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.

Occupational exposure to endotoxin-rich swine barn air induces respiratory diseases and loss of lung function. Barn exposure induces recruitment of pulmonary intravascular monocytes/macrophages (PIMMs) and subsequent increased host sensitivity to <i>Escherichia coli</i> LPS challenge. Therefore, to further clarify the biology of PIMMs we examined the role of recruited PIMMs in a rat <i>Escherichia coli</i>-induced lung inflammation model. Following sepsis, lung inflammation was induced with recruitment of PIMMs and subsequently, <i>Escherichia coli</i> LPS challenge exacerbated the lung inflammation with localization of multiple inflammatory cytokines in PIMMs to suggest their possible involvement in modulating lung inflammation in this model.<p> In order to delineate mechanisms of barn air induced lung dysfunction, a rat model of occupational exposure was characterized to show that one and five exposures to the barn environment induced acute lung inflammation and increased airway hyperresponsiveness (AHR). Following 20 exposures, AHR was dampened to indicate adaptive responses. Barn air contains high levels of endotoxin which led us to investigate its role in lung inflammation and AHR. Exposure of mice with either a functional TLR4 (WT) or non-functional TLR4 (mutants) to barn air revealed dependence of lung inflammation but not AHR on a functional TLR4.<p> I investigated whether exposure to barn air alters host responses to a subsequent microbial challenge. Following one day barn exposure and <i>Escherichia coli</i> LPS challenge, lung inflammation was exacerbated with increased granulocytes and IL-1β levels compared to one day barn exposed rats without <i>Escherichia coli</i> LPS challenge. However, increased granulocytes and IL-1β levels in barn exposed and <i>Escherichia coli</i> LPS challenged rats were not different from control rats treated with <i>Escherichia coli</i> LPS indicating a lack of priming effect of barn exposure. However, above results are suggestive of an underlying risk of increased lung inflammation following secondary microbial infection in naïve barn workers.<p> Lastly, I investigated the expression and activity of novel signalling molecules called <i>N</i>-myristoyltransferase and calcineurin in barn air and <i>E. coli</i> LPS induced lung inflammation models. Following one day barn exposure, increased protein expression but not activity of <i>N</i>-myristoyltransferase and calcineurin was shown. However, there is a need to identify the specific role of these two molecules in barn air induced lung inflammation. To conclude, animal models of barn exposure are useful tools to understand mechanisms of lung inflammation and AHR. However, there is still a need to examine endotoxin-independent nature of AHR and roles of other molecules of the innate immune system in regulating barn air induced effects.

Occupational exposure to endotoxin-rich swine barn air induces respiratory diseases and loss of lung function. Barn exposure induces recruitment of pulmonary intravascular monocytes/macrophages (PIMMs) and subsequent increased host sensitivity to <i>Escherichia coli</i> LPS challenge. Therefore, to further clarify the biology of PIMMs we examined the role of recruited PIMMs in a rat <i>Escherichia coli</i>-induced lung inflammation model. Following sepsis, lung inflammation was induced with recruitment of PIMMs and subsequently, <i>Escherichia coli</i> LPS challenge exacerbated the lung inflammation with localization of multiple inflammatory cytokines in PIMMs to suggest their possible involvement in modulating lung inflammation in this model.<p> In order to delineate mechanisms of barn air induced lung dysfunction, a rat model of occupational exposure was characterized to show that one and five exposures to the barn environment induced acute lung inflammation and increased airway hyperresponsiveness (AHR). Following 20 exposures, AHR was dampened to indicate adaptive responses. Barn air contains high levels of endotoxin which led us to investigate its role in lung inflammation and AHR. Exposure of mice with either a functional TLR4 (WT) or non-functional TLR4 (mutants) to barn air revealed dependence of lung inflammation but not AHR on a functional TLR4.<p> I investigated whether exposure to barn air alters host responses to a subsequent microbial challenge. Following one day barn exposure and <i>Escherichia coli</i> LPS challenge, lung inflammation was exacerbated with increased granulocytes and IL-1β levels compared to one day barn exposed rats without <i>Escherichia coli</i> LPS challenge. However, increased granulocytes and IL-1β levels in barn exposed and <i>Escherichia coli</i> LPS challenged rats were not different from control rats treated with <i>Escherichia coli</i> LPS indicating a lack of priming effect of barn exposure. However, above results are suggestive of an underlying risk of increased lung inflammation following secondary microbial infection in naïve barn workers.<p> Lastly, I investigated the expression and activity of novel signalling molecules called <i>N</i>-myristoyltransferase and calcineurin in barn air and <i>E. coli</i> LPS induced lung inflammation models. Following one day barn exposure, increased protein expression but not activity of <i>N</i>-myristoyltransferase and calcineurin was shown. However, there is a need to identify the specific role of these two molecules in barn air induced lung inflammation. To conclude, animal models of barn exposure are useful tools to understand mechanisms of lung inflammation and AHR. However, there is still a need to examine endotoxin-independent nature of AHR and roles of other molecules of the innate immune system in regulating barn air induced effects.

Asthma is a chronic respiratory disease with a high prