be pro-inflammatory, but also 14 under-represented MGS, mainly known to be beneficial. We also explored local consequences of IgA deficiency, particularly whether IgM could replace IgA at host/bacterial interface. Using a combination of bacterial flow sorting and DNA sequencing, we therefore analysed the composition of IgM-coated microbiomes observed in sIgAd. We show that IgM only partially supply IgA deficiency, as not all typical IgA targets can also be opsonized by IgM, but nevertheless contribute to maintain Actinobacteria diversity. IgA deficiency is associated with a skewed circulating CD4+ T cell profile towards TH17, as well as markers of bacterial translocation. Finally, sIgAd is associated with a perturbation of the minimal bacterial network. Altogether our results suggest that human IgA deficiency is associated with a mild dysbiosis associated to systemic inflammation despite the presence of IgM

La maladie de Berger est la glomérulonéphrite la plus fréquente avec une estimation selon laquelle 1% de la population mondiale serait touchée. L’agent causal est une immunoglobuline (Ig)A anormale (polymérique et hypogalactosylée) qui se dépose dans le mésangium et provoque un dysfonctionnement rénal (protéinurie, hématurie) et des lésions glomérulaires. Dans 25% des cas, la maladie évolue sur 20 ans vers l’insuffisance rénale terminale. Des évidences, de plus en plus nombreuses, montrent que l’environnement microbien, en particulier bactérien, commensal ou pathogène, a un impact important sur le développement de la maladie. Au cours de ma thèse, j’ai d’abord étudié l’effet d’une molécule de la paroi bactérienne, le lipopolysaccharide (LPS), sur la production des IgA dans les muqueuses chez la souris normale. Les résultats que j’ai obtenus et ceux publiés permettent de proposer que la stimulation chronique des muqueuses par l’environnement microbien conduit à une augmentation de la production d’IgA néphrotoxiques, qui, du fait d’un déficit de leur récepteur pIgR mucosal, sont anormalement dirigées vers la circulation plûtôt que dans la lumière es muqueuses. Dans une seconde partie de mon travail, j’ai étudié l’effet du LPS sur le développement de la maladie de Berger dans un modèle de souris 1KI. Ces souris génétiquement modifiées produisent de l’IgA humaine et développent spontanément des dépôts mésangiaux d’IgA mais n’ont pas de protéinurie, d’hématurie ou de lésions glomérulaires. Nos résultats montrent que le LPS provoque une forte hématurie dans les souris 1KI lorsque celles-ci expriment le récepteur des IgA humaines, CD89, à la surface des polymorphonucléaires neutrophiles. En conclusion, mon travail de thèse a permis de mettre en lumière un impact de l’environnement microbien sur pIgR et sur les polymorphonucléaires neutrophiles dont la déficience ou l’activation pourrait contribuer au développement de la maladie de Berger. / IgA nephropathy is the most frequent glomerulonephritis worldwide. It features mesangial immunoglobulin (Ig)A deposits and proteinuria, hematuria and glomerular histological lesions. In 25% patients, it evolves, within 20 years, towards the end stage renal disease. Microbial environment, through the interaction with mucosa, is believed to play a crucial role in the development of the disease. The objectives of my work were to evaluate the effect of the microbial compound lipopolysaccharide (LPS) on the production of the nephritogenic IgA in the mucosa of mice and on the development of IgA nephropathy in the murine model 1KI that spontaneously develops mesangial IgA deposits without the other signs of the disease. Our results and those previously published suggest that the chronic stimulation of mucosa by microbiota leads to the increased production of nephritogenic IgA in the mucosa. These IgA, thanks to a deficient mucosal receptor pIgR in patients with IgA nephropathy, may be abnormally routed in the blood. We also showed that LPS provokes hematuria in the 1KI mice, when they express a specific IgA receptor, CD89, on the surface of polymorphonuclear neutrophils. Altogether our findings highlight the impact of microbial environment on pIgR and on polymorphonuclear neutrophils and thus, potentially, on IgA nephropathy.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22833号 / 医博第4672号 / 新制||医||1047(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 竹内 理, 教授 濵﨑 洋子, 教授 椛島 健治 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Introduction: Nonsteroidal anti-inflammatory drugs (NSAIDs) are one of the most used drugs in equine medicine, mainly used to treat inflammation, endotoxemia, pain or fever. NSAIDs inhibit cyclooxygenases which induce to synthesize prostanoids. But NSAIDs have side effects to renal functions too. Objectives: The current study was carried out to investigate the effects of the most common used NSAIDs on urinary parameters in horses. Materials and Methods: Thirty healthy horses were used as a control group and 20 horses with left dorsal displacement, left ventral impaction or lameness of using either flunixin meglumine (FM), metamizole (MZ) or phenylbutazone (PHZ) have been assigned to groups 1, 2 or 3, respectively. Creatinine, urea nitrogen, glucose, protein and electrolytes were measured in serum and urine including GGT using an automatic analyzer. Fractional excretions (FE) of sodium, chloride, potassium, calcium, magnesium and inorganic phosphate, in addition to urinary protein (U-Pro):U-Cr and urinary gamma glutamyl transferase (U-GGT):U-Cr ratios were calculated. Urinary mucus and IgA concentrations were measured and their ratios to the urinary creatinine were calculated. The data were statistically analyzed using Shapiro-Wilks test, descriptive statistics, Kruskal-Wallis one-way analysis of variance and Dunn’s test. Significance was set at P £ 0.05. Results: The FEMg was significantly higher in group 3 (P < 0.033) compared to the control group. The U-GGT:U-Cr ratio was also significantly higher in group 3 (P < 0.001) compared with the control group. The U-Pro:U-Cr ratio was significantly higher in groups 1 and 2 (P < 0.007 and P < 0.001, respectively) than in the control group. PHZ group had a significantly increase in mucus:U-Cr ratio (P < 0.005). Significant increases were observed regarding the IgA:U-Cr ratio in groups 1 (P < 0.007) and 2 (P < 0.014). Conclusions: Long-term use of PHZ has an influence on the renal ascending limb of the loop of Henle, and all these drugs could have effects on the proximal tubules. Phenylbutazone causes an increase in urinary mucus secretion, probably as a protective mechanism against the necrotic effect in renal pelvis of PHZ. Parameters such as U-Pro:U-Cr and U-GGT:U-Cr ratios and FEMg are helpful in detecting these renal abnormalities.

