Les effets anti-allergiques des bactéries lactiques sont suggérés par plusieurs études épidémiologiques, des essais cliniques et des modèles expérimentaux d’allergie. Cependant, les propriétés immunomodulatrices des bactéries lactiques sont sous-exploitées par les stratégies vaccinales développées pour combattre l’allergie et les mécanismes empruntés par ces bactéries pour moduler l’allergie restent peu caractérisés.<p>Dès lors, nous avons caractérisé les propriétés immunomodulatrices qu’exerce Lactobacillus plantarum NCIMB8826, une bactérie lactique modèle, sur la cellule dendritique étant donné le rôle déterminant de cette cellule sur la réponse allergique. Nous montrons que L. plantarum induit une forte sécrétion d’IL-12 p40, d’IL-12 p70, de TNF-a mais une faible production d’IL-10. Cette faculté à induire la sécrétion de cytokines polarisantes dépend de TLR2, de TLR9, de MyD88, de NF-kB, des MAPKs (en particulier JNK, p38 et ERK 1/2), de la composition de l’acide lipotéichoïque de L. plantarum et de CD14. Nous montrons aussi que l’ADN génomique de L. plantarum est un agoniste de TLR9 et que CD14 et CD36 facilitent la liaison de la cellule dendritique avec L. plantarum.<p>Ensuite, nous avons évalué le potentiel vaccinal d’une coadministration L. plantarum + Der p 1 dans un modèle murin d’allergie à Der p 1. Cette formulation vaccinale prévient la production d’IgE Der p 1-spécifique et atténue l’éosinophilie pulmonaire tout en stimulant une forte production d’anticorps IgG2a Der p 1-spécifiques et d’IFN-g par les cellules spléniques. Ces effets bénéfiques nous ont conduit à élaborer une bactérie lactique recombinante dérivée de L. plantarum produisant Der p 1 pour la vaccination contre l’allergie aux acariens. La forme antigénique que nous avons réussi à faire produire par L. plantarum correspond à une protéine de fusion entre la Maltose Binding Protein de E. coli et ProDer p 1 (le zymogène de Der p 1), la présence de ce partenaire de fusion étant indispensable à la production de ProDer p 1. En prophylaxie, la vaccination par cette bactérie recombinante prévient la production d’anticorps IgE-Der p 1-spécifiques et stimule la production d’anticorps IgG2a spécifiques, reproduisant les effets de la coadministration L. plantarum + Der p 1. Elle réduit de manière drastique la production d’IL-5 des cellules spléniques et des cellules ganglionnaires médiastinales et prévient l’éosinophilie pulmonaire mais n’a pas d’effet sur l’hyperréactivité bronchique. Der p 1 étant un des allergènes d’acarien les plus immunodominants, cet ensemble de données montre donc que cette bactérie recombinante constitue un vaccin prophylactique prometteur pour la prévention de l’allergie aux acariens. Des résultats préliminaires obtenus à partir de cellules dendritiques humaines et lymphocytes T autologues montrent la forte capacité de cette bactérie recombinante à induire le développement d’une réponse Th1 fortement polarisée (production d’IFN-g en l’absence de production d’IL-4 et d’IL-5), ce qui suggère que l’utilisation de cette bactérie recombinante pourrait être envisagée pour le traitement de l’allergie chez l’homme. <p> / Doctorat en Sciences agronomiques et ingénierie biologique / info:eu-repo/semantics/nonPublished

<p>Regardless of their origin or pathology, many, if not all, diseases have long been regarded as complex. Yet, despite the progression in the understanding of complexity and the development of systems biology, the majority of biomedical research has been derived from qualitative principles. In comparison to the ethical, temporal and logistical limitations of human experimentation, <em>in vivo</em> animal models have served to provide a more advantageous means to elucidate the underlying disease mechanisms. However, given the additional limitations presented by such models, <em>in silico </em>models have emerged as an effective complement, and, in some cases, a replacement for <em>in vivo</em> experimentation. The <em>in silico </em>models presented in this thesis were developed using mathematical and computational methods to investigate the evolution of two complex, diverse diseases from a systems biology perspective: allergic asthma and cancer.</p> <p>We generated two novel <em>in silico</em> models of allergic asthma aimed at clarifying some dynamic aspects of allergic responses. Experimentally, we utilized an <em>in vivo</em> murine model of chronic exposure to the most pervasive aeroallergen worldwide, house dust mite (HDM), for up to 20 weeks, equivalent to at least 20 human years. Using a range of HDM concentrations, experimental data were collected to study local and systemic effects. The first model applied empirical mathematical techniques to establish equations for airway inflammation and HDM-specific immunoglobulins using an iterative approach of experimentation and validation. Using the equations generated, we showed that the model was able to accurately predict and simulate data. The model also demonstrated the non-linear relationship between HDM exposure and both airway inflammation and allergic sensitization and identified system thresholds.</p> <p>The second model used mechanistic mathematical techniques to investigate the trafficking of eosinophils as they migrated from bone marrow to the blood and, ultimately, to the lungs. Making use of a limited data set, the model determined the effect of individual processes on the system. We identified eosinophil production, survival and death as having the greatest impacts, while migration played a relatively minor role. Furthermore, the model was used to simulate knockout models and the use of antibodies <em>in silico</em>.</p> <p>In the context of cancer growth and metastasis, we developed a theoretical model demonstrating the spatio-temporal development of a tumour in two-dimensions. The model was encoded to create a computer graphic simulation program, which simulated the effects of various parameters on the size and shape of a tumour. Through simulations, we demonstrated the importance of the diffusion process in cancer growth and metastasis.</p> <p>Ultimately, we believe the greatest benefit of each <em>in silico</em> model is the ability to provide an understanding of each respective disease recognized as dynamic and formally complex, but predominantly studied in reductionist, static or un-integrated approaches.</p> / Doctor of Philosophy (Medical Science)

<p>NB: I had another committee member, Dr. Mark Larché; and would like to have his name included in the document.</p> <p>Thank you.</p> / <p><strong>Introduction: </strong>Increasing incidence of chronic immune diseases are mirrored by changing disease risk factors, which include maternal stress during pregnancy. To date, no studies have investigated the impact of prenatal stress challenge (PNS) on the fetal immune system. Fetal liver and bone marrow represent major sources of hematopoietic stem cells (HSC) at mid gestation, which differentiate and mature in the thymus. Disturbance of immune development may cause immune impairment in later life. Further, progesterone is recognized as a critical part of feto-maternal interaction. This study aimed to determine if PNS interferes with normal fetal immune development in mice and the impact of progesterone supplementation on stress effects. <strong>Methods: </strong>DBA/2J-mated BALB/c dams were sorted into three groups: control, PNS (gestation days (GDs) 12.5 and 14.5) and PNS plus progesterone supplementation (DHD). Fetal tissue was collected on GDs 16.5 and 18.5. Flow cytometric analysis examined frequency and phenotype of fetal immune cell populations: HSC in fetal liver and bone marrow, and different stages of T cell maturation and regulatory T (Treg) cells in the thymus. Fetal tails were collected to determine fetal sex by PCR analysis. <strong>Results: </strong>PNS induced a decrease in organ size on GD16.5, which was not seen on GD18.5 and was reversed by DHD treatment. PNS altered the percentage and absolute number of HSC within the liver and bone marrow populations, on GD16.5 and 18.5. There was a significant lag in T cell maturation as demonstrated by the altered expression of CD3 and skewed CD3-:CD3+ ratio. There was a significant decrease in Treg cells within CD3+ thymic cells in response to PNS. PNS effects in the thymus were ameliorated by DHD treatment. There was no PNS-induced sex bias. <strong>Conclusions: </strong>These results indicate that PNS compromises the developing fetal immune system, which could account for impaired immune responses in adults with chronic immune disease, and provide evidence for a therapeutic role of progesterone supplementation.</p> / Master of Science (MSc)

<p>Obstructive lung diseases limit airflow and gas exchange and have a major impact on a patient’s long-term health. Asthma and chronic obstructive pulmonary disease (COPD) are the most prevalent obstructive lung diseases and represent a major burden on healthcare systems worldwide. It is now accepted that the pathologies associated with these diseases are heterogeneous in nature, and as the function of the lung is determined by its three-dimensional structure, methods to volumetrically evaluate the lung are important tools in furthering the study of these pathologies.