Introdução: Embora a fisiopatogenia da rinossinusite crônica (RSC) ainda não esteja totalmente elucidada, em virtude da sua heterogeneidade e multifatorialidade, existe um crescente corpo de evidências apontando que as bactérias exerçam um papel significativo na gênese ou perpetuação da inflamação crônica. Uma das possíveis formas de atuação são os biofilmes bacterianos, comumente encontrados em pacientes com RSC e que estão relacionados com má evolução clínica. Ainda, existem evidências de que algumas espécies bacterianas, especialmente o Staphylococcus aureus (S. aureus), são capazes de invadir as células epiteliais e permanecerem viáveis em seu interior. Por fim, tem se demonstrado que pacientes RSC com pólipo nasal (RSCcPN) revelam alta associação com a presença de superantígenos estafilocócicos na mucosa respiratória, responsáveis pela estimulação acentuada de respostas inflamatórias locais. Apesar de essas diferentes formas bacterianas estarem bem descritas na RSC, não se sabe ainda com clareza como elas estão associadas nesses indivíduos. Objetivos: Avaliar a associação entre a presença de biofilmes, bactérias intracelulares e superantígenos estafilocócicos em pacientes com RSC (com e sem pólipo nasal), comparados com o grupo controle. Casuística e Métodos: Avaliou-se a prevalência de biofilmes bacterianos, bactérias intracelulares e presença de superantígenos bacterianos em indivíduos com RSCcPN, sem pólipo nasal (RSCsPN) e controles, analisando a associação de distribuição de prevalência desses diferentes grupos (teste exato de Fisher, nível de significância quando p<0,05). Os biofilmes foram definidos por características morfológicas à microscopia eletrônica de varredura (MEV), as bactérias intracelulares foram analisadas por microscopia eletrônica de transmissão (MET) e hibridização fluorescente in situ (FISH) para S. aureus, e superantígenos de S. aureus A-E foram quantificados pela técnica de ELISA (Enzime Linked Imunosorbent Assay). Foram incluídos 90 indivíduos, divididos em três grupos: 1) 38 pacientes com RSCcPN, 2) 26 com RSCsPN e 3) 26 controles. Resultados: Quarenta e dois por cento dos pacientes com RSCcPN (16/38), assim como os com RSCsPN (11/26) apresentaram amostras positivas para biofilmes bacterianos, mas não observou essa positividade no grupo controle (0/26). A análise para bactérias intracelulares demonstrou a presença em 31,5% de pacientes com RSCcPN (12/38), 19,2% em RSCsPN (5/26) e 0% nos controles (0/26). No estudo por FISH, 58% dos pacientes com RSCcPN (18/31) apresentaram positividade para S. aureus intracelular, seguido de 54% nos com RSCsPN (13/24) e em nenhum caso dos 24 analisados do grupo controle. Na avaliação por ELISA, apenas um paciente com RSCcPN foi positivo para a presença de superantígenos estafilocócicos. A avaliação da associação de biofilme bacteriano na superfície mucosa à MEV com bactéria intracelular à MET e com S. aureus intracelular por FISH nos dois diferentes grupos de RSC com e sem pólipo nasal, não mostrou diferença estatisticamente significativa. Conclusão: Foi observada uma maior prevalêcia de biofilmes e bactérias intracelulares em indivíduos com RSC com ou sem pólipo nasal, comparado a Resumo controles. Não houve diferença significativa dentre os grupos de RSC, com e sem pólipo nasal para a presença de biofilmes e bactérias intracelulares. Não houve associação entre a presença de biofilme e bactéria intracelular em pacientes com RSC. Os achados do presente estudo indicam que tanto biofilmes na superfície mucosa quanto microrganismos intracelulares podem estar envolvidos na fisiopatogenia da RSC. / Introduction: Although the pathophysiology of chronic rhinosinusitis (CRS) has not yet been fully elucidated, due to its heterogeneity and multifactorial etiology, there is a growing body of evidence that bacteria play a significant role in the genesis or perpetuation of chronic inflammation. One of the possible forms of acting are bacterial biofilms, which are commonly found in patients with CRS, and are associated with poor clinical outcomes in these patients. In addition to biofilms, there are some evidence pointing out that some bacterial species, especially Staphylococcus aureus (S. aureus), are able to invade into epithelial cells and remain viable intracellulary. Finally, it has been demonstrated that patients with CRS with nasal polyps (CRSwNP) have a high association with the presence of staphylococcal superantigens in the respiratory mucosa, responsible for the stimulation of marked local inflammatory responses. Although these different bacterial forms are well described in CRS, it is still unclear how they are associated in these individuals. Objectives: To evaluate the correlation between the presence of biofilms, intracellular bacteria expression and S. aureus superantigens in CRS patients (with and without nasal polyposis) compared to a control group. Casuistic and Methods: We evaluated the prevalence of bacterial biofilms, intracellular bacteria and the presence of bacterial superantigens in individuals with CRSwNP, without nasal polyp (CRSsNP) and controls, evaluating the association of prevalence distribution of these different groups (Fisher exact test, level of significance set at p<0.05). The biofilms were defined by morphological characteristics by scanning electron microscopy, intracellular bacteria were analyzed by transmission electron microscopy and fluorescence in situ hybridization (FISH) for S. aureus, and S. aureus A-E superantigens were quantified by ELISA. Ninety individuals were included, divided into 38 patients with CRSwNP, 26 patients with CRSsNP and 26 control patients. Results: 42% of patients with CRSwNP (16/38) as well as those with CRSsNP (11/26) presented positive samples for bacterial biofilms, while none of the control patients (0/26) had positive samples. The analysis for intracellular bacteria showed the presence in 31.5% of patients with CRSwNP (12/38), 19.2% in CRSsNP (5/26) and 0% in control patients (0/26). In the FISH study, 58% of patients with CRSwNP (18/31) presented intracellular S. aureus positivity, followed by 54% in patients with CRSsNP (13/24) and in none of the 24 analyzed in the control group. In the ELISA evaluation, only one patient with CRSwNP was positive for the presence of staphylococcal superantigens. The evaluation of the association of bacterial biofilm on the mucosal surface (SEM) with intracellular bacteria (MET) and with intracellular S. aureus by FISH in the two different groups of CRS (with and without nasal polyps) did not show a statistically significant difference. Conclusion: We found a higher prevalence of biofilms and intracellular bacteria in individuals with CRS, either with and without nasal polyps. There was no significant difference between the groups of CRS, with and without nasal polyp, for the presence of biofilms or intracellular bacteria. There was no significant diference on the association of biofilms and intracellular bacteria on pacientes with CRS. Our data indicate that both biofilms on the mucosal surface and intracellular microorganisms may be involved in the pathophysiology of CRS.

Introdução: Embora a fisiopatogenia da rinossinusite crônica (RSC) ainda não esteja totalmente elucidada, em virtude da sua heterogeneidade e multifatorialidade, existe um crescente corpo de evidências apontando que as bactérias exerçam um papel significativo na gênese ou perpetuação da inflamação crônica. Uma das possíveis formas de atuação são os biofilmes bacterianos, comumente encontrados em pacientes com RSC e que estão relacionados com má evolução clínica. Ainda, existem evidências de que algumas espécies bacterianas, especialmente o Staphylococcus aureus (S. aureus), são capazes de invadir as células epiteliais e permanecerem viáveis em seu interior. Por fim, tem se demonstrado que pacientes RSC com pólipo nasal (RSCcPN) revelam alta associação com a presença de superantígenos estafilocócicos na mucosa respiratória, responsáveis pela estimulação acentuada de respostas inflamatórias locais. Apesar de essas diferentes formas bacterianas estarem bem descritas na RSC, não se sabe ainda com clareza como elas estão associadas nesses indivíduos. Objetivos: Avaliar a associação entre a presença de biofilmes, bactérias intracelulares e superantígenos estafilocócicos em pacientes com RSC (com e sem pólipo nasal), comparados com o grupo controle. Casuística e Métodos: Avaliou-se a prevalência de biofilmes bacterianos, bactérias intracelulares e presença de superantígenos bacterianos em indivíduos com RSCcPN, sem pólipo nasal (RSCsPN) e controles, analisando a associação de distribuição de prevalência desses diferentes grupos (teste exato de Fisher, nível de significância quando p<0,05). Os biofilmes foram definidos por características morfológicas à microscopia eletrônica de varredura (MEV), as bactérias intracelulares foram analisadas por microscopia eletrônica de transmissão (MET) e hibridização fluorescente in situ (FISH) para S. aureus, e superantígenos de S. aureus A-E foram quantificados pela técnica de ELISA (Enzime Linked Imunosorbent Assay). Foram incluídos 90 indivíduos, divididos em três grupos: 1) 38 pacientes com RSCcPN, 2) 26 com RSCsPN e 3) 26 controles. Resultados: Quarenta e dois por cento dos pacientes com RSCcPN (16/38), assim como os com RSCsPN (11/26) apresentaram amostras positivas para biofilmes bacterianos, mas não observou essa positividade no grupo controle (0/26). A análise para bactérias intracelulares demonstrou a presença em 31,5% de pacientes com RSCcPN (12/38), 19,2% em RSCsPN (5/26) e 0% nos controles (0/26). No estudo por FISH, 58% dos pacientes com RSCcPN (18/31) apresentaram positividade para S. aureus intracelular, seguido de 54% nos com RSCsPN (13/24) e em nenhum caso dos 24 analisados do grupo controle. Na avaliação por ELISA, apenas um paciente com RSCcPN foi positivo para a presença de superantígenos estafilocócicos. A avaliação da associação de biofilme bacteriano na superfície mucosa à MEV com bactéria intracelular à MET e com S. aureus intracelular por FISH nos dois diferentes grupos de RSC com e sem pólipo nasal, não mostrou diferença estatisticamente significativa. Conclusão: Foi observada uma maior prevalêcia de biofilmes e bactérias intracelulares em indivíduos com RSC com ou sem pólipo nasal, comparado a Resumo controles. Não houve diferença significativa dentre os grupos de RSC, com e sem pólipo nasal para a presença de biofilmes e bactérias intracelulares. Não houve associação entre a presença de biofilme e bactéria intracelular em pacientes com RSC. Os achados do presente estudo indicam que tanto biofilmes na superfície mucosa quanto microrganismos intracelulares podem estar envolvidos na fisiopatogenia da RSC. / Introduction: Although the pathophysiology of chronic rhinosinusitis (CRS) has not yet been fully elucidated, due to its heterogeneity and multifactorial etiology, there is a growing body of evidence that bacteria play a significant role in the genesis or perpetuation of chronic inflammation. One of the possible forms of acting are bacterial biofilms, which are commonly found in patients with CRS, and are associated with poor clinical outcomes in these patients. In addition to biofilms, there are some evidence pointing out that some bacterial species, especially Staphylococcus aureus (S. aureus), are able to invade into epithelial cells and remain viable intracellulary. Finally, it has been demonstrated that patients with CRS with nasal polyps (CRSwNP) have a high association with the presence of staphylococcal superantigens in the respiratory mucosa, responsible for the stimulation of marked local inflammatory responses. Although these different bacterial forms are well described in CRS, it is still unclear how they are associated in these individuals. Objectives: To evaluate the correlation between the presence of biofilms, intracellular bacteria expression and S. aureus superantigens in CRS patients (with and without nasal polyposis) compared to a control group. Casuistic and Methods: We evaluated the prevalence of bacterial biofilms, intracellular bacteria and the presence of bacterial superantigens in individuals with CRSwNP, without nasal polyp (CRSsNP) and controls, evaluating the association of prevalence distribution of these different groups (Fisher exact test, level of significance set at p<0.05). The biofilms were defined by morphological characteristics by scanning electron microscopy, intracellular bacteria were analyzed by transmission electron microscopy and fluorescence in situ hybridization (FISH) for S. aureus, and S. aureus A-E superantigens were quantified by ELISA. Ninety individuals were included, divided into 38 patients with CRSwNP, 26 patients with CRSsNP and 26 control patients. Results: 42% of patients with CRSwNP (16/38) as well as those with CRSsNP (11/26) presented positive samples for bacterial biofilms, while none of the control patients (0/26) had positive samples. The analysis for intracellular bacteria showed the presence in 31.5% of patients with CRSwNP (12/38), 19.2% in CRSsNP (5/26) and 0% in control patients (0/26). In the FISH study, 58% of patients with CRSwNP (18/31) presented intracellular S. aureus positivity, followed by 54% in patients with CRSsNP (13/24) and in none of the 24 analyzed in the control group. In the ELISA evaluation, only one patient with CRSwNP was positive for the presence of staphylococcal superantigens. The evaluation of the association of bacterial biofilm on the mucosal surface (SEM) with intracellular bacteria (MET) and with intracellular S. aureus by FISH in the two different groups of CRS (with and without nasal polyps) did not show a statistically significant difference. Conclusion: We found a higher prevalence of biofilms and intracellular bacteria in individuals with CRS, either with and without nasal polyps. There was no significant difference between the groups of CRS, with and without nasal polyp, for the presence of biofilms or intracellular bacteria. There was no significant diference on the association of biofilms and intracellular bacteria on pacientes with CRS. Our data indicate that both biofilms on the mucosal surface and intracellular microorganisms may be involved in the pathophysiology of CRS.

The nonobese diabetic mouse (NOD) is an excellent animal model to study type 1 diabetes. As with some humans, disease in the NOD mouse is effected by a combination of genetic and environmental factors. At least 20 insulin dependent diabetes (Idd) susceptibility loci have been identified so far, both in humans and in the NOD mouse. In this thesis, the overall aim has been to understand the genetic basis of diabetes in the NOD mouse by assessing immunogically-related phenotypes. As lymphocytes are the main players in the onset and progression to overt diabetes, we searched for physiological abnormalities in T and B cells, which could contribute to the breakdown of tolerance to pancreatic antigens. Ultimately, we postulate that abnormalities in the T or B cell compartments, under the genetic control of a previously defined diabetes susceptibility regions (Idds) could unravel the biological mechanisms underlying diabetes susceptibility and facilitate the identification of etiological polymorphisms involved in the disease. NOD T cells are defective in upregulating CTLA-4 upon in vitro activation. Previous studies have shown that this defect is, at least in part, controlled by gene(s) in the Idd5 region on chromosome 1. In paper I, we provide evidence that defective upregulation of the CTLA-4 in NOD T cells is not controlled by the Idd5.1 and Idd5.2 regions, but rather by genes linked to the telomeric region of chromosome 1 and to the Idd3 locus, for which the prime candidate gene is Il-2. Interestingly, we could restore some of the defective CTLA-4 expression in NOD T cells by the addition of exogenous IL-2 during T cell activation in vitro. In paper II, we show that NOD thymocytes are resistant to superantigen-mediated negative selection and that this trait is under control of the Idd5.2 region. Interestingly, it appears to operate in a T cell non-autonomous manner. In paper III, we describe a competitive advantage of NOD thymocytes to mature when they co-develop with B6 thymocytes in embryo aggregation chimeras. These results imply that defects exist in the positive/negative selection mechanisms in the NOD thymus. Apart from T cells, B cells also play an important role in the initiation of diabetes in NOD mice, probably as antigen presenting cells. In paper IV, we report that the genetic basis of an enlarged marginal zone (MZ) B cell population observed in the NOD mice is linked to the Idd9/Idd11 region. Together, these findings contribute to the dissection of the molecular mechanisms underlying diabetes pathogenesis, and shed light on the contribution of central and peripheral tolerance mechanisms to this process.

Les vaccins à base de cellules dendritiques (DCs) constituent une avenue très populaire en immunothérapie du cancer. Alors que ces cellules peuvent présenter des peptides exogènes ajoutés au milieu, l’efficacité de chargement de ces peptides au le complexe majeur d'histocompatibilité (CMH) de classe II est limitée. En effet, la majorité des molécules du CMH II à la surface des DCs sont très stable et l’échange de peptide spontané est minime. Confinée aux vésicules endosomales, HLA-DM (DM) retire les peptides des molécules du CMH II en plus de leur accorder une conformation réceptive au chargement de peptides. Il est possible, cependant, de muter le signal de rétention de DM de façon à ce que la protéine s’accumule en surface. Nous avons émis l’hypothèse que ce mutant de DM (DMY) sera aussi fonctionnel à la surface que dans la voie endosomale et qu’il favorisera le chargement de peptides exogènes aux DCs. Nous avons utilisé un vecteur adénoviral pour exprimer DMY dans des DCs et avons montrer que la molécule augmente le chargement de peptides. L’augmentation du chargement peptidique par DMY est autant qualitatif que quantitatif. DMY améliore la réponse T auxiliaire (Th) du coté Th1, ce qui favorise l’immunité anti-cancer. Du côté qualitatif, le chargement de peptides résulte en des complexes peptide-CMHII (pCMH) d’une conformation supérieure (conformère). Ce conformère (Type A) est le préféré pour la vaccination et DMY édite avec succès les complexes pCMH à la surface en éliminant ceux de type B, lesquels sont indésirables. La fonction de DM est régulée par HLA-DO (DO). Ce dernier inhibe l’habilité de DM à échanger le peptide CLIP (peptide dérivée de la chaîne invariante) en fonction du pH, donc dans les endosomes tardifs. Mes résultats indiquent que la surexpression de DO influence la présentation des superantigènes (SAgs) dépendants de la nature du peptide. Il est probable que DO améliore indirectement la liaison de ces SAgs au pCMH dû à l’accumulation de complexe CLIP-CMH, d’autant plus qu’il neutralise la polarisation Th2 normalement observée par CLIP. Ensemble, ces résultats indiquent que DMY est un outil intéressant pour renforcer le chargement de peptides exogènes sur les DCs et ainsi générer des vaccins efficaces. Un effet inattendu de DO sur la présentation de certains SAgs a aussi été observé. Davantage de recherche est nécessaire afin de résoudre comment DMY et DO influence la polarisation des lymphocytes T auxiliaires. Cela conduira à une meilleure compréhension de la présentation antigénique et de son étroite collaboration avec le système immunitaire. / Dendritic cell peptide-based vaccines are the most common immunotherapy approach in cancer therapy. While, in principle, dendritic cells (DCs) could be loaded efficiently by exogenously added tumor peptides, their loading efficacy is severely reduced due to low number of peptide-receptive MHC II on cell surface. Most surface MHC II molecules are either occupied by endogenous peptides or are inactive due to a conformation that is not receptive for free peptides. In MHC II antigen presentation pathway, HLA-DM (DM) in acidic endosomal vesicles removes the self-peptides and grants a peptide receptive conformation to MHC II. Mutating of an intracellular sorting motif in DM, renders its accumulation on cell surface. We hypothesized that the mutant DM (DMY) is functional on cell surface and can generate peptide receptive MHC II on surface of DCs for exogenous peptide loading. By using an adenoviral vector that expresses DMY, we found that DMY is functional on surface of DCs. DMY supplied peptide receptive MHC II on surface of DCs and improved exogenous peptide loading. The improvement of peptide loading by DMY is both quantitative and qualitative. DMY improves helper T cell (Th) response in Th1 direction that favors anti-cancer immunity. The qualitative improvement of peptide loading extends to loading of superior conformational isomer (conformer) of peptide-MHC complexes. This superior conformer (type A) is the favourite type for vaccination approaches and DMY successfully edits peptide-MHC conformers on cell surface level by eliminating undesirable one (type B). Function of DM is regulated by HLA-DO (DO) and it is well accepted that in acidic pH of late endosomes, DO inhibits function of DM by preventing removal of class II associated invariant chain peptide (CLIP) from peptide binding groove of MHC II. My results indicate that DO overexpression, changes binding of peptide-dependent superantigens to MHC II molecules. Superantigens (SAgs) are small microbial proteins that bind out side peptide binding groove of MHC II. DO probably enhances binding of peptide-dependent SAgs by forcing the accumulation of CLIP on the cell surface of antigen presenting cells. DO also neutralizes Th2 polarization by CLIP. Collectively, these results indicate that DMY is a valuable tool for improvement of exogenous peptide loading in DCs vaccines. An unnoticed effect of DO on SAgs binding was also recognized. Further investigations are needed to clarify the mechanisms by which, DMY and DO influence Th polarization. This would provide a better understanding of antigen presentation pathway and its interaction with immune system.

No description available.