The cytokines GM-CSF, IL-3 and IL-5 are central regulators of haemopoietic cell functions and are pivotal in the regulation of haemopoiesis and inflammatory responses of myeloid cells. In particular, these cytokines have been shown to perform essential functions in host defence against foreign pathogens through their ability to regulate innate immune responses in myeloid cells. As key regulators of such important processes, these cytokines play an important role in human inflammatory pathologies such as rheumatoid arthritis, asthma, multiple sclerosis and psoriasis as well as a number of leukemias such as JML and CMML. GM-CSF, IL-3 and IL-5 signal through receptors containing α subunits specific to each cytokine and a common β subunit (βc). Cytokine stimulation leads to tyrosine phosphorylation of the βc and promotes specific responses such as proliferation, survival and activation of haemopoietic cells. Mouse knockout studies identified a key function of these cytokines in the activation of effector functions of myeloid cells, including production of reactive oxygen species (ROS) and phagocytosis. These earlier studies provide a link between cytokine signalling and inflammation, but the molecular mechanisms by which βc activation regulates effector cell functions, and the receptor motifs involved, are unknown. The aim of this thesis was to address two broad questions with regard to βc signalling: (1) Does βc regulate specific cellular responses by phosphotyrosine-independent mechanisms? (2) What are the molecular mechanisms by which βc initiates signalling to promote specific biological responses such as activation of effector cell functions? To address the first question, we have focussed on Serine 585, a potential 14-3-3 binding site which lies in the cytoplasmic potion of huβc. Out results show that the mutation huβc S585G disrupted the interaction of 14-3-3ζ with βc, whilst not affecting receptor tyrosine phosphorylation. Both mouse and human βc were shown to interact with 14-3-3 proteins, indicating that this interaction is conserved between these species. Significantly, a huβc S585G mutant was unable to promote haemopoietic cell survival in response to IL-3. These results identify a new mechanism by which cytokine receptors are able to couple to downstream signalling pathways that regulate cell survival. An approach was developed and optimised to analyse specific GM-CSF-mediated responses in monocytes/macrophages expressing wildtype or mutant huβc, (including huβc S585G that was defective in regulating survival). Bone marrow-derived muβc -/-;muβIL-3 -/- monocytes/macrophages were retrovirally transduced with constructs expressing wildtype or mutant huβc, along with huGMRα, then purified by FACS. Two assays were established to measure effector functions in the transduced monocyte/macrophages; (1) a flow cytometry assay for ROS production, and (2) an assay for phagocytosis. The capacity for GM-CSF to prime (i.e. enhance effector functions) ROS production and phagocytosis was investigated in huGMRα-transduced monocytes/macrophages. Our results have identified two key residues in the cytoplasmic domain of βc subunit: Tyrosine 577 (required for huβc interaction with the adaptor protein Shc) and serine 585 (required for 14-3-3 association), that are essential for the ability of GM-CSF to regulate key effector functions in monocytes/macrophages. These novel findings are significant in that they establish a molecular link between the GM-CSF/IL-3/IL-5 receptor and the regulation of both haemopoietic cell survival and inflammatory responses, and therefore have important implications in our understanding of inflammatory diseases such as rheumatoid arthritis and asthma. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1317007 / Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 2008

Durch Immunregulation und -modulation sorgt das Immunsystem dafür, daß von außen in den Organismus gelangende Agentien nicht zu dauerhaften Schäden führen. Wesentliche Funktionen des Immunsystems stützen sich dabei auf die zelluläre Immunität. Gerät dieses komplizierte Regelwerk aus dem Gleichgewicht, können schwere Erkrankungen autoimmuner oder atopischer Genese resultieren. Der erste Teil der Arbeit befaßt sich mit zwei Aspekten der zellulären Immunantwort. Allergische Immunantworten sind durch die Typ 2 T Zellantwort charakterisiert. Für die Induktion einer Typ 2 Antwort wird Interleukin-4 benötigt, dessen Herkunft nicht geklärt ist. NK1.1 positive T Zellen als Quelle des initialen IL-4 konnten durch in vivo und in vitro Messung von allergie-spezifischen Parametern ausgeschlossen werden. Der MHC-Komplex präsentiert T Zellen Antigene in Form von Peptiden. Pathologische T Zellantworten können durch fortwährende Antigenpräsentation unterhalten werden. Durch Untersuchungen zur molekularen Charakteristik der MHC - Peptid Interaktion ließen sich Bindungsmotive so verfeinern, daß Peptide mit sehr starker Bindung an den MHC ohne gleichzeitige