O objetivo deste trabalho foi estudar o efeito da endotoxina durante a sensibilização com extrato de Blomia tropicalis (Bt) Para induzir a alergia pulmonar experimental, camundongos foram sensibilizados s.c. com Bt adsorvido ao alum em presença ou não de LPS e desafiados i.n. com Bt. Os animais sensibilizados e desafiados com de Bt apresentaram intensa inflamação pulmonar eosinofílica, AHR, grande produção de muco, aumento na produção de citocinas do tipo 2 no lavado broncoalveolar (BAL) e aumento de IgE. Quando o LPS foi adicionado, observou-se inibição no influxo de eosinófilos e aumento no influxo de neutrófilos no BAL. O LPS também inibiu IL-4 e IL5 e aumentou IFN<font face=\"Symbol\">g e IL-17. Surpeendentemente, o LPS não afetou a AHR, nem a produção de IgE. Utilizando animais nocautes, verificou-se que a inibição eosinofílica induzida pelo LPS é dependente de IFN<font face=\"Symbol\">g e MyD88 enquanto que o aumento de neutrófilos é independente de IFN<font face=\"Symbol\">g e dependente da sinalização dos TLR2, TLR4 e CD14. Além disso, na ausência de TLR2, TLR4 ou CD14, o LPS suprimiu a alergia pulmonar experimental. Em conclusão, os resultados indicam que camundongos sensibilizados com Bt e LPS co-adsorvidos ao alum desenvolvem um fenótipo de asma não clássico, dependente da sinalização pelos TLR. / The aim of this work was to study the effects of endotoxin during sensitization with Blomia tropicalis extract (Bt). To induce experimental allergic airways, mice were sensitized s.c. with Bt with or without LPS co-adsorbed onto alum, and challenged i.n. with Bt. Wild type mice sensitized and challenged with HDM showed eosinophilic lung inflammation, Arway hyperactivity (AHR), mucus hyperproduction, increased levels of type 2 cytokines in the bronchoalveolar lavage (BAL) fluid, and augmented total IgE. When LPS was added at the sensitization, we observed an inhibition of eosinophil influx and an increase in neutrophil counts in the BAL. Moreover, LPS inhibited IL-4 and IL-5 production and increased IFN-<font face=\"Symbol\">g and IL-17 production. Surprisingly, LPS did not affect AHR or IgE production. Using knockout mice, we found that the eosinophilic inhibition induced by LPS was dependent on IFN<font face=\"Symbol\">g and MyD88, while the neutrophilic increase was independent of IFN<font face=\"Symbol\">g and dependent on TLR2, TLR4 and CD14 signaling. Moreover, in the absence of TLR2, TLR4 or CD14, LPS suppressed AAD. In conclusion, our results indicate that mice sensitized with Bt and LPS co-adsorbed to alum develop a non-classical asthma-like phenotype that is dependent on TLR signaling.

Avaliamos os mecanismos regulatórios desencadeados pela imunização materna na resposta alérgica da prole OVA. A imunização materna com OVA promoveu alterações como o aumento da expressão dos receptores FcgRIIb nos linfócitos B esplênicos dos neonatos aos 3 dias de idade (d.i.), o que se manteve até os 20 d.i. Com a imunização das proles no período neonatal a imunização materna inibiu a produção de anticorpos IgE anti-OVA. Além disso, foi observado na população de linfócitos B da prole o aumento da expressão dos receptores FcgRIIb e CD44. A transferência passiva de IgG de mães imunes no pós-natal mostrou uma inibição da produção de IgE, e no período pré-natal foi capaz de reduzir a expressão das moléculas CD40 e CD23 nos linfócitos B e a secreção de IL-10 em linfócitos T CD4 na prole no período neonatal. As evidências mostram que a imunização pré-concepcional com OVA induz mecanismos que regulam a resposta IgE da prole imunizada no período neonatal, o que foi parcialmente observado com a transferência passiva de anticorpos IgG durante o período pré e pós-natal. / To evaluate the regulatory mechanisms triggered by maternal immunization in the immune response of the offspring, the effect of preconceptional immunization with OVA was evaluated. Maternal immunization with OVA led to early alteration with increased expression of FcgRIIb in B lymphocytes from 3 days old pups. Offspring from immune mother showed diminished percentage of CD4 T cells IL-4+. The immunization of offspring during neonatal period showed that maternal immunization inhibits the production of anti-OVA IgE antibodies. The evaluation of CD4 T cell population revealed diminished IL-4+ cells. Passive IgG transfer from immune mother during neonatal period showed inhibition in the IgE synthesis, during pregnancy showed capacity to reduce the expression of CD40 molecules in B cells from neonatal pups. These evidences show that maternal OVA immunization down regulates the IgE response of offspring including phenotypic and functional alteration in B and CD4 T cells. These alterations were partially observed with IgG transfer during pregnancy or after birth.

B cells get help in the antibody response by presenting processed antigen to helper T cells. We asked whether the antigen presenting B cell must induce T helper functions before receiving help, or whether B cell activation is a direct consequence of T cell antigen recognition on the B cell surface. Although antigen-dependent increases in B cell c-myc expression occur as early as two hours after conjugation, the B cell response depends on induction of a contact-dependent helper function in the T cell, which is inhibitable by cyclosporin A. Induction but not delivery of contact help is blocked by anti-class II MHC antibody, indicating that the delivery of T cell help is not Ag dependent or MHC restricted. Also, contact with activated helper T cells induces a different pattern of immediate early gene expression from signals transduced through the B cell antigen receptor. Egr-1 is rapidly upregulated in response to mitogenic signals induced by receptor crosslinking on murine B lymphocytes, and its expression closely correlates with B cell proliferation in several models of B cell activation and tolerance. We compared egr-1 expression during B cell stimulation with Fab'2 and IgG anti-Ig, since it is known that Fab'2 anti-Ig is mitogenic while IgG is not, due to a dominant inhibitory effect of crosslinking the B cell FcγRII to membrane Ig. While mitogenic doses of Fab'2 anti-Ig induce large and rapid increases in egr-1 expression, intact anti-Ig results in only small increases in egr-1 mRNA, comparable to that seen with submitogenic concentrations of Fab'2 anti-Ig. However, when IL-4 is added as a comitogen to induce B cell proliferation with submitogenic concentrations of Fab'2 anti-Ig or IgG anti-Ig, no concomitant increases in egr-1 are observed. The regulation of egr-1 therefore, is similar to that of c-myc in this system, since neither correlates with IL-4 induced DNA synthesis.

Diisocyanates (dNCO) such as methylene diphenyl diisocyanate (MDI) are used primarily as cross-linking agents in the production of polyurethane products such as paints, elastomers, coatings and adhesives, and are the most frequently reported cause of chemically induced immunologic sensitization and occupational asthma (OA). Immune mediated hypersensitivity reactions to dNCOs include allergic rhinitis, asthma, hypersensitivity pneumonitis and allergic contact dermatitis. There is currently no simple diagnosis for the identification of dNCO asthma due to the variability of symptoms and uncertainty regarding the underlying mechanisms. Immunological sensitization due to dNCO exposure is traditionally thought to require initial conjugation of the dNCO to endogenous proteins to generate neoantigens, which trigger production of dNCO specific T lymphocytes and ultimately dNCO specific IgE. Testing for dNCO-specific IgE, for diagnosis of dNCO asthma is however, only specific (96-98%) but not sensitive (18-27%). The low prevalence of detectable dNCO specific IgE has been attributed to both assay limitations and a potential IgE-independent dNCO asthma mechanism(s). The identity of the conjugated proteins responsible for the sensitization also remains unknown. It is also not clear whether dNCOs bind to extracellular, cell membrane, or intracellular proteins as a way of triggering non-IgE asthma. Standardization and optimization of immunoassays used to screen for dNCO specific antibodies in sera is important if its utility as a dNCO asthma diagnostic tool is to be achieved. This will potentially improve sensitivity and allow comparison of results across studies. Current studies on assays of dNCO-specific IgE and IgG lack or have limited characterization of the conjugates used. Diisocyanates bound to hemoglobin (Hb), human serum albumin (HSA), and THP-1 proteins were quantified by HPLC with fluorescence detection. Proteomic tandem mass spectrometry (MS) was used to delineate TDI and MDI specific amino acid binding sites on Hb as well as identification of proteins from MDI exposed THP-1 cells. The trinitrobenzene sulfonic acid assay (TNBS) and SDS gel electrophoresis were used to evaluate extent of intra and intermolecular cross-linking in dNCO-HSA conjugates. Binding of monoclonal antibodies (mAbs) to dNCO bound proteins in enzyme-linked immunosorbent assay (ELISA) was used to evaluate antigenicity of dNCO-protein conjugates. The amount of dNCO binding to HSA and Hb increased with the concentration of the dNCO used for conjugation. All the dNCOs reacted with HSA more than with Hb. Eight binding sites were observed with both MDI and TDI on Hb. The N-terminal valines of both the alpha and beta subunits on Hb, lysine 40 of the alpha subunit and lysine 61 of the beta subunit were common binding sites for both TDI and MDI. Lysine 7 of the alpha subunit and lysines 8, 65 and 66 of the beta subunit were unique to MDI. On the other hand, lysines 11, and 16 of the alpha subunit and lysines 17 and 144 of the beta subunit were unique to TDI. Protein bound MDI was detected in a dose-dependent manner in membrane and cytoplasm fractions of MDI exposed THP-1 cells. MDI was also detected in 11 of the 13 cytoplasmic protein bands. The extent of MDI intracellular protein binding was not affected by cytochalasin D, a chemical that binds actin filaments and inhibits active uptake into cells. The extent of cross-linking shown using the TNBS assay was found to increase with amount of dNCO used. Clear bands from both intra and intermolecular cross-linking were observed on all dNCO-Hb/HSA SDS gels. Using ELISA, both TDI-Hb and TDI-HSA conjugates were reactive to monoclonal antibodies produced against TDI conjugated HSA indicating that dNCO-Hb is also antigenic. The best characterization of dNCO-protein conjugates is achieved by the quantitative determination of conjugated dNCO per mole of protein as well as determining the extent of dNCO cross-linking. Although HSA is more reactive to dNCOs than other serum proteins such as Hb, contribution from other serum proteins to development of OA should not be overlooked as dNCO-Hb was found to be reactive to dNCO specific mAbs. dNCO-conjugated proteins identified in the soluble fraction of MDI exposed THP-1 cells were all of intracellular origin suggesting that MDI can cross the cell membrane and react with intracellular proteins. The entry of MDI into live cells is a passive process, as the extent of intracellular binding was not affected by cytochalasin D. The present study support the potential involvement of dNCO-haptenated membrane and intracellular proteins in development of non-IgE dNCO asthma.

<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)

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

A crotoxina (CTX) é a principal toxina do veneno de serpentes Crotalus durissus terrificus, caracterizada como <font face=\"Symbol\">b-neurotoxina com ação principalmente pré-sináptica, inibindo a liberação de acetilcolina nas junções neuro-musculares. No entanto, vários dados de literatura têm mostrado que, adicionalmente à atividade neurotóxica, a CTX apresenta efeitos imunomodulatórios antiinflamatório, analgésico e anti-tumoral. Apesar destes dados, não foi ainda determinada a ação desta toxina sobre modelos experimentais de alergia, como por exemplo, a inflamação alérgica pulmonar. Este trabalho teve por objetivo investigar o possível efeito inibitório da CTX sobre a gênese e o desenvolvimento de resposta inflamatória pulmonar induzida em ratos pela sensibilização com Ovalbumina (OVA), bem como a sua possível interferência na alergia já estabelecida. Em conjunto, os resultados sugerem que a CTX interfere com a inflamação pulmonar acarretada pela sensibilização e desafio por OVA, modulando mecanismos indutores e efetores da resposta alérgica. / Crotoxin (CTX) is the main toxin of the venom of South American rattlesnake Crotalus durissus terrificus, corresponding to 65% of the protein content of the crude venom. CTX is a <font face=\"Symbol\">b-neurotoxin that displays mainly pre-synaptic action, inhibiting the release of acetylcholine in the neuromuscular junctions. However, some experimental and clinical data have shown that, in addition to the neurotoxic activity, CTX displays immunomodulatory, antiinflammatory, analgesic and anti-tumoral properties. Despite these observations, the possible inhibitory action of this toxin in experimental models of allergy, such as lung allergic inflammation, was not determined yet. Therefore, this study aims to investigate the inhibitory effect of CTX on the genesis and development of lung inflammation induced in rats by sensitization with Ovalbumin (OVA). Together, these results suggest that CTX inhibits lung inflammation induced by sensitization and challenge with OVA, interfering with the initiators and effectors mechanisms of allergic inflammation.