Das Multiple Myelom (MM) ist eine hämatologische Erkrankung, welche sich durch eine Akkumulation von malignen Plasmazellen im Knochenmark auszeichnet und eine gestörte Hämatopoiese und Osteolyse zur Folge hat. Komplexe molekulare Interaktionen zwischen MM-Zellen und der Mikroumgebung/Nische im Knochenmark (bone marrow microenvironment, BMM) führen zu einer Aktivierung von verschiedenen Wachstums-, Überlebens- und anti-apoptotischen Signalwegen, die zur Entstehung bzw. Wirkstoffresistenz von MM-Zellen beitragen. IL-6R/STAT3, Ras/MAPK und PI3K/Akt sind die drei wichtigsten Signalwege, die mit dem Wachstum und der Entwicklung des MM assoziiert sind. Auf der anderen Seite sind Myelomzellen insensitiv gegenüber einer Blockade des IL6R/STAT3-Signalweges bzw. des Ras/MAPK-Signalwegs in der Gegenwart von Knochenmarksstromazellen (bone marrow stroma cells, BMSCs), was die Entbehrlichkeit dieser beiden Signalwege unter Ko-Kultur-Bedingungen nahelegt. Interessanterweise aber induziert die gleichzeitige Unterbrechung der IL6R/STAT3 und Ras/MAPK Signalwege Apoptose in MM-Zellen. Ziel der Arbeit war die Identifizierung und Analyse von Zielgenen, die von beiden Signalwegen und nicht durch einen Signalweg alleine reguliert werden. Genexpressionsanalysen zeigten eine deutliche Herunterregulierung der Proteine Hsp90alpha und Hsp90beta nach einer gleichzeitigen Inhibition der IL6R/STAT3 und Ras/MAPK Signalwege. In Hinblick auf die zentrale Rolle von Hsp90 in der Tumorbiologie fokussiert sich die vorliegende Arbeit auf die Erforschung der Rolle von Hsp90 im Multiplen Myelom. Die siRNA-vermittelte Herunterregulation der Proteinexpression von Hsp90-Proteinen zeigte, daß das Ausschalten von HsP90alpha alleine nur zu einer moderaten Apoptoseinduktion in INA-6- und MM.1s-Zellen führte. Die gleichzeitige Herunterregulation von HsP90beta hingegen führte zu einer Verstärkung dieses Effektes und deutet darauf hin, daß beide Proteine miteinander kooperieren. Die pharmakologische Inhibition der Hsp90-Funktion mittels eines neuen Hsp90-Inhibitors (17-DMAG) führte zu einer Verringerung von phospho-ERK1/2, zur Degradation von STAT3 und zu einem verminderten Überleben von MM-Zellen. Die pro-apoptotischen Effekte der gestörten Hsp90-Funktion konnten weder durch BMSCs und Osteoklasten noch durch ECs (??) abgeschwächt werden, obwohl für ECs beschrieben wurde, daß sie zum Wachstum und Überleben von MM-Zellen beitragen können. Diese Beobachtungen deuten auf einen positiven Rückkopplungskreislauf zwischen HsP90alpha/beta und den wichtigsten Signalwegen hin, welcher das Überleben von MM-Zellen gewährleistet. Desweiteren zeigten immunhistologische Analysen, daß Hsp90-Proteine im Vergleich zu MGUS (??) bzw. normalen Plasmazellen in MM-Plasmazellen hochreguliert sind. Zusammengefasst zeigen die Ergebnisse der vorliegenden Arbeit die essentielle Rolle von Hsp90-Proteinen für die Überlebensfähigkeit von MM-Zellen. Ein neuer Mechanismus der Hsp90-Regulation durch das Zusammenwirken der Signalwege IL6R/STAT3 und Ras/MAPK in MM-Zellen konnte gezeigt werden. Darüber hinaus deuten die Ergebnisse darauf hin, daß ein positiver Rückkopplungskreislauf zwischen Hsp90-Proteinen und den wichtigsten Signalwegen existiert, welcher zum Wachstum und zur Entwicklung von MM-Zellen beiträgt. Die Inhibition der Hsp90-Funktion durch den pharmakologischen Inhibitor 17-DMAG führte zum Absterben von MM-Zellen und der pro-apoptotische Effekt der Hsp90-Depletion konnte nicht durch unterstützende BMM-Zellen aufgehoben werden. Diese Beobachtungen untermauern die multifunktionelle Rolle von Hsp90 in der MM-Biologie und zeigen die Wichtigkeit der Entwicklung neuer therapeutischer Wirkstoffe zur Inhibition der Hsp90-Funktion bei der Behandlung des MM. / Multiple myeloma (MM) is a haematological malignancy characterised by the accumulation of malignant plasma cells in the bone marrow leading to impaired haematopoiesis and osteolytic bone destruction. Intricate molecular interactions between MM cells and the BMM activate a diverse set of growth, survival and anti-apoptotic signaling cascades that mediate tumor progression and drug resistance. IL-6R/STAT3, Ras/MAPK and PI3K/Akt are the three major signal transduction pathways that are associated with MM growth and progression. However, myeloma cells have shown independence from IL-6R/STAT3 blockade or insensitivity towards Ras/MAPK pathway inhibition in the presence of BMSCs, indicating the dispensability of both in co-culture conditions. Interestingly, concomitant disruption of both IL-6R/STAT3 and Ras/MAPK pathways was successful to drive MM cells into significant apoptosis. This study aimed to identify and analyse the downstream target genes that are regulated by both pathways and not by either pathway alone. Gene expression profiling revealed prominent downregulation of Hsp90alpha and Hsp90beta proteins after combined inhibition of the IL-6R/STAT3 and Ras/MAPK pathways. Owing to the important role played by Hsp90 in cancer biology, this study was narrowed down to investigate the role of Hsp90 in MM. Specific siRNA-mediated knockdown of Hsp90 proteins showed that although