Erkennungssequenz für den T Zellrezeptor entwickelt werden konnten. Peptide dieser Art könnten zur Blockierung einer pathologischen T Zellantwort genutzt werden. Für viele immunologische Erkrankungen ist die Beteiligung genetischer Faktoren beschrieben worden. Der zweite Teil der Arbeit befaßt sich mit der Bedeutung genetischer Disposition bei Allergien im Mausmodell. Homozygote Inzuchtstämme konnten als High- und Low-Responder für den Phänotyp "allergische Soforttypreaktion der Haut" gegenüber Birkenpollenextrakt definiert werden. Die Phänotypisierung der F1 Generation wies auf dominante Vererbung hin, die informative Rückkreuzung auf die Beteiligung von mindestens zwei Genen für die Ausprägung des Merkmals. Wie die Analyse MHC congener Mäuse zeigte, entspricht einer dieser Loci dem MHC Komplex. Durch eine genomweite Kartierung mit Mikrosatelliten wurde als weiterer Kandidat der IL-5 Rezeptor identifiziert. Die detaillierte Analyse des Gens in High-und Low-Respondern weist auf eine Anzahl funktionell bedeutsamer Polymorphismen hin. Dabei imponiert das Low-Responder Allel als Suszeptibilitätsallel. Die Unterschiede haben Auswirkung auf Transkription/Translation und Spleißvorgänge, die zu quantitativer Differenz der Genprodukte führt. Die Daten weisen damit auf einen regulatorischen Mechanismus hin, da die Proteinstruktur des Rezeptors bei High- und Low-Respondern identisch ist. / Immune deviations can lead to serious autoimmune or atopic disorders. Two possible candidates for such pathological immune responses have been investigated: (i) the MHC class I allele HLA-B27, which is strongly linked to ankylosing spondylitis and reactive arthritis. Its presentation of potentially pathogenic peptides and therapeutical modifications of peptidic ligands have been studied. (ii) Interleukin-4 (IL-4), which is the key player in the induction and maintenance of allergic immune responses. A subpopulation of natural killer (NK1.1) cells have been discussed as a source of initial IL-4. Through experiments with NK1.1 deficient mice we could demonstrate, that such immune resonses are not dependent on the presence of NK1.1 cells. Both, autoimmunity and atopy belong to the large group of multifactorial diseases, i. e. genetic and environmental factors influence the expression of the various phenotypes. To dissect the genetics of allergic diseases systematically, a mouse model of immmediate cutaneous hypersensitivity (ICHS) upon birch pollen sensitization has been etablished. Phenotyping of the F1 progeny of high- and low-responder mice revealed ICHS as a dominant trait with incomplete penetrance. Mice from a backcross to the low-responder strain did split into the three classes of high-, intermediate and low-response. Genotyping of these mice revealed a strong candidate gene on chromosome 6: the interleukin-5 receptor (IL-5R). Analysis of the IL-5R gene resulted in the detection of genetic variance of the high- and low-responder allele in non-coding regions with functional relevance. Additional regulatory variance is implicated through differential alternative splicing of the three receptor isoforms. A second gene cluster contributes to the expression of the allergic phenotype: MHC class II alleles, as has been demonstrated for other immunologic disorders in mice and humans.

A tuberculose é uma doença crônica granulomatosa caracterizada por um déficit de imunidade antígeno específica do hospedeiro, cuja resposta imune é ativamente regulada por citocinas. No Brasil há mais de 50 milhões de habitantes infectados pelo Mycobacterium tuberculosis. O objetivo foi avaliar a linfoproliferação e a produção de citocinas por células mononucleares do sangue periférico (PBMC) estimuladas por quatro diferentes antígenos do M. tuberculosis, um complexo, o antígeno sonicado, e três purificados, ESAT-6, antígeno 85B e antígeno HBHA, eventuais candidatos à vacina anti-tuberculose. Para avaliação da produção de IFN-g e IL-10 foram utilizados dois métodos: Elispot e Elisa à partir de sobrenadante de cultura de PBMC. Para essas avaliações, os pacientes com tuberculose ativa (TB-A) foram comparados a dois subgrupos de indivíduos controles. O primeiro subgrupo foi constituído por indivíduos saudáveis PPD+ e o segundo por indivíduos curados de um episódio de tuberculose (TB-C). Nossos resultados de linfoproliferação e de Elisa revelaram diminuição da resposta linfoproliferativa e da produção de IFN-g dos pacientes em comparação com os indivíduos PPD+, enquanto os indivíduos TB-C apresentaram em geral resultados intermediários. Observou-se também que as respostas à PHA não diferiam significativamente entre os grupos, ressaltando a natureza antígeno específica da hiporreatividade na tuberculose. Adicionalmente, verificamos maior reatividade ao antígeno complexo, sonicado, que aos antígenos purificados, e entre estes, a reatividade foi maior para ESAT-6 e 85B que para HBHA, A resposta ao HBHA pode ter sido eventualmente subestimada por razões técnicas, como utilização