</p> <p>Three-dimensional imaging methodologies, such as computed tomography (CT) and single photon emission computed tomography (SPECT), are used clinically in the diagnosis of lung disease, but results are not commonly quantified. In addition, asthma and COPD develop slowly over time and diagnosis normally takes place after the underlying pathologies are well established. Experimental models in small animals, such as rats and mice, allow for the study of disease pathogenesis in a controlled setting and development of quantitative imaging practices for these models provides translational tools for relating results back to the clinic.</p> <p>In this thesis, CT densitometry and ventilation/perfusion (V/Q) SPECT are explored as methods to investigate models of asthma and COPD. CT densitometry is shown to be capable of quantifying allergic inflammation in an asthma model but is of less use in a model of COPD, predominantly due to the relative amounts of inflammation present. However, V/Q imaging is shown to be quite sensitive to the effects of cigarette smoke in a model of COPD and has been used to better understand how pathologies associated with COPD contribute to gas exchange limitation in the lung.</p> <p>The models, imaging techniques, and analysis methods described in this work provide insight into chronic obstructive lung disease and allow for future investigations into how pathologies effect gas exchange. Further, the characterization of the models described in this thesis allows for drug efficacy studies to be performed, both on established and novel treatments. Future research into asthma and COPD will benefit further from the use of threedimensional imaging methodologies because they provide volumetric information on structure and function and can act as a translational bridge between clinical disease and preclinical animal models.</p> / Doctor of Philosophy (Medical Science)

Two instruments were developed and administered to fifteen female and three male rheumatoid arthritics (mean age 44) previously hospitalized for allergy treatment by ecological methods. The Physical Functioning Ability Scale assessed functional capacity according to common daily activities, and the Rehabilitation Questionnaire surveyed adjustment problems subjects encountered after hospital discharge. ANOVA was performed on the functionality scale comparing dimensions of dependence, pain, and difficulty with categorical activities of mobility, work, and personal care, Significant F values (p(.05) were obtained for interaction and dimensions, but not for categories. Functional independence from human/mechanical assistance was particularly noted. Recommendation for future research concerns replicating this study using a control group of rheumatoid arthritics treated by traditional medical approaches.

L’allergie de type I, une réaction de type hypersensibilité immédiate (HIS), a considérablement augmenté au cours des dernières décades dans les pays industrialisés. Le rôle des lymphocytes T régulateurs (Treg) dans le contrôle des maladies allergiques a été récemment apprécié. Cependant, il n’existe actuellement aucune connaissance précise si et comment les CD4+ Treg peuvent réguler l’allergie de type I. Le but du projet était d’étudier la contribution de cellules Treg dans le contrôle de l’allergie de type I chez la souris et chez l’homme. En utilisant un modèle murin d’HSI aux protéines de lait de vache, ß-lactoglobuline(BLG), nous avons montré que l’absence de Treg augmentait les réponses des cytokines Th1et Th2 et des anticorps IgE, IgG1 et IgG2a spécifique de la BLG. De plus, l’absence de Treg a augmenté la sévérité de l’anaphylaxie chez les souris sensibilisées lors de l’épreuve orale avec la BLG, avec une augmentation concomitante de la protéase des mastocytes muqueux 1(mMCP-1) dans le sérum. La contribution des cellules Treg dans un modèle murin d’anaphylaxie systémique passive (PSA) a été également étudiée. Une anaphylaxie plus sévère a été observée chez les souris déficientes en CHM de classe II ainsi que chez des souris B6 traitées avec un anti-CD4. En revanche, la déplétion sélective des Foxp3+ Treg chez des souris Tg DEREG n’a pas d’effet sur l’anaphylaxie.La capacité des Treg à contrôler la dégranulation des basophiles a été étudiée chez l’homme. Les données préliminaires ont montré que les Treg ont été incapables de contrôler la dégranulation des basophiles chez les donneurs sains. En revanche, une légère mais reproductible down-regulation de la dégranulation/activation des basophiles a été observée dans l’allergie induite par les médicaments chez l’homme. Dans l’ensemble, notre travail met en évidence un nouveau rôle encore non identifié des Treg dans le contrôle de l’HSI et souligne que chez la souris et l’homme, les Treg peuvent dans certaines conditions, limiter la sévérité de l’HSI en agissant sur les mastocytes et/ou basophiles effecteurs / Type I allergy, an immediate type hypersensitivity reaction (ITH), has dramatically increased during the past decades affecting up to 30% of the population in industrialized countries. The role of regulatory T cells (Treg) in the control of allergic diseases has been recently appreciated. However, there is currently no precise knowledge of whether and howCD4+ Treg can regulate type I allergy. The aim of this project was to investigate the contribution of Treg in the control of type I allergy. Using a murine model of ITH to cow’s milk protein the ß-lactoglobulin (BLG), we showed that lack of Treg increased BLG-specific Th2 and Th1 cytokine response and BLG-specific IgE, IgG1 and IgG2a antibodies.Furthermore, absence of Treg enhanced the severity of the anaphylaxis with concomitant increase in serum mucosal mast cell protease 1 (mMCP-1) in sensitized mice upon oral challenge with BLG. Furthermore, we investigated the contribution of Treg in a murine model of passive systemic anaphylaxis (PSA). More sever anaphylaxis was observed in both MHCclass II KO mice and after anti-CD4 mAb treatment. Alternatively, selective depletion of Foxp3+ Treg in DEREG Tg mice did not show changes in anaphylaxis.The capacity of Treg to control basophils degranulation was investigated in humans. Preliminary data showed that Treg were unable to control basophils degranulation in healthy donors. In contrast, a slight butreproducible downregulation of basophils activation/ degranulation was observed in allergic individuals. Taken together, our work points out to a novel as yet unidentified role of Treg in the control of ITH and emphasizes that both mouse and human Treg can, in certain conditions, limit the severity of ITH by acting on mast cells and/or basophils effectors of ITH

At birth, the immune system is immature and the gut microbiota influences immune maturation. Staphylococcus aureus (S. aureus) and lactobacilli are part of the neonatal gut microbiota and have seemingly opposite effects on the immune system. S. aureus is a potent immune activator and early-life colonization associates with higher immune responsiveness later in life. Lactobacilli-colonization associates with reduced allergy-risk and lower immune responsiveness. Further, lactobacilli modulate immune-activation and have probiotic features. Here, we investigated S. aureus-induced activation of human lymphocytes, including T regulatory cells (Tregs), conventional T-cells (CD4+ and CD8+), unconventional T-cells (γδ T-cells and MAIT-cells) and NK-cells from children and adults, together with the modulatory effect of lactobacilli on immune-activation. Further, early-life colonization with these bacteria was related to lymphocyte-maturation, plasma cytokine- and chemokine-levels and allergy.  S. aureus cell free supernatant (CFS) and staphylococcal enterotoxin (SE) A induced an increased percentage of FOXP3+ Tregs and of CD161+, IL-10+, IFN-γ+ and IL-17A+ Tregs (Paper I). The same pattern was observed in children with a lower degree of activation, possibly due to lower CD161-expression and poor activation of naive T-cells (Paper II). S. aureus-CFS induced IFN-γ-expression, proliferation and cytotoxic capacity in conventional and unconventional T-cells, and NK-cells. SEA, but not SEH, induced activation of unconventional T-cells and NK-cells by unknown mechanism(s) (Paper III, extended data). Lactobacilli-CFS reduced S. aureus-induced lymphocyte activation without the involvement of IL-10, Tregs or monocytes, but possibly involving lactate (Paper III). Early-life colonization with S. aureus associated with increased percentages of CD161+ and IL-10+ Tregs while lactobacilli-colonization negatively correlated with the percentage of IL-10+ Tregs later in life (Paper II). Allergic disease in childhood associated with double allergic heredity, being born wintertime and with higher plasma levels of TH2-, TH17- and TFH-related chemokines early in life. Lactobacilli-colonization associated with lower prevalence of allergy, reduced chemokine-levels and increased levels of IFN-γ in plasma (Paper IV).    This thesis provides novel insights into S. aureus- and SE-mediated activation of Tregs, unconventional T-cells and NK-cells and suggests an overall impairment of immune-responsiveness towards this bacterium in children. Further, S. aureus-colonization may influence the maturation of peripheral Tregs. Our data show that lactobacilli potently dampen lymphocyte-activation in vitro and that colonization associates with Treg-responsiveness, altered plasma cytokine- and chemokine-levels and with remaining non-allergic, thereby supporting the idea of lactobacilli as important immune-modulators. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.</p><p> </p>