knockdown of Hsp90beta was sufficient to induce moderate apoptosis in INA-6 and MM.1s cells, the effect was more pronounced when both Hsp90 proteins were targeted, indicating co-operation between them. Pharmacological inhibition of Hsp90 function by using a novel Hsp90 inhibitor (17-DMAG) down-regulated the levels of pERK1/2 and led to degradation of STAT3 and decreased viability of MM cells. The pro-apoptotic effects of compromised Hsp90 function could not be alleviated by either BMSCs, OCs or ECs, which are well-known to support myeloma growth and survival. These observations point to the existence of a positive feedback loop consisting of Hsp90alpha/beta and major signaling pathways supporting MM cell survival. Furthermore, immunohistochemical analysis unveiled the up-regulated status of Hsp90 proteins in MM PCs as compared to MGUS or normal PCs. Taken together, the results of this study explain the critical contribution of Hsp90 proteins to MM cell survival. A novel mechanism of Hsp90 regulation by co-operation between the IL-6R/STAT3 and Ras/MAPK pathways was discovered in myeloma cells. There is also strong evidence of the existence of a positive feedback loop between Hsp90alpha/beta proteins and major signaling pathways supporting MM growth and progression. Inhibition of Hsp90 function by using the Hsp90 inhibitory drug 17-DMAG proved to be lethal for myeloma cells and the pro-apoptotic effects of Hsp90 blockade could not be reversed by the presence of cells from the supportive BMM. These observations highlight a multi-functional role of Hsp90 in MM biology and strongly strengthen the notion that therapeutic strategies targeting Hsp90 may open new perspectives for anti-myeloma drug development.

A rejeição ao aloenxerto mediada por mecanismos celulares ou humorais representa uma importante complicação no pós-transplante renal. Estudos anteriores demonstraram que o depósito de C4d peritubular é um marcador de rejeição mediada por anticorpos. A técnica padrão ouro para a pesquisa de C4d é a imunofluorescência em criostato. No entanto, o manuseio do material congelado implica em algumas limitações custo-operacionais, particularmente em nosso meio. Nos casos de rejeição mediada por anticorpos é de relevância patogenética a análise da participação de linfócitos B e plasmócitos, pois são as células responsáveis pela produção de anticorpos. Considerando que até o momento o envolvimento de linfócitos B e plasmócitos no processo de rejeição foi pouco investigado, no presente estudo também será analisada a expressão de CD20 e CD138 em biópsias renais, para caracterização destes componentes celulares. Portanto, o objetivo do presente estudo foi analisar a detecção do fragmento C4d por meio de 4 diferentes técnicas, além de analisar o infiltrado de linfócitos B e plasmócitos em biópsias de enxerto renal, correlacionando estes achados com o C4d peritubular. Foram analisadas 107 biópsias de 82 pacientes submetidos a transplante renal. A pesquisa do C4d foi realizada utilizando-se as técnicas de imunofluorescência [IF] (em cortes de criostato e de parafina) e imuno-histoquímica [IH] (em cortes de criostato e de parafina), enquanto que as pesquisas dos linfócitos B e plasmócitos foram realizadas pela técnica de IH em cortes de parafina utilizando-se os anticorpos anti-CD20 e anti-CD138, específicos para linfócitos B e plasmócitos, respectivamente. Com relação à detecção de C4d, as técnicas com maior índice de concordância com a IF-criostato, considerada padrão-ouro, foram IH-criostato (75,6% dos casos apresentaram resultados coincidentes, r=0,72; p<0,0001) e IF-parafina (73%, r=0,59; p=0,0001), enquanto a taxa de concordância na técnica de IH-parafina foi de apenas 51,4% (r=0,35; p=0,03). Analisando a evolução clínica, a sobrevida do enxerto renal em 3 anos pós-biópsia foi menor no grupo C4d positivo comparado ao grupo C4d negativo (67% vs 96%, respectivamente, p=0,01). A prevalência de linfócitos B (CD20+) foi de 54% das biópsias de enxerto renal. A análise histológica do infiltrado de linfócitos B revelou 2 padrões distintos de infiltrado: padrão de células isoladas (74%) e padrão nodular (26%). O padrão nodular esteve associado a uma menor sobrevida do enxerto renal em 3 anos pós-biópsia (61% vs 89% no grupo CD20 negativo; p=0,03; e 61% vs 87% no grupo padrão de células isoladas; p=0,03). A prevalência de plasmócitos (CD138+) em biópsias de enxerto renal foi de 59%. O infiltrado plasmocitário não esteve associado a uma pior evolução clínica do transplante. A análise da correlação entre C4d peritubular, linfócitos B e plasmócitos demonstrou que o número de células CD20+ e CD138+ foi significativamente maior nos casos C4d positivos comparados aos casos C4d negativos (CD20+: 155±53 vs 26±7 cels/mm2, respectivamente; p=0,001; CD138+: 46±22 vs 4±1 cels/mm2, respectivamente; p=0,002). O presente estudo concluiu que o depósito peritubular de C4d e o infiltrado de linfócitos B, em especial o padrão nodular, estão associados a uma evolução clínica desfavorável do transplante renal. Outra conclusão importante é que há uma associação positiva entre os infiltrados de linfócitos B e de plasmócitos com o C4d peritubular, sugerindo um possível papel destas células responsáveis pela produção de anticorpos na ativação do sistema complemento in situ. Finalmente, as técnicas de IH-criostato e IF-parafina podem ser consideradas técnicas alternativas à técnica de IF-criostato para a detecção do C4d / Allograft rejection mediated by cellular or humoral mechanisms represents an important complication after kidney transplantation. Capillary C4d deposition was recognized as a specific and independent prognostic marker of antibody mediated rejection. The gold standard technique for C4d detection is the immunofluorescence in cryostat sections. However, this technique involves some operating and costs limitations, particularly in Brazil. In antibody mediated rejection, the analysis of B lymphocytes and plasma cells is of pathogenetic relevance since these cells are responsible for antibody production. Considering that the involvement of B lymphocytes and plasma cells in the rejection process is not clear, in this study the CD20 and CD138 expression in kidney biopsies will be also analyzed. Therefore, the aim of the present study was to analyze the C4d detection by 4 different techniques and the infiltration of B lymphocytes and plasma cells in renal allograft biopsies, correlating these findings with the capillary C4d deposition. One hundred and seven biopsies of 82 renal transplant patients were analyzed. C4d was evaluated by immunofluorescence (IF) and immunohistochemistry (IHC) techniques in frozen and paraffin sections (obtained from the same patients), whereas B-lymphocytes and plasma cells were evaluated by immunohistochemistry in paraffin sections using specific antibodies anti-B Lymphocytes (CD20) and anti-plasma cells (CD138). Regarding the C4d detection, the techniques with higher concordance rate with frozen-IF, considered the gold standard, were the frozen-IHC technique (85.4% of cases showed coincident results, r=0.72; p<0.0001) and the paraffin-IF technique (73%, r=0.59; p=0.0001), whereas the concordance rate in the paraffin-IHC technique was only 51.4% (r=0.35; p=0.03). The clinical follow up analysis demonstrate that C4d positive group was associated with a poor graft survival at 3 years post-diagnosis (67% vs 96% in C4d negative group; p=0.01). The histological analysis of mature B cells (CD20+) infiltrate showed 2 distinct patterns: scattered cells and clusters. The cluster pattern was associated with poor graft survival at 3 years (61% vs 89% in the CD20 negative group; p=0.03; and 61% vs 87% in the CD20+ scattered pattern group; p=0.03). The plasma cells infiltrate was not associated with a worse clinical transplant outcome. The analysis of the capillary C4d and B lymphocytes correlation demonstrate that the number of CD20+ and CD138+ cells was significantly higher in C4d positive cases (CD20+: 155±53 vs 26±7 cells/mm2, respectively; p=0.001; CD138+: 46±22 vs 4±1 cells/mm2, respectively; p=0.002). This study concluded that C4d capillary and mature B cells (clusters pattern) are associated with worse graft survival. Another important conclusion is a positive association between B lymphocytes (mature B cells and plasma cells) and capillary C4d, suggesting a possible role of these cells, responsible for antibodies production, in the in situ complement system activation. Finally, the frozen-IHC and paraffin-IF techniques may be considered alternative to frozen-IF technique for C4d detection

La polyarthrite rhumatoïde (PR) est une maladie auto-immune chronique. Elle est caractérisée par une inflammation persistante touchant de multiples petites articulations, causant douleurs, rougeurs, gonflements et déformations. Des études menées auprès de patients et d’animaux ont démontré que certains auto-anticorps, cytokines et enzymes tissue-déstructives sont des médiateurs importants dans le développement de la PR. Au cours des deux dernières décennies, les traitements de fond (DMARDs en anglais) ont été démontrés très efficaces pour traiter la PR. D'autre part, des effets secondaires ont été rapportés pour ces traitements, par exemple l'augmentation du risque d'infections opportunistes. L’objectif de ce travail est d’acquérir des connaissances sur le rôle du TL1A (TNF-like molécule 1 A; TNFSF15) et son partenaire Nob1 (Pno1 ; YOR145c) dans la pathogenèse de la PR afin de découvrir de nouveaux médicaments contre ces molécules dans l'avenir. TL1A est un membre de la famille du TNF. Il déclenche des signaux co-stimulateurs via le récepteur de mort 3 (DR3) et induit la prolifération ainsi que la production des cytokines pro inflammatoires par les lymphocytes. Des données multiples suggèrent l'implication de la cascade TL1A-DR3 dans plusieurs maladies auto-immunes. Donc, nous avons proposé les hypothèses suivantes:1) la production locale de TL1A dans les articulations est un composant d’un cercle vicieux qui aggrave la PR; 2) dans la PR, la production de TL1A dans les organes lymphoïde augmente la production d’auto-anticorps pathogénique. Au cours de ce travail, nous avons démontré que la TL1A aggrave la maladie chez les souris où l’arthrite a été induite par le collagène (AIC). Par ailleurs, nous avons constaté que l’expression de TL1A est élevée dans les tissus atteints de PR ainsi que dans les ganglions lymphatiques drainant de la souris AIC. Mécaniquement, nous avons découvert que la TL1A est induite par le TNF-α et IL-17 produits par les cellules T in vitro. Ces résultats montrent directement que les TL1A-DR3 jouent un rôle essentiel dans la pathogenèse de la PR. De plus, afin de poursuivre notre étude, la TL1A a été génétiquement supprimée dans les souris (TL1A KO). Nous avons montré que les souris TL1A KO n’ont aucune anomalie apparente et aucun dysfonctionnement du système immunitaire dans des conditions normales. Cependant, ces souris manifestent des AIC améliorées et une réduction significative des niveaux d'anticorps, anti-collagène du type II i dans le sérum. Nous avons trouvé que les ganglions lymphatiques de drainage (dLNs) de souris KO étaient plus petites avec une cellularité inférieure comparativement aux souris WT de 14 jours après l’immunisation. De plus, nous avons découvert que le DR3 a été exprimé par les cellules plasmatiques dans l’étape de la différenciation terminale et ces cellules surviennent mieux en présence de TL1A. La conclusion de cette étude apporte des nouvelles connaissances sur le rôle de TL1A qui amplifie les réponses humorales d’AIC. Nous avons suggéré que TL1A pourrait augmenter la réponse d’initiation d'anticorps contre collagène II (CII) ainsi que prolonger la survie des cellules plasmatiques. Une autre molécule qui nous intéresse est Pno1. Des études antérieures menées chez la levure ont suggéré que Pno1 est essentielle pour la néogénèse du protéasome et du ribosome Le protéasome étant crucial pour la différenciation terminale des cellules plasmatiques pendant les réponses humorales chez les mammifères, nous avons donc supposé que Pno1 joue un rôle dans la production d'anticorps pathogenique dans la PR via la voie du protéasome. Nous avons donc généré des souris génétiquement modifiées pour Pno1 afin d’étudier la fonction de Pno1 in vivo. Cependant, une mutation non-sens dans le Pno1 provoque une létalité embryonnaire à un stade très précoce chez les souris. D'autre part, une réduction de 50% de Pno1 ou une surexpression de Pno1 n’ont aucun effet ni sur le fonctionnent des cellules T et B, ni sur les activités du protéasome ainsi que sur la réponse humorale dans l’AIC. Ces résultats suggèrent que Pno1 est une molécule essentielle sans redondance. Par conséquent, il n’est pas une cible appropriée pour le développement de médicaments thérapeutiques. En conclusion, nos études ont révélé que la TL1A n’est pas essentielle pour maintenir les fonctions du système immunitaire dans des conditions normales. En revanche, il joue un rôle critique dans la pathogenèse de la PR en favorisant l'inflammation locale et la réponse humorale contre des auto-antigènes. Par conséquent, une inhibition de la TL1A pourrait être une stratégie thérapeutique pour le traitement de la PR. Au contraire, Pno1 est essentiel pour la fonction normale des cellules. Une délétion totale pourrait entraîner des conséquences graves. Il n’est pas une cible appropriée pour développer des médicaments de la PR. / Rheumatoid Arthritis (RA) is a chronic autoimmune disease characterized by persistent inflammation of multiple small joints, which manifests pain, redness, swelling, and deformation. Studies with patients and animal models have found that autoantibodies, cytokines and tissue-destructive enzymes are important mediators of the pathogenesis of RA. In the past two decades, biologic disease-modifying antirheumatic drugs (DMARDs) have achieved great success in the treatment of RA. On the other hand, they are also associated with adverse effect like increasing the chance of opportunistic infections. The aim of present work was to investigate the roles of TNF-like molecule 1A (TL1A; TNFSF15) and partner of Nob1 (Pno1; YOR145c) in the pathogenesis of RA for developing novel drugs based on these molecules in the future. TL1A is a member of the TNF superfamily. It triggers costimulatory signals though death receptor 3 (DR3) and induces the proliferation and pro-inflammatory cytokine production in lymphocytes. Multiple lines of evidence suggest the implication of TL1A-DR3 signaling in several autoimmune diseases. Therefore, We hypothesized that 1) local TL1A production in the joints is a component of a vicious circle aggravating RA; 2) in RA, TL1A production in lymphoid organs enhances pathogenic autoantibody production. We demonstrated that the TL1A aggravates disease in murine collagen-induced arthritis (CIA). Moreover, we found elevated TL1A expression in RA-affected tissues, as well as in the draining lymph nodes (dLNs) of CIA mice. Mechanistically, we discovered that TL1A induces TNF-α and IL-17 production by T cells in vitro. These findings provided direct evidence that TL1A-DR3 signaling plays a critical role in the pathogenesis of RA. TL1A knockout (TL1A KO) mice were generated to further our study. We showed that TL1A KO mice have no visual anomaly, and no malfunction