The Flex Representation Method (FRM) leverages unique computational advantages of splines to address limitations in the process of building CAE simulation models from CAD geometric models. Central to the approach is the envelope CAD domain that encapsulates a CAD model. An envelope CAD domain can be of arbitrary topological and geometric complexity. Envelope domains are constructed from spline representations, like U-splines, that are analysis-suitable. The envelope CAD domain can be used to approximate none, some, or all of the features in a CAD model. This yields additional simulation modeling options that simplify the model-building process while leveraging the properties of splines to control the accuracy and robustness of computed solutions. Modern integration techniques are adapted to envelope domains to maintain accurate solutions regardless of the CAD envelope chosen. The potential of the method is illustrated through several carefully selected benchmark problems.

No description available.

La régulation de l'homéostasie intestinale est de la plus haute importance en raison de la constante exposition de l'intestin aux antigènes alimentaires et à la flore commensale. La perturbation de la flore intestinale est souvent associée à diverses maladies telles que l'allergie, l'obésité et certaines maladies inflammatoires. La plupart des individus sont tolérant aux antigènes alimentaires et ne développe pas de réponse immunitaire sauf en cas de prédisposition génétique ou d'exposition à un environnement défavorable. La réponse allergique se caractérise par la production d'IgE stimulé par les lymphocytes Th2. Les symptômes allergiques sont très variés et affectent plusieurs parties de l'organisme. La plupart des travaux de recherche se sont focalisé jusqu'à présent sur le rôle des cellules de l'immunité adaptative dans le développement de l'allergie en sous-estimant le rôle majeur des cellules épithéliales et des cellules de l'immunité innée. L'objectif de ce projet est de comprendre comment les cellules épithéliales intestinales modulent la réponse immunitaire à distance vers la muqueuse pulmonaire ou la peau après stimulation allergique. L'ingestion de l'antigène associé à l'adjuvant de la toxine cholérique permet d'étudier la réponse allergique chez l'animal. Nous avons démontré sur ce modèle animal que l'absence de la kinase inhibitrice IKKb dans la voie de signalisation du facteur de transcription NF-kB altère la composition de la flore intestinal d'une part et transforme la réponse immunitaire inflammatoire au niveau pulmonaire et de la peau grâce à la présence d'IgA et de lymphocyte Th17 d'autre part. En adéquation avec les observations cliniques rapportées chez les patients allergiques (allergies alimentaires, asthme, dermatite atopique), nos résultats identifient IKKb dans la cellule épithéliale intestinale comme cible potentielle pour traiter les allergies alimentaires. De futurs efforts devront être faits pour développer de nouvelles stratégies thérapeutiques qui considèrent la muqueuse intestinale, la production d'IgA et l'importance des bactéries commensales dans le traitement des allergies. / Immune homeostasis is of paramount importance in the gastrointestinal tract, which is constantly exposed to ingested antigens and commensal microbiota. The gut microbiota can be perturbed by endogenous or exogenous factors and it is now established that microbial dysbiosis is associated with allergy, obesity, and inflammatory diseases. Ingestion of food antigens generally fails to promote brisk immune responses but rather results in a state of immune tolerance. However, aberrant immune responses can develop in individuals with a genetic predisposition. Food allergies are generally regarded as pathologic responses to food antigens mediated by excessive Th2 responses and antigen-specific IgE antibody responses. Clinical manifestations of food allergies are very broad and symptoms can affect different organs. While past research on allergy focused on the role of cells and molecules involved in adaptive immunity, epithelial cells lining the sites of antigen entry and innate immune responses have recently emerged as important players in allergy. This project was undertaken to understand the mechanisms employed by intestinal epithelial cells (IECs) to shape immune responses to allergens and influence allergic manifestations in distant mucosal sites such as the airways or the skin. Oral administration of food antigen with cholera toxin as adjuvant in experimental animals is a well-accepted model to study allergic sensitization to food antigens. Using this model, we show that a localized impairment of the canonical NF-κB pathway through deletion of IkB kinase (IKKβ) in IECs alters the gut microbiota during oral allergic sensitization and regulates the magnitude of allergic inflammatory responses at distant sites of the airway and the skin through enhancement of IgA Abs and Th17 responses. Consistent with the clinical observations linking atopic diseases (food allergy, allergic asthma, atopic dermatitis), our results identify IKKβ in IECs as a potential therapeutic target for treatment of food allergies and subsequent disease. They also suggest that future efforts for controlling allergic responses in the airways and the skin could include strategies that use the gut microbiota and promote IgA Ab responses and prevent IL-17 responses.