Trabalhadores expostos a animais de laboratório possuem elevado risco de desenvolvimento de reações alérgicas a proteínas animais, conhecidas como alergia a animais de laboratório. A constante eliminação de proteínas dos animais através da saliva, suor e principalmente pela urina faz com que os laboratórios e biotérios tornem-se lugares propícios para o desenvolvimento de reações alérgicas. É de conhecimento que a atopia, definida como teste cutâneo de hipersensibilidade imediata (TCHI) a qualquer alérgeno, é um dos principais fatores de risco para alergia a animais de laboratório. Ao se estudar atopia como fator de risco, acreditamos que seria mais efetivo analisar os efeitos da sensibilização a alérgenos ocupacionais em comparação aos comuns, pois nossa hipótese é de que, assim, chegamos ao principal marcador de risco. O presente estudo teve como objetivo investigar a associação entre sensibilização alérgica ocupacional e a presença de desfechos clínicos. Especulamos que a contínua exposição ocupacional ao alérgeno ocupacional sensibilizante faça com que a sensibilização ocupacional seja melhor marcador de risco para tais reações alérgicas do que a sensibilização comum. O presente estudo transversal foi realizado em duas universidades brasileiras, onde trabalhadores e estudantes eram expostos a pelo menos um dos seguintes animais: rato, camundongo, cobaia, coelho e hamster. Os voluntários foram submetidos à TCHI, com 5 alérgenos denominados ocupacionais (alérgenos de rato, camundongo, cobaia, coelho e hamster) e 11 alérgenos chamados comuns (alérgenos de ácaros, gato, cachorro, baratas, fungos e mistura de gramíneas). Os indivíduos foram alocados em grupos de acordo com o resultado do TCHI: grupo não sensibilizado (TCHI negativo para todos os alérgenos testados), grupo sensibilização comum (TCHI positivo para qualquer alérgeno comum) e grupo sensibilização ocupacional (TCHI positivo para qualquer alérgeno ocupacional). Os indivíduos responderam questões sobre características da exposição a animais, sintomas, doenças alérgicas e respiratórias, e também foram submetidos à espirometria e teste de broncoprovocação com manitol para detectar hiperreatividade brônquica (HRB). Razão de prevalência (RP) foi estimada usando regressão de Poisson. Dados de 453 indivíduos foramanalisados. O grupo não sensibilizado foi composto por 237 indivíduos, o grupo sensibilização comum, composto por 142 indivíduos e o grupo sensibilização ocupacional, composto por 74 indivíduos. A sensibilização ocupacional associou-se ao aumento de risco para os seguintes desfechos: sintomas cutâneos (RP: 1,36; intervalo de confiança de 95% (IC95%): 1,01-1,85), sibilo (RP: 1,75; IC95%: 1,21-2,53), rinite (RP: 1,25; IC95%: 1,11- 1,40), dispneia noturna (RP: 2,40; IC95%: 1,31-4,40), HRB (RP: 2,47; IC95%: 1,50-4,09) e asma confirmada (RP: 2,65; IC95%: 1,45-4,85), quando comparado ao grupo sensibilização comum. Além disso, a sobreposição de asma, rinite e sintomas cutâneos no mesmo indivíduo foi mais frequente no grupo sensibilização ocupacional com valor de 16,2% versus 4,9% do grupo sensibilização comum e 1,6% do grupo não sensibilizado (p<0,01). Concluímos que a sensibilização alérgica ocupacional esteve associada com aumento de prevalência dos desfechos estudados, quando comparada à sensibilização comum, exceto para eczema ou alergia cutânea. Acreditamos que a realização periódica de TCHI com alérgenos ocupacionais poderia contribuir para detecção de risco elevado para doenças alérgicas e respiratórias entre trabalhadores expostos a animais de laboratório. / Workers exposed to laboratory animals have a high risk of developing allergic reactions to laboratory animals, known as laboratory animal allergy (LAA). Animals constantly eliminate proteins through saliva, sweat and especially urine, which makes laboratories and animal rooms favorable to the development of allergic reactions. We know that atopy, defined as a positive skin prick test (SPT) to any allergen, is a major risk factor for LAA. When considering atopy as a risk factor, we believe that it would be more effective to analyze the effects of occupational sensitization than the effects of common sensitization, thus our hypothesis is that occupational sensitization would lead to the main risk biomarker. The present study aimed to investigate the association between occupational allergic sensitization and the presence of clinical outcomes. We speculate that the continuous occupational exposure to the sensitizing occupational allergen will make occupational sensitization a better marker of risk than common sensitization. The present cross-sectional study was carried out in two Brazilian universities, where workers and students were exposed