of immune system under a normal circumstance. However, they display ameliorated CIA and significantly reduced anti-Collagen II antibody levels in sera. We found that the draining lymph nodes (dLNs) from KO mice were smaller in size and lower in cellularity compared with their WT counterparts 14 days after immunization. Furthermore, we discovered that terminally differentiated plasma cells express DR3 and they survive better in the presence of TL1A. Our findings in this study present novel knowledge about the role of iii TL1A promoting the humoral responses in CIA; we suggest that TL1A could elevate the initial Ab response against Collagen II (CII), as well as prolong the survival of plasma cells producing such pathogenic Abs. Another molecule we were interested in present study is Pno1. Previous studies conducted in yeast suggest that Pno1 is essential to the proteasome and ribosome neogenesis. Since proteasome is crucial for the terminal differentiation of plasma cells during the humoral response in mammals, we hypothesized that Pno1 plays a role in the pathogenic Ab production in RA by affecting the proteasome assembly. For this purpose, we generated pno1 gene- modified mice to investigate the function of Pno1 in vivo. However, null-mutation in pno1 causes embryonic lethality in mice at a very early stage. On the other hand, a half amount reduction or overexpression of Pno1 is neither harmful nor useful to the T and B cell function, proteasome activities as well as humoral immune responses in CIA. These findings suggest that Pno1 is a vital molecule with no redundancy and is absolutely required for cell function, but animals can function normally with a small fraction of the normal Pno1 expression level. Thus, it might not be an appropriate target for developing therapeutic drugs. In conclusion, our studies suggest that TL1A seems not essential in maintaining the immune functions under normal circumstances, but plays critical roles in the pathogenesis of RA by promoting local inflammation and humoral immune responses against autoantigens. Therefore, inhibiting TL1A could be a propitious therapeutic strategy for treating RA. In contrast, Pno1 is vital to the normal cell function, and its disruption could cause disastrous consequences. Thus, it might not be a good drug target for treating RA.

Dissertation (Ph.D.)--University of South Florida, 2006. / Includes vita. Includes bibliographical references (leaves 119-141). Also available online.

La réponse humorale à immunoglobulines A (IgA) constitue un des principaux mécanismes immunologiques permettant de maintenir l'homéostasie intestinale. L'initiation de la réponse IgA se déroule dans les tissus lymphoides associés à l'intestin, où la reconnaissance des antigènes intestinaux entraine l'activation des lymphocytes B naïfs, la commutation isotypique vers IgA et leur différenciation en plasmocytes. Mon travail de thèse a consisté à étudier la contribution du foie et des cellules dendritiques plasmacytoides (pDC) dans la réponse IgA intestinale. L'utilisation de deux modèles murins permettant la déplétion sélective des pDC nous a permis de démontrer que, en dépit de données publiées montrant leur capacité à engager la réponse IgA in vitro, les pDC ne sont pas nécessaires in vivo pour l'induction ou le maintien de la réponse IgA homéostatique. Nous montrons ensuite que le foie abrite une population importante de plasmocytes à IgA. Chez la souris, nous montrons que ces cellules possèdent des caractéristiques phénotypiques distinctes des plasmocytes de l'intestin et proviennent de lymphocytes B récemment activés dans les plaques de Peyer. A l'homéostasie, ces plasmocytes hépatiques secrètent des IgA dirigées contre les bactéries de la flore intestinale. Enfin, dans un modèle murin de consommation chronique d'alcool, nous montrons une corrélation entre une augmentation de cette population cellulaire, une élévation sérique des IgA et des dépôts d'IgA hépatiques, deux désordres fréquemment observés chez les patients atteints d hépatopathies alcooliques. Nos données indiquent donc que le foie constitue un site effecteur alternatif de la réponse / IgA humoral response is one of the main mechanisms by which immune homeostasis is maintained in the intestine. The IgA response is initiated in gut-associated lymphoid tissues, where recognition of intestinal antigens drives naïve B cell activation, IgA class-switch recombination and plasma cell differentiation. My thesis work addressed the contribution of the liver and plasmacytoid dendritic cells (pDCs) in intestinal IgA response. By using two complementary mouse models allowing for selective depletion of pDCs, we have demonstrated that, in contrast to published work showing their ability to drive IgA response in vitro, pDCs are dispensable in vivo for the induction and the maintenance of homeostatic intestinal IgA responses.Then, we showed that the liver contains an important population of IgA plasma cells. In mice, we demonstrated that these cells harbor distinct phenotypic characteristics in comparison to intestinal IgA plasma cells, and are derived from B cells recently activated in Peyer’s patches. At homeostasis, hepatic IgA plasma cells secrete IgA directed against bacteria from intestinal flora. Finally, in a mouse model of chronic ethanol consumption, we found a correlation between an increase in hepatic igA plasma cell population, elevation of serum iGA and IgA deposits in liver sinusoids, two disorders frequently observed in alcoholic liver disease patients. Thus, our results indicate that the liver constitutes an alternative effector site for IgA response initiated in the intestine

A rejeição ao aloenxerto mediada por mecanismos celulares ou humorais representa uma importante complicação no pós-transplante renal. Estudos anteriores demonstraram que o depósito de C4d peritubular é um marcador de rejeição mediada por anticorpos. A técnica padrão ouro para a pesquisa de C4d é a imunofluorescência em criostato. No entanto, o manuseio do material congelado implica em algumas limitações custo-operacionais, particularmente em nosso meio. Nos casos de rejeição mediada por anticorpos é de relevância patogenética a análise da participação de linfócitos B e plasmócitos, pois são as células responsáveis pela produção de anticorpos. Considerando que até o momento o envolvimento de linfócitos B e plasmócitos no processo de rejeição foi pouco investigado, no presente estudo também será analisada a expressão de CD20 e CD138 em biópsias renais, para caracterização destes componentes celulares. Portanto, o objetivo do presente estudo foi analisar a detecção do fragmento C4d por meio de 4 diferentes técnicas, além de analisar o infiltrado de linfócitos B e plasmócitos em biópsias de enxerto renal, correlacionando estes achados com o C4d peritubular. Foram analisadas 107 biópsias de 82 pacientes submetidos a transplante renal. A pesquisa do C4d foi realizada utilizando-se as técnicas de imunofluorescência [IF] (em cortes de criostato e de parafina) e imuno-histoquímica [IH] (em cortes de criostato e de parafina), enquanto que as pesquisas dos linfócitos B e plasmócitos foram realizadas pela técnica de IH em cortes de parafina utilizando-se os anticorpos anti-CD20 e anti-CD138, específicos para linfócitos B e plasmócitos, respectivamente. Com relação à detecção de C4d, as técnicas com maior índice de concordância com a IF-criostato, considerada padrão-ouro, foram IH-criostato (75,6% dos casos apresentaram resultados coincidentes, r=0,72; p<0,0001) e IF-parafina (73%, r=0,59; p=0,0001), enquanto a taxa de concordância na técnica de IH-parafina foi de apenas 51,4% (r=0,35; p=0,03). Analisando a evolução clínica, a sobrevida do enxerto renal em 3 anos pós-biópsia foi menor no grupo C4d positivo comparado ao grupo C4d negativo (67% vs 96%, respectivamente, p=0,01). A prevalência de linfócitos B (CD20+) foi de 54% das biópsias de enxerto renal. A análise histológica do infiltrado de linfócitos B revelou 2 padrões distintos de infiltrado: padrão de células isoladas (74%) e padrão nodular (26%). O padrão nodular esteve associado a uma menor sobrevida do enxerto renal em 3 anos pós-biópsia (61% vs 89% no grupo CD20 negativo; p=0,03; e 61% vs 87% no grupo padrão de células isoladas; p=0,03). A prevalência de plasmócitos (CD138+) em biópsias de enxerto renal foi de 59%. O infiltrado plasmocitário não esteve associado a uma pior evolução clínica do transplante. A análise da correlação entre C4d peritubular, linfócitos B e plasmócitos demonstrou que o número de células CD20+ e CD138+ foi significativamente maior nos casos C4d positivos comparados aos casos C4d negativos (CD20+: 155±53 vs 26±7 cels/mm2, respectivamente; p=0,001; CD138+: 46±22 vs 4±1 cels/mm2, respectivamente; p=0,002). O presente estudo concluiu que o depósito peritubular de C4d e o infiltrado de linfócitos B, em especial o padrão nodular, estão associados a uma evolução clínica desfavorável do transplante renal. Outra conclusão importante é que há uma associação positiva entre os infiltrados de linfócitos B e de plasmócitos com o C4d peritubular, sugerindo um possível papel destas células responsáveis pela produção de anticorpos na ativação do sistema complemento in situ. Finalmente, as técnicas de IH-criostato e IF-parafina podem ser consideradas técnicas alternativas à técnica de IF-criostato para a detecção do C4d / Allograft rejection mediated by cellular or humoral mechanisms represents an important complication after kidney transplantation. Capillary C4d deposition was recognized as a specific and independent prognostic marker of antibody mediated rejection. The gold standard technique for C4d detection is the immunofluorescence in cryostat sections. However, this technique involves some operating and costs limitations, particularly in Brazil. In antibody mediated rejection, the analysis of B lymphocytes and plasma cells is of pathogenetic relevance since these cells are responsible for antibody production. Considering that the involvement of B lymphocytes and plasma cells in the rejection process is not clear, in this study the CD20 and CD138 expression in kidney biopsies will be also analyzed. Therefore, the aim of the present study was to analyze the C4d detection by 4 different techniques and the infiltration of B lymphocytes and plasma cells in renal allograft biopsies, correlating these findings with the capillary C4d deposition. One hundred and seven biopsies of 82 renal transplant patients were analyzed. C4d was evaluated by immunofluorescence (IF) and immunohistochemistry (IHC) techniques in frozen and paraffin sections (obtained from the same patients), whereas B-lymphocytes and plasma cells were evaluated by immunohistochemistry in paraffin sections using specific antibodies anti-B Lymphocytes (CD20) and anti-plasma cells (CD138). Regarding the C4d detection, the techniques with higher concordance rate with frozen-IF, considered the gold standard, were the frozen-IHC technique (85.4% of cases showed coincident results, r=0.72; p<0.0001) and the paraffin-IF technique (73%, r=0.59; p=0.0001), whereas the concordance rate in the paraffin-IHC technique was only 51.4% (r=0.35; p=0.03). The clinical follow up analysis demonstrate that C4d positive group was associated with a poor graft survival at 3 years post-diagnosis (67% vs 96% in C4d negative group; p=0.01). The histological analysis of mature B cells (CD20+) infiltrate showed 2 distinct patterns: scattered cells and clusters. The cluster pattern was associated with poor graft survival at 3 years (61% vs 89% in the CD20 negative group; p=0.03; and 61% vs 87% in the CD20+ scattered pattern group; p=0.03). The plasma cells infiltrate was not associated with a worse clinical transplant outcome. The analysis of the capillary C4d and B lymphocytes correlation demonstrate that the number of CD20+ and CD138+ cells was significantly higher in C4d positive cases (CD20+: 155±53 vs 26±7 cells/mm2, respectively; p=0.001; CD138+: 46±22 vs 4±1 cells/mm2, respectively; p=0.002). This study concluded that C4d capillary and mature B cells (clusters pattern) are associated with worse graft survival. Another important conclusion is a positive association between B lymphocytes (mature B cells and plasma cells) and capillary C4d, suggesting a possible role of these cells, responsible for antibodies production, in the in situ complement system activation. Finally, the frozen-IHC and paraffin-IF techniques may be considered alternative to frozen-IF technique for C4d detection

Le sepsis est un problème mondial de santé publique du fait de son incidence croissante et de sa mortalité importante. Après une première phase très inflammatoire, les patients septiques présentent une profonde immunodépression, objectivée par un risque accru d’infections secondaires et de décès. Dans ce contexte, des thérapeutiques immunostimulantes sont actuellement testées. Afin d’identifier des cibles thérapeutiques pertinentes et des biomarqueurs permettant l’individualisation des traitements, la description exhaustive des mécanismes immunosuppresseurs mis en jeu est primordiale. Les lymphocytes B ont été peu étudiés au cours du sepsis. Des données récentes ont révélé l’existence de cellules B régulatrices dans différents contextes cliniques. Ainsi, nous avons fait l’hypothèse que des lymphocytes B / plasmocytes régulateurs pouvaient être impliqués dans l’établissement de l’immunodépression induite par le sepsis.Chez les patients en choc septique, nous avons montré que les lymphocytes B présentent une perte de leur fonction de prolifération et un phénotype d’épuisement cellulaire. Ils produisent de l’IL-10, cytokine suppressive (Journal of Immunology 2018). Enfin, une plasmocytose circulante apparait, confirmée par cytométrie de masse (Scientific Reports 2018). Dans un modèle murin de sepsis, ces plasmocytes inhibent la prolifération des lymphocytes T ex vivo et expriment des marqueurs phénotypiques suggérant pour la première fois la présence de plasmocytes régulateurs dans le sepsis.L’importance clinique des plasmocytes régulateurs reste à définir (mécanismes régulateurs, liens avec les paramètres cliniques, association avec les réactivations virales…). Elle pourrait conduire à l’établissement de nouveaux biomarqueurs pour le suivi immunitaire des patients et à de nouvelles approches thérapeutiques / Sepsis in a major health problem associated with rising incidence and high mortality rate. In septic patients, an initial proinflammatory response causing life-threatening organ failures is followed by the development of a deep immunosuppression, associated with increased risk of secondary infections and death. In this context, immunostimulating therapies are currently tested. However, success of this strategy requires identification of relevant therapeutical targets and biomarkers allowing individualisation of treatments. B lymphocytes have been poorly studied in sepsis. Moreover, existence of regulatory B cells was recently revealed in several clinical contexts. Thus, we made the assumption that regulatory B lymphocytes or plasma cells could be implied in sepsis-induced immunosuppression establishment.In septic shock patients, we showed that B cells present with decreased proliferation capacity and exhausted-like profile, and are able to produce immunosuppressive IL-10 (Journal of Immunology 2018). We also observed blood plasmacytosis in patients, confirmed by mass cytometry analysis (Scientific Reports 2018). In a murine model of sepsis, the ability of plasma cells to inhibit T cell proliferation ex vivo, and their phenotypic profile suggest for the first time the existence of regulatory plasma cells in sepsis.The role of regulatory plasma cells within sepsis-induced immunosuppression has to be further explored in patients (underlying regulatory mechanisms, associations with clinical outcomes and viral reactivations…). It could highlight novel biomarkers for patients’ monitoring and innovative therapeutical approaches

La prévalence de Streptococcus suis, notamment du sérotype 2, à l’échelle mondiale pose de grands problèmes à l’industrie porcine ainsi qu’à la santé publique. La compréhension des mécanismes immunitaires permettant alors de lutter contre cette bactérie devient un atout majeur dans le développement des vaccins. S. suis dispose cependant d’un puissant arsenal pour contrer ces mécanismes. Enveloppé d’une capsule polysaccharidique (CPS), cette bactérie résiste à la phagocytose, à moins que certaines cellules soient en mesure de produire des anticorps opsonisants. Par chance, au sein de la rate, en périphérie du follicule, une zone nommée la « zone marginale » regroupe des lymphocytes B spécialisés dans la réponse aux bactéries encapsulées et aux antigènes polysaccharidiques. Ceci sans l’intervention des lymphocytes T auxiliaires, contrairement aux lymphocytes B se trouvant à l’intérieur du follicule, qui interviennent dans une réponse plus dirigée contre des antigènes protéiques. L’objectif général de ce mémoire est donc l’étude de la réponse anticorps dite « extrafolliculaire » que les LB-MZ sont suspectés d’orchestrer lors d’une infection à S. suis. En premier lieu nous avons déterminé la cinétique de différentiation en plasmocytes des LB-MZ (LB-MZ) lors de l’infection par S. suis. Puis nous avons étudié la fonctionnalité et le type d’anticorps produits par ces LB-MZ. Les présents travaux ont démontré toute l’importance des LB-MZ dans l’élimination de la bactérie ou dans le ralentissement de sa dissémination systémique durant les premiers stades de l’infection, notamment grâce à un environnement propice à la différenciation en plasmocytes. Les principaux anticorps produits à cet effet étaient de classe IgM et dirigés contre des antigènes de la CPS. Nos résultats éclairent un peu plus la voie, et permettent d’imaginer des options pour le développement des vaccins qui activeraient spécifiquement cette réponse extrafolliculaire efficace. / The worldwide prevalence of Streptococcus suis, particularly serotype 2, poses major problems for the pig industry and public health. Understanding the immune mechanisms involved in combating this bacterium is becoming a major asset in the development of vaccines. However, S. suis has a powerful arsenal at its disposal to counter these mechanisms. Enveloped in a polysaccharide capsule (CPS), this bacterium resists phagocytosis unless certain cells can produce opsonising antibodies. Fortunately, within the spleen, at the periphery of the follicle, a zone known as the "marginal zone" contains B lymphocytes specialised in responding to encapsulated bacteria and polysaccharide antigens. This is without the intervention of T helper lymphocytes, unlike the B lymphocytes inside the follicle, which are involved in a response more directed against protein antigens. The general objective of this thesis is therefore to study the so-called 'extrafollicular' antibody response that LB-MZ are thought to orchestrate during S. suis infection. We first determined the kinetics of LB-MZ differentiation into plasma cells during S. suis infection. We then studied the functionality and type of antibodies produced by these LB-MZs. This work has demonstrated the importance of LB-MZ in eliminating the bacterium or slowing down its systemic dissemination during the early stages of infection, thanks to an environment conducive to differentiation into plasma cells. The main antibodies produced for this purpose were IgM class antibodies directed against CPS antigens. Our results shed a little more light on the pathway, and allow us to imagine options for the development of vaccines that would specifically activate this effective extrafollicular response.

Das Verständnis der Immunpathogenese des systemischen Lupus erythematodes (SLE) hilft, das komplexe Krankheitsgeschehen zu verstehen und neue Therapiestrategien zu entwickeln. Die Krankheitsmanifestationen des SLE sind im Wesentlichen Folge von Autoantikörpern, Immunkomplexen und Zytokinen. Insbesondere die Neigung zu unterschiedlichen Autoantikörpern macht das Wesen der Erkrankung aus; die genauen Spezifitäten der Autoantikörper führen zu ganz unterschiedlichen Organmanifestationen. Diese Übersichtsarbeit stellt den klinisch relevanten Stand des Wissens zur SLE-Pathogenese dar – mit dem Ziel, ein für den klinischen Einsatz nützlichesModell zu etablieren, das auch hilft, die neuen Therapieansätze einzuordnen.

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