de dose sub-ótima ou perda da atividade biológica. Em relação ao Elispot para IFN-g, não pudemos observar diferenças entre os grupos, tanto quando se considerou o número total de spots, como quando se contou apenas spots com diâmetro > 65 mm, apresentando portanto uma sensibilidade aparentemente menor comparado aos outros 2 métodos. A comparação entre os métodos revelou pouca correlação entre seus resultados, que pode ser eventualmente explicado pela diferente contribuição das populações celulares (T CD4+ e T CD8+) para cada uma das provas munológicas. Finalmente, a análise da produção de IL-10 medida por Elisa no sobrenadante de cultura e por spots de IL10, também não revelou diferenças entre os grupos. Convém notar que o Elisa detectou baixas concentrações de IL-10 nos sobrenadantes, porém o Elispot demonstrou número elevado de spots e boa correlação entre as resposta aos antígenos. Em conclusão, nossos resultados sugerem que métodos \'clássicos\', e já estabelecidos, como linfoproliferação e Elisa, persistem válidos para se avaliar a imunidade celular, e que em nossas condições laboratoriais, a técnica de Elispot não representou, até o momento, uma melhora na qualidade da avaliação imunológica. / Tuberculosis is a chronic granulomatous disease characterized by a deficit of the antigen-specific immunity of the host, whose immune response is actively regulated by cytokines. In Brazil there are 50 million people infected with Mycobacterium tuberculosis. The objective of the present work was to evaluate the lymphoproliferative response e the IFN-g response by peripheral blood mononuclear cells (PBMC) indiced with 4 different antigens isolated from Mycobacterium tuberculosis: a complex, crude, the sonicate antigen, and 3 other, purified ones, Esat-6, 85B, and HBHA, the last 3 eventual candidates to the design of a vaccine against tuberculosis. We used 2 methods to evaluate the IFN-g and IL-10 productions, namely Elispot and Elisa of supernatant of PBMC cultures. We studied a group of active tuberculosis patients (TB-A), and compared them with controls individuals comprising 2 groups, one made of healthy PPD+ individuals and the second one of individuals who have been cured from an episode of tuberculosis in the past (TB-C). Our results of lymphoproliferation and Elisa revealed decrease in the lymphoproliferative and IFN-g responses by patients\' PBMC as compared to the PPD+ group, with the TB-C group in general presenting intermediate results. We also observed that the responses to the mitogen PHA were not statisically different among the groups, denoting the antigen-specific nature of the immune deficit in tuberculosis. In addition, we verified that stronger reactivity to the complex antigen than with the purified antigens, and, among the latter, the reactivity was stronger with Esat-6 and 85B as compared to HBHA, Reactivity to HBHA may have been understimated due to technical reasons, such as loss of .the biological activity of the molecule or use of a sub-optimal dose. By using the Elispot for IFN-g we were not able to detect differences among the groups, even when we counted all spots formed or spots with more than > 65 mm in diameter. Thus our Elispot for IFN-g apparently showed lower sensitivity than the other 2 methods. Furthermore, comparisons between the methods revealed low correlation between their results, a finding that may be explained by the differning contribution of different subpopulations (T CD4+ and T CD8+) to each of the results. Finally, analysis of the production of IL-10 as measured by Elisa in the culture supernatants as well as by Elispot revealed no differences among the groups. It is noteworthy that the levels of IL-10 detected by Elisa were low, but the Elispot revealed high number of spots and a good correlation between the antigen responses. In conclusion, we may say that our well standardized \'classical\' methods Elisa and lymphoproliferation persist useful to evaluate cellular immunity responses, and that the Elispot technique, up to now and in our laboratorial conditions, did not represent an improvement in the quality of the immunological evaluation.

A tuberculose é uma doença crônica granulomatosa caracterizada por um déficit de imunidade antígeno específica do hospedeiro, cuja resposta imune é ativamente regulada por citocinas. No Brasil há mais de 50 milhões de habitantes infectados pelo Mycobacterium tuberculosis. O objetivo foi avaliar a linfoproliferação e a produção de citocinas por células mononucleares do sangue periférico (PBMC) estimuladas por quatro diferentes antígenos do M. tuberculosis, um complexo, o antígeno sonicado, e três purificados, ESAT-6, antígeno 85B e antígeno HBHA, eventuais candidatos à vacina anti-tuberculose. Para avaliação da produção de IFN-g e IL-10 foram utilizados dois métodos: Elispot e Elisa à partir de sobrenadante de cultura de PBMC. Para essas avaliações, os pacientes com tuberculose ativa (TB-A) foram comparados a dois subgrupos de indivíduos controles. O primeiro subgrupo foi constituído por indivíduos saudáveis PPD+ e o segundo por indivíduos curados de um episódio de tuberculose (TB-C). Nossos resultados de linfoproliferação e de Elisa revelaram diminuição da resposta linfoproliferativa e da produção de IFN-g dos pacientes em comparação com os indivíduos PPD+, enquanto os indivíduos TB-C apresentaram em geral resultados intermediários. Observou-se também que as respostas à PHA não diferiam significativamente entre os grupos, ressaltando a natureza antígeno específica da hiporreatividade na tuberculose. Adicionalmente, verificamos maior reatividade ao antígeno complexo, sonicado, que aos antígenos purificados, e entre estes, a reatividade foi maior para ESAT-6 e 85B que para HBHA, A resposta ao HBHA pode ter sido eventualmente subestimada por razões técnicas, como utilização de dose sub-ótima ou perda da atividade biológica. Em relação ao Elispot para IFN-g, não pudemos observar diferenças entre os grupos, tanto quando se considerou o número total de spots, como quando se contou apenas spots com diâmetro > 65 mm, apresentando portanto uma sensibilidade aparentemente menor comparado aos outros 2 métodos. A comparação entre os métodos revelou pouca correlação entre seus resultados, que pode ser eventualmente explicado pela diferente contribuição das populações celulares (T CD4+ e T CD8+) para cada uma das provas munológicas. Finalmente, a análise da produção de IL-10 medida por Elisa no sobrenadante de cultura e por spots de IL10, também não revelou diferenças entre os grupos. Convém notar que o Elisa detectou baixas concentrações de IL-10 nos sobrenadantes, porém o Elispot demonstrou número elevado de spots e boa correlação entre as resposta aos antígenos. Em conclusão, nossos resultados sugerem que métodos \'clássicos\', e já estabelecidos, como linfoproliferação e Elisa, persistem válidos para se avaliar a imunidade celular, e que em nossas condições laboratoriais, a técnica de Elispot não representou, até o momento, uma melhora na qualidade da avaliação imunológica. / Tuberculosis is a chronic granulomatous disease characterized by a deficit of the antigen-specific immunity of the host, whose immune response is actively regulated by cytokines. In Brazil there are 50 million people infected with Mycobacterium tuberculosis. The objective of the present work was to evaluate the lymphoproliferative response e the IFN-g response by peripheral blood mononuclear cells (PBMC) indiced with 4 different antigens isolated from Mycobacterium tuberculosis: a complex, crude, the sonicate antigen, and 3 other, purified ones, Esat-6, 85B, and HBHA, the last 3 eventual candidates to the design of a vaccine against tuberculosis. We used 2 methods to evaluate the IFN-g and IL-10 productions, namely Elispot and Elisa of supernatant of PBMC cultures. We studied a group of active tuberculosis patients (TB-A), and compared them with controls individuals comprising 2 groups, one made of healthy PPD+ individuals and the second one of individuals who have been cured from an episode of tuberculosis in the past (TB-C). Our results of lymphoproliferation and Elisa revealed decrease in the lymphoproliferative and IFN-g responses by patients\' PBMC as compared to the PPD+ group, with the TB-C group in general presenting intermediate results. We also observed that the responses to the mitogen PHA were not statisically different among the groups, denoting the antigen-specific nature of the immune deficit in tuberculosis. In addition, we verified that stronger reactivity to the complex antigen than with the purified antigens, and, among the latter, the reactivity was stronger with Esat-6 and 85B as compared to HBHA, Reactivity to HBHA may have been understimated due to technical reasons, such as loss of .the biological activity of the molecule or use of a sub-optimal dose. By using the Elispot for IFN-g we were not able to detect differences among the groups, even when we counted all spots formed or spots with more than > 65 mm in diameter. Thus our Elispot for IFN-g apparently showed lower sensitivity than the other 2 methods. Furthermore, comparisons between the methods revealed low correlation between their results, a finding that may be explained by the differning contribution of different subpopulations (T CD4+ and T CD8+) to each of the results. Finally, analysis of the production of IL-10 as measured by Elisa in the culture supernatants as well as by Elispot revealed no differences among the groups. It is noteworthy that the levels of IL-10 detected by Elisa were low, but the Elispot revealed high number of spots and a good correlation between the antigen responses. In conclusion, we may say that our well standardized \'classical\' methods Elisa and lymphoproliferation persist useful to evaluate cellular immunity responses, and that the Elispot technique, up to now and in our laboratorial conditions, did not represent an improvement in the quality of the immunological evaluation.

Introdução: A rinite alérgica (RA) e a rinossinusite crônica (RSC) são doenças inflamatórias nasais com prevalência alta e crescente. Estima-se que 15,5% dos norte-americanos tenham RSC, e estudo recente encontrou prevalência de 5,51% na cidade de São Paulo, enquanto a RA acomete entre 10 e 20% da população mundial. Apesar de terem mecanismos fisiopatológicos distintos, em ambas, há recrutamento de células de defesa, principalmente linfócitos T, e produção de citocinas inflamatórias. Esses mediadores variam não apenas entre as doenças, mas também entre as populações acometidas, e seu conhecimento é importante para o diagnóstico correto e direcionamento da terapia escolhida. Objetivos: Mapear os mediadores inflamatórios presentes no lavado nasal e no condensado do ar exalado na RSC e na RA, avaliando possíveis biomarcadores da doença, e analisar o endotipo inflamatório dos pacientes estudados. Além disso, avaliar a qualidade de vida, o nível de obstrução nasal, a qualidade do sono dos pacientes afetados, o transporte mucociliar e coletar material para análise de pH, contagem de células totais e seu diferencial. Pacientes e métodos: Estudo exploratório prospectivo em corte transversal, sendo os pacientes divididos em quatro grupos: 1) Grupocontrole com pacientes sem queixas; 2) Pacientes com RA com prick test positivo; 3) Pacientes com RSC com polipose; e 4) Pacientes com RSC sem polipose. Todos os pacientes responderam a quatro questionários: 20-Item Sino-Nasal Outcome Test (SNOT-20p), Nasal Obstruction Symptom Evaluation (NOSE), o Índice de Qualidade do Sono de Pittsburgh (Pittsburgh Sleep Quality Index - PSQI-BR) e o questionário para triagem e diagnóstico da asma da European Community Respiratory Health Survey (ECRHS). Foi realizado exame físico, incluindo endoscopia nasal (escore de Lund- Kennedy modificado) e, nos pacientes com RSC, avaliação da tomografia computadorizada (TC) de face (escore de Lund-Mackay). Foi também avaliado o transporte mucociliar por meio do teste da sacarina. Coletou-se o condensado do ar exalado para análise do pH e lavado nasal para avaliação do pH, da presença de citocinas e da celularidade (total e diferencial). Foi avaliada a presença de IL-4, IL-5, IL-8, IL-17A, IL-22, TNF-Alfa e IFN-Gama no lavado nasal e IL-5, IL-17A, IL-22 e IFN-Gama no condensado do ar exalado. Resultados: Os pacientes com RSC apresentaram escores significativamente piores nos questionários de obstrução nasal (NOSE, p < 0,01) e qualidade de vida (SNOT-20p, p < 0,01) quando comparados aos controles, e tanto os pacientes com RSC quanto com RA apresentaram pior qualidade do sono (PSQI-BR, p < 0,01). O escore de extensão de Lund- Mackay foi mais elevado nos pacientes com RSC com polipose (p < 0,02). O teste da sacarina apresentou tempo mais prolongado no grupo RSC com polipose (p < 0,01). O pH do condensado do ar exalado não diferiu entre os grupos. O grupo RSC com polipose apresentou tanto diferença do pH (p < 0,01) quanto da contagem de células totais do lavado nasal (p < 0,01) quando comparado ao grupo-controle, porém sem diferença na contagem diferencial. IFN? do condensado foi mais elevado no grupo RA em comparação ao grupo C (p=0,05), enquanto IL-5 foi mais alto no grupo RSC com polipose quando comparado ao grupo RSC sem polipose (p=0,02). Os pacientes foram, então, divididos em endotipos, segundo os grupos descritos por Tomassen et al., sendo que endotipos são os subtipos da doença definidos funcionalmente e patologicamente por mecanismos moleculares distintos. Conclusão: Pacientes com RSC apresentaram escores piores nos questionários de qualidade de vida (SNOT-20p e NOSE), o que, em parte, poderia ser atribuído à pior qualidade do sono apresentada tanto por estes pacientes quanto pelos portadores de RA. O teste da sacarina evidenciou pior transporte mucociliar nos pacientes com RSC com polipose. Não houve diferença do pH do condensado do ar exalado, sugerindo que, apesar de interessante na avaliação das vias aéreas inferiores, este pode não ser um bom teste para análise das vias aéreas superiores. Foram encontradas alterações significantes tanto do pH quanto da contagem de células totais do lavado nasal do grupo com RSC com polipose, sem, no entanto, haver diferença na contagem diferencial. Dos 17 pacientes com RSC com perfil completo de citocinas, 12 se encaixam em endotipos já descritos, sendo que, dos cinco restantes, dois apresentam o mesmo perfil, podendo indicar um novo subgrupo / Introduction: Allergic rhinitis (AR) and chronic rhinosinusitis (CRS) are nasal inflammatory diseases with high and increasing prevalence. It is estimated that 15.5% of Americans have CRS, and a recent study found a prevalence of 5.51% in the city of São Paulo, while AR affects between 10 and 20% of the world population. Although they have distinct pathophysiological mechanisms, in both there is recruitment of defense cells, mainly T lymphocytes, and production of inflammatory cytokines. These mediators vary not only between diseases but also among affected populations, and their knowledge is important for the correct diagnosis and targeting of the therapy chosen. Objectives: To map the inflammatory mediators present in the nasal wash and the exhaled breath condensate in the CRS and AR, evaluating possible biomarkers of this diseases, and to analyze the inflammatory endotype of the patients studied. In addition, assess quality of life, level of nasal obstruction, sleep quality of affected patients, mucociliary transport and collect samples for pH analysis, total cell count and its differential. Patients and Methods: Prospective cross-sectional exploratory study, divided into four groups: 1) Control group with patients without complaints 2) Patients with AR with prick test positive 3) Patients with CRS with polyps 4) Patients with CRS without polyps. All patients responded to four questionnaires: 20-Item Sino-Nasal Outcome Test (SNOT-20p), Nasal Obstruction Symptom Evaluation (NOSE), Pittsburgh Sleep Quality Index (PSQI-BR) and European Community Respiratory Health Survey (ECRHS) questionnaire for screening and diagnosis of asthma. A physical examination was performed, including nasal endoscopy (modified Lund-Kennedy score) and, in patients with CRS, a computerized tomography (Lund-Mackay score) evaluation. The mucociliary transport was also evaluated through the saccharin test. The exhaled breath condensate was collected for pH analysis and the nasal wash for evaluation of pH, cytokines and cellularity (total and differential). The presence of IL-4, IL-5, IL-17, IL-17A, IL-22, TNF-Alfa and IFN-Gama in the nasal wash and IL-5, IL-17A, IL-22 and IFN-Gama in the exhaled breath condensate. Results: Patients with CRS had significantly worse scores in the nasal obstruction questionnaire (NOSE, p < 0.01) and quality of life questionnaire (SNOT-20p, p < 0.01) when compared to controls, and both patients with CRS and AR presented worse sleep quality (PSQI-BR, p < 0.01). The Lund-Mackay extension score was higher in patients with CRS with polyps (p < 0.02). The saccharin test showed longer time in the CSR group with polyps (p < 0.01). The pH of the exhaled breath condensate did not differ between groups. The CRS with polyps group presented both pH difference (p < 0.01) and higher total nasal wash cell count (p < 0.01) when compared to the control group, but with no difference in the differential count (p = 0.05), while IL-5 was higher in the CRS group with polyps when compared to the RSC without polyps group (p = 0.02). Patients were then divided into endotypes according to the groups described by Tomassen et al, Endotypes are the subtypes of the disease defined functionally and pathologically by distinct molecular mechanisms. Conclusions: Patients with CRS presented worse scores on quality of life questionnaires (SNOT-20p and NOSE), which could be attributed in part to the poorer quality of sleep presented by both patients with CRS and AR. The saccharin test evidenced worse mucociliary transport in patients with CRS with polyps when compared with control group. There was no difference in the pH of the exhaled breath condensate, suggesting that, although interesting in the evaluation of the lower airways, this may not be a good test for analyzing the upper airways. Significant alterations were found in both pH and total nasal wash cell count in the CSR group with polyposis, but there was no difference in the differential count. Of the 17 patients with CRS with complete cytokine profile, 12 fit into already described endotypes, and of the remaining five, two have the same profile, which may indicate a new subgroup

Introdução: A rinite alérgica (RA) e a rinossinusite crônica (RSC) são doenças inflamatórias nasais com prevalência alta e crescente. Estima-se que 15,5% dos norte-americanos tenham RSC, e estudo recente encontrou prevalência de 5,51% na cidade de São Paulo, enquanto a RA acomete entre 10 e 20% da população mundial. Apesar de terem mecanismos fisiopatológicos distintos, em ambas, há recrutamento de células de defesa, principalmente linfócitos T, e produção de citocinas inflamatórias. Esses mediadores variam não apenas entre as doenças, mas também entre as populações acometidas, e seu conhecimento é importante para o diagnóstico correto e direcionamento da terapia escolhida. Objetivos: Mapear os mediadores inflamatórios presentes no lavado nasal e no condensado do ar exalado na RSC e na RA, avaliando possíveis biomarcadores da doença, e analisar o endotipo inflamatório dos pacientes estudados. Além disso, avaliar a qualidade de vida, o nível de obstrução nasal, a qualidade do sono dos pacientes afetados, o transporte mucociliar e coletar material para análise de pH, contagem de células totais e seu diferencial. Pacientes e métodos: Estudo exploratório prospectivo em corte transversal, sendo os pacientes divididos em quatro grupos: 1) Grupocontrole com pacientes sem queixas; 2) Pacientes com RA com prick test positivo; 3) Pacientes com RSC com polipose; e 4) Pacientes com RSC sem polipose. Todos os pacientes responderam a quatro questionários: 20-Item Sino-Nasal Outcome Test (SNOT-20p), Nasal Obstruction Symptom Evaluation (NOSE), o Índice de Qualidade do Sono de Pittsburgh (Pittsburgh Sleep Quality Index - PSQI-BR) e o questionário para triagem e diagnóstico da asma da European Community Respiratory Health Survey (ECRHS). Foi realizado exame físico, incluindo endoscopia nasal (escore de Lund- Kennedy modificado) e, nos pacientes com RSC, avaliação da tomografia computadorizada (TC) de face (escore de Lund-Mackay). Foi também avaliado o transporte mucociliar por meio do teste da sacarina. Coletou-se o condensado do ar exalado para análise do pH e lavado nasal para avaliação do pH, da presença de citocinas e da celularidade (total e diferencial). Foi avaliada a presença de IL-4, IL-5, IL-8, IL-17A, IL-22, TNF-Alfa e IFN-Gama no lavado nasal e IL-5, IL-17A, IL-22 e IFN-Gama no condensado do ar exalado. Resultados: Os pacientes com RSC apresentaram escores significativamente piores nos questionários de obstrução nasal (NOSE, p < 0,01) e qualidade de vida (SNOT-20p, p < 0,01) quando comparados aos controles, e tanto os pacientes com RSC quanto com RA apresentaram pior qualidade do sono (PSQI-BR, p < 0,01). O escore de extensão de Lund- Mackay foi mais elevado nos pacientes com RSC com polipose (p < 0,02). O teste da sacarina apresentou tempo mais prolongado no grupo RSC com polipose (p < 0,01). O pH do condensado do ar exalado não diferiu entre os grupos. O grupo RSC com polipose apresentou tanto diferença do pH (p < 0,01) quanto da contagem de células totais do lavado nasal (p < 0,01) quando comparado ao grupo-controle, porém sem diferença na contagem diferencial. IFN? do condensado foi mais elevado no grupo RA em comparação ao grupo C (p=0,05), enquanto IL-5 foi mais alto no grupo RSC com polipose quando comparado ao grupo RSC sem polipose (p=0,02). Os pacientes foram, então, divididos em endotipos, segundo os grupos descritos por Tomassen et al., sendo que endotipos são os subtipos da doença definidos funcionalmente e patologicamente por mecanismos moleculares distintos. Conclusão: Pacientes com RSC apresentaram escores piores nos questionários de qualidade de vida (SNOT-20p e NOSE), o que, em parte, poderia ser atribuído à pior qualidade do sono apresentada tanto por estes pacientes quanto pelos portadores de RA. O teste da sacarina evidenciou pior transporte mucociliar nos pacientes com RSC com polipose. Não houve diferença do pH do condensado do ar exalado, sugerindo que, apesar de interessante na avaliação das vias aéreas inferiores, este pode não ser um bom teste para análise das vias aéreas superiores. Foram encontradas alterações significantes tanto do pH quanto da contagem de células totais do lavado nasal do grupo com RSC com polipose, sem, no entanto, haver diferença na contagem diferencial. Dos 17 pacientes com RSC com perfil completo de citocinas, 12 se encaixam em endotipos já descritos, sendo que, dos cinco restantes, dois apresentam o mesmo perfil, podendo indicar um novo subgrupo / Introduction: Allergic rhinitis (AR) and chronic rhinosinusitis (CRS) are nasal inflammatory diseases with high and increasing prevalence. It is estimated that 15.5% of Americans have CRS, and a recent study found a prevalence of 5.51% in the city of São Paulo, while AR affects between 10 and 20% of the world population. Although they have distinct pathophysiological mechanisms, in both there is recruitment of defense cells, mainly T lymphocytes, and production of inflammatory cytokines. These mediators vary not only between diseases but also among affected populations, and their knowledge is important for the correct diagnosis and targeting of the therapy chosen. Objectives: To map the inflammatory mediators present in the nasal wash and the exhaled breath condensate in the CRS and AR, evaluating possible biomarkers of this diseases, and to analyze the inflammatory endotype of the patients studied. In addition, assess quality of life, level of nasal obstruction, sleep quality of affected patients, mucociliary transport and collect samples for pH analysis, total cell count and its differential. Patients and Methods: Prospective cross-sectional exploratory study, divided into four groups: 1) Control group with patients without complaints 2) Patients with AR with prick test positive 3) Patients with CRS with polyps 4) Patients with CRS without polyps. All patients responded to four questionnaires: 20-Item Sino-Nasal Outcome Test (SNOT-20p), Nasal Obstruction Symptom Evaluation (NOSE), Pittsburgh Sleep Quality Index (PSQI-BR) and European Community Respiratory Health Survey (ECRHS) questionnaire for screening and diagnosis of asthma. A physical examination was performed, including nasal endoscopy (modified Lund-Kennedy score) and, in patients with CRS, a computerized tomography (Lund-Mackay score) evaluation. The mucociliary transport was also evaluated through the saccharin test. The exhaled breath condensate was collected for pH analysis and the nasal wash for evaluation of pH, cytokines and cellularity (total and differential). The presence of IL-4, IL-5, IL-17, IL-17A, IL-22, TNF-Alfa and IFN-Gama in the nasal wash and IL-5, IL-17A, IL-22 and IFN-Gama in the exhaled breath condensate. Results: Patients with CRS had significantly worse scores in the nasal obstruction questionnaire (NOSE, p < 0.01) and quality of life questionnaire (SNOT-20p, p < 0.01) when compared to controls, and both patients with CRS and AR presented worse sleep quality (PSQI-BR, p < 0.01). The Lund-Mackay extension score was higher in patients with CRS with polyps (p < 0.02). The saccharin test showed longer time in the CSR group with polyps (p < 0.01). The pH of the exhaled breath condensate did not differ between groups. The CRS with polyps group presented both pH difference (p < 0.01) and higher total nasal wash cell count (p < 0.01) when compared to the control group, but with no difference in the differential count (p = 0.05), while IL-5 was higher in the CRS group with polyps when compared to the RSC without polyps group (p = 0.02). Patients were then divided into endotypes according to the groups described by Tomassen et al, Endotypes are the subtypes of the disease defined functionally and pathologically by distinct molecular mechanisms. Conclusions: Patients with CRS presented worse scores on quality of life questionnaires (SNOT-20p and NOSE), which could be attributed in part to the poorer quality of sleep presented by both patients with CRS and AR. The saccharin test evidenced worse mucociliary transport in patients with CRS with polyps when compared with control group. There was no difference in the pH of the exhaled breath condensate, suggesting that, although interesting in the evaluation of the lower airways, this may not be a good test for analyzing the upper airways. Significant alterations were found in both pH and total nasal wash cell count in the CSR group with polyposis, but there was no difference in the differential count. Of the 17 patients with CRS with complete cytokine profile, 12 fit into already described endotypes, and of the remaining five, two have the same profile, which may indicate a new subgroup

Le cancer de la vessie est un cancer fréquent et extrêmement onéreux par patient puisque plusieurs patients subissent des récidives de cancer et ont recours parfois à des chirurgies complexes. Il est donc important de diagnostiquer efficacement ces cancers lors de la prise en charge initiale du patient. En effet, la procédure standard d’imagerie pour la détection du cancer est la cystoscopie de la vessie guidée par lumière blanche, toutefois cette méthode ne permet pas de bien distinguer les cellules qui sont propices à l’invasion musculaire des cellules de cancer de la vessie non infiltrant. Ce mémoire propose d’utiliser un nouvel immunoconjugué fluorescent ciblant la sous-unité alpha du récepteur de l’interleukine 5, un nouveau biomarqueur spécifique aux cellules du cancer de la vessie infiltrant, afin d’effectuer la cystoscopie de la vessie guidée par fluorescence. Pour ce faire, un protocole de conjugaison du fluorochrome cyanine-5 (Cy5) à un anticorps monoclonal a été développé. De plus, un protocole de conjugaison d’un peptide Cell Accumulator (ACCUM) sur cet anticorps fluorescent (A14-Cy5-ACCUM) a été optimisé. Ensuite, la capacité de cet immunoconjugué à marquer les cellules humaines de cancer de la vessie infiltrantes du muscle (MIBC), HT1376, a été testée. Par la suite, un nouveau modèle orthotpique murin de MIBC humain permettant la validation préclinique prochaine de l’A14-Cy5-ACCUM a été développé. Une banque de plasma et sérum sanguin, et d’urine de patients sains et atteints de cancer de la vessie a été compilé. Cette biobanque contient 111 échantillons de plasma sanguin et d’urine qui pourront être utilisé afin de tester l’hypothèse selon laquelle le niveau d’interleukine-5 sanguin pourrait être un facteur pronostique pour la progression du cancer de la vessie. Ce projet jette les bases pour l’évaluation potentielle de la cystoscopie guidée par fluorescence lors de la prise en charge initiale des patients atteints de cancer de la vessie afin d’améliorer la survie sans progression et la survie à long terme des patients atteints de MIBC. / Abstract: Bladder cancer is a frequent and extremely costly cancer when evaluated on a per-patient basis because of its high recurrence rate and patients undergoing complex medical procedures. It is of utmost importance to better identify the aggressiveness of this cancer at initial diagnosis. The standard procedure for bladder cancer detection is still white-light guided cystoscopy, which relies mostly on physicians experience in regard to identifying invasive malignancies. This memoir proposes the use of a new fluorescent immunoconjugate, targeting the alpha subunit of interleukin-5 receptor (IL-5R[apha]), a new biomarker specific to muscle-invasive bladder cancer (MIBC) cells for fluorescence-guided cystoscopy. To do so, a conjugation protocol to fluorescently label a monoclonal antibody with cyanine-5 fluorophores has been developped. Then, a conjugation protocol to attach Cell Accumulator (ACCUM) peptides to this fluorescent immunoconjugate (A14-Cy5-ACCUM) has been optimized. Moreover, the ability of A14-Cy5-ACCUM to stain MIBC cell line HT1376 has been tested. Most importantly, a novel orthotpic rat model of human MIBC for the future preclinical validation of fluorescence-guided cystoscopy in rat bladder has been developped. Finally, a new bladder cancer tissue repository at the CHUS has been established. This repository contains a total of 111 plasma and urine patient samples that will be helpful to evaluate if interleukin-5 blood levels could be used as a prognosis marker for bladder cancer progression. This project laid the basis for the potential evaluation of fluorescence-guided cystoscopy during initial diagnosis of bladder cancer patients to improve their disease-free and long-term survival.