to at least one of the following animals: rat, mouse, guinea pig, rabbit and hamster. The volunteers were submitted to SPT with 5 occupational allergens (rat, mouse, guinea pig, rabbit and hamster allergens) and 11 common allergens (mite, cat, dog, cockroaches, fungi and grass mix allergens). Individuals were allocated into groups according to the outcome of SPT: non-sensitized group (negative SPT for all allergens tested), common sensitization group (positive SPT for any common allergen) and occupational sensitization group (positive SPT for any occupational allergen). Individuals answered questions about characteristics of animal exposure, symptoms, allergic and respiratory diseases, and also underwent spirometry and bronchial challenge test with mannitol to detect bronchial hyperresponsiveness (BHR). Prevalence ratio (PR) was estimated using Poisson regression. Data from 453 individuals were analyzed. Occupational sensitization was associated with an increased risk for skin symptoms (PR: 1.36, 95% confidence interval (95% CI: 1.01-1.85), wheezing (RP: 1.75, 95% CI: 1.21-2.53), rhinitis (RP: 1.25, 95% CI: 1.11-1.40), nocturnal dyspnoea (RP: 2.40, 95% CI: 1.31-4.40),BHR (PR: 2.47, 95% CI: 1.50-4.09) and confirmed asthma (PR: 2.65; 95% CI: 1.45-4.85) when compared to the common sensitization group. In addition, overlap of asthma, rhinitis, and skin symptoms in the same individual was more frequent in the occupational sensitization group, 16.2% versus 4.9% in the common sensitization group and 1.6% in the non-sensitized group (p <0.01). We concluded that occupational allergic sensitization was associated with an increased prevalence of all studied outcomes when compared to common sensitization, except for eczema or skin allergy. We believe that periodic SPT with occupational allergens could contribute to the detection of high risk for allergic and respiratory diseases among workers exposed to laboratory animals.

A asma afeta aproximadamente 300 milhões de pessoas no mundo e é a maior causa de internação hospitalar em crianças nos países desenvolvidos. Essa doença é incurável e por vezes refratária ao tratamento em um número significativo de pacientes. As taxas de prevalência de tabagismo entre pacientes asmáticos são semelhantes aos da população em geral e o impacto da fumaça de cigarro nestes pacientes ainda é clinicamente controverso. O objetivo deste trabalho foi traçar o perfil inflamatório e analisar o papel das células dendríticas sobre a imunomodulação provocada pela exposição à fumaça do cigarro na inflamação alérgica pulmonar induzida previamente por ovalbumina (OVA) em um modelo murino. Primeiramente avaliamos in vivo a ação da fumaça de cigarro na inflamação pulmonar alérgica crônica avaliando a responsividade brônquica, o remodelamento pulmonar, a produção de anticorpos antígeno-específicos, o perfil de células inflamatórias pulmonares e sistêmicas e a produção de citocinas inflamatórias e moduladoras. Em seguida, realizamos estudo in vitro do perfil de maturação, migração e inflamatório de células dendríticas expostas a OVA e/ou a extrato de fumaça de cigarro. Nosso estudo mostrou que a sensibilização e desafios inalatórios com OVA levaram à inflamação pulmonar de característica Th2 com aumento de responsividade brônquica, remodelamento, altos níveis de IgE e de citocinas pró-inflamatórias como IL-4, IL-5 e IL-13. A exposição à fumaça de cigarro, surpreendentemente, levou a uma redução dos níveis de IL-4, IL-5 e IL-13, e simultaneamente reduziu os níveis de citocinas anti-inflamatórias como IL- 10 e TGF-beta em animais sensibilizados e desafiados com antígeno. Foi observada nestes animais, uma redução no número de eosinófilos no lavado broncoalveolar e aumento no número de neutrófilos no pulmão. A combinação da inflamação alérgica com exposição à fumaça de cigarro levou a um aumento do recrutamento e ativação de células dendríticas linfoides nos linfonodos mediastinais, que mostrou relação direta com aumento do influxo de células T CD8+ e ativação das mesmas no pulmão. A inflamação alérgica juntamente com a exposição à fumaça de cigarro, levou a uma redução no recrutamento de células dendríticas plasmocitoides além de reduzir o recrutamento de células T regulatórias. In vitro, mostramos que o extrato de fumaça de cigarro combinado ao antígeno aumenta a capacidade migratória e fagocítica do antígeno pelas BMDCs. No entanto, houve redução da expressão gênica para IL-13 neste mesmo grupo. Concluímos que neste modelo de inflamação pulmonar alérgica crônica combinada com a exposição à fumaça de cigarro leva a uma descaracterização do perfil inflamatório característico da resposta Th2 com a redução do recrutamento de eosinófilos, redução dos níveis de IL-4, IL-5 e IL-13 aliados a um aumento do número de neutrófilos, o que pode estar relacionado ao aumento do recrutamento e ativação de células dendríticas linfoides bem como de células T CD8+ e redução local de células dendríticas plasmocitoides. Mostramos ainda que a fumaça de cigarro juntamente com o antígeno leva as células dendríticas a aumentarem sua capacidade fagocítica porém, reduzir sua capacidade pró-inflamatória pela expressão gênica reduzida de IL-13 / Asthma affects approximately 300 million people worldwide and it is the major cause of hospitalization among children in developed countries. This disease is often refractory to treatment in a high number of patients. The prevalence rates of smoking among asthmatic patients are similar to the general population and the impact of cigarette smoke is also clinically controversial. The main goal of this study is to outline, in a murine model, the inflammatory profile and to analyze the role of dendritic cells on the immunomodulation caused by exposure to cigarette smoke in ovalbumin (OVA)-induced pulmonary allergic inflammation. First, we evaluated in vivo the action of cigarette smoke on chronic allergic pulmonary inflammation, evaluating the bronchial responsiveness, pulmonary remodeling, the production of antigen-specific antibodies, pulmonary and systemic inflammatory cell profile and the production of inflammatory and modulating cytokines. Next, we performed an in vitro study of the maturation, migration and inflammatory profile of dendritic cells exposed to OVA and/or cigarette smoke extract. Our study showed that sensitization and challenge with OVA led to Th2-type lung inflammation with increased bronchial responsiveness, remodeling, high levels of IgE and proinflammatory cytokines such as IL-4, IL-5 and IL-13. Exposure to cigarette smoke has surprisingly led to a reduction in levels of IL-4, IL-5 and IL-13, and simultaneously reduced levels of anti-inflammatory cytokines such as IL-10 and TGF-beta in animals sensitized and challenged with the antigen. We also observed a reduction in the number of eosinophils in bronchoalveolar lavage fluid and an increase in the number of neutrophils in the lung of these animals. The combination of allergic inflammation with exposure to cigarette smoke led to increased recruitment and activation of lymphoid dendritic cells in the mediastinal lymph nodes, which showed to be direct related with increased activation and influx of CD8+ T cells in lung. Allergic inflammation combined with cigarette smoke led to a decrease of plasmacytoid dendritic cells a well as regulatory T cells. In vitro, we showed that cigarette smoke extract combined with antigen increased migratory and phagocytic capacity of BMDCs. However, there was a reduction of IL-13 gene expression in this same group. We conclude that in this model of chronic pulmonary allergic inflammation combined with exposure to cigarette smoke leads to a mischaracterization of the characteristic inflammatory profile of the Th2 response with the reduction of eosinophil recruitment, reduction of levels of IL-4, IL-5 and IL-13 allied to increased number of neutrophils, which is related to increased recruitment and activation of lymphoid dendritic cells as well as CD8+ T cells and local decrement of plasmacytoid dendritic cells. We further show that cigarette smoke combined with antigen increases dendritic cell phagocytic capacity however, reduces its pro-inflammatory capacity by the reduced gene expression of IL-13

INTRODUÇÃO: Há evidências consistentes a partir de estudos epidemiológicos de que os profissionais de limpeza têm um risco elevado de desenvolver asma. Os determinantes deste risco não são totalmente conhecidos. Esses trabalhadores estão expostos a agentes ocupacionais de baixo e alto peso molecular, tanto a agentes sensibilizantes, como a irritantes. É importante produzir evidências de que este risco está relacionado ao trabalho e não às condições sociais ou outros fatores concorrentes, conhecer a anormalidade patológica subjacente, e investigar os possíveis agentes. O acúmulo deste conhecimento permitirá a proposição de medidas para substituição ou controle do uso dos agentes envolvidos e prevenção da ocorrência de novos casos desnecessariamente. Além disso, o uso de novas técnicas não invasivas, como a citologia do escarro e A FeNO, poderá facilitar o diagnóstico precoce dos casos. Desta maneira, este estudo pretende avaliar se o ambiente de trabalho induz inflamação pulmonar em trabalhadores assintomáticos, antes da alteração das provas funcionais e a eficácia do escarro induzido e da FeNO NO como marcadores de inflamação pulmonar precoce entre trabalhadores de limpeza profissional não doméstica. MÉTODO: Os trabalhadores foram avaliados através da comparação da citologia do escarro, valores da FeNO, espirometria e PFE, realizados durante o período de trabalho e após as férias. A amostra foi caracterizada através do questionário de triagem do estudo de saúde respiratória da Comunidade Européia, questionário de sintomas respiratórios e a pontuação no ISAAC. RESULTADOS: Em nosso estudo, encontramos um aumento significativo dos valores do VEF1 após o período de férias, (pré 2,76 ± 0,57 e pós 2,94 ± 0,61; p < 0,05) apesar de estar dentro da normalidade, em ambos os períodos. A média das medidas do PFE também mostrou-se maior durante o período de férias em comparação ao período de trabalho, embora não estatisticamente significante (pré 366,6 ± 54,1 e pós 386,4 ± 62,9 e p > 0,05). Encontramos uma redução dos valores da medida da FeNO após as férias (pré 16,3 ± 9,7 e pós 13,8 ± 7,8 p < 0,05) e redução de eosinófilos (pré 0,019 ± 0,05 e pós 0,003 ± 0,01 p < 0,05), linfócitos (pré 0,16 ± 0,35 e pós 0,01 ± 0,09 p < 0,05) e macrófagos (pré 0,421 ± 0,47 e pós 0,235 ± 0,30 p < 0,05) na citologia do escarro induzido, realizada após o período de férias. CONCLUSÃO: Demonstramos que o ambiente ocupacional ao qual são expostos os trabalhadores de limpeza profissional não doméstica provoca inflamação nas vias aéreas de trabalhadores assintomáticos. Esta inflamação pode ser aferida por métodos não invasivos como escarro induzido e FeNo, antes do aparecimento de alterações nas provas funcionais, embora estes métodos ainda necessitem de padronização. São necessários novos estudos para quantificar a exposição ao cloro e sua relação com inflamação, assim como para padronizar o uso do escarro induzido e da FeNO no diagnóstico de doenças ocupacionais entre trabalhadores de limpeza, além de medidas preventivas e educativas nesta população / There is consistent evidence from epidemiological studies that the cleaning professionals have a high risk of developing asthma. The determinants of this risk are not fully known. These workers are exposed to occupational agents of low and high molecular weight, both the sensitizing agents, such as irritant. It is important to produce evidence that this risk is related to work and not social conditions or other competitive factors, know the underlying pathological abnormality, and investigate possible agents. The accumulation of this knowledge will allow proposing measures to replace or control the use of the agents involved and preventing the occurrence of new cases unnecessarily. In addition, the use of new non-invasive techniques, such as sputum cytology and the FeNO may facilitate early diagnosis of cases. Thus, this study aims to assess if the work environment induces lung inflammation in asymptomatic workers, before the change of functional tests and the effectiveness of induced sputum and exhaled NO as early lung inflammation markers between professional cleaning workers. METHOD: Workers were evaluated by comparing the sputum cytology, FeNO values, spirometry and PEF, made during the work period and after the holidays. The sample was characterized by screening questionnaire of respiratory health study of the European Community, questionnaire of respiratory symptoms and a score in ISAAC. RESULTS: In our study, we found a significant increase in FEV1 values after the vacational period, (pre 2.76 ± 0.57 and 2.94 ± 0.61; post; p < 0.05) despite of being within the normal range in both periods. The average peak flow measurements also was higher during the vacational period compared to the period of work, although not statistically significant (366.6 ± 54.1 pre and post 386.4 ± 62.9; p > 0.05). We found a reduction of the exhaled measured values of NO after the holidays (pre and post 16.3 ± 9.7, 13.8 ± 7.8; p < 0.05), reduction of eosinophils (pre and post 0.05 ± 0.019, 0.003 ± 0.01; p < 0.05), lymphocytes (pre and post 0.16 ± 0.35, 0.01 ± 0.09; p < 0.05) and macrophages (pre and post 0.421 ± 0.47 0.235 ± 0 30 p < 0.05) in induced sputum cytology, performed after the holiday period. CONCLUSION: We demonstrate that the occupational environment to which professional cleaning non-domestic workers are exposed causes inflammation in the airways of asymptomatic workers. This inflammation can be measured by non-invasive methods such as induced sputum and FeNo, before the onset of changes in functional tests, although these methods still require standardization. Further studies are needed to quantify the exposure to chlorine and its relation to inflammation, as well as to standardize the use of induced sputum and exhaled nitric oxide in the diagnosis of occupational diseases among cleaning workers, and preventive and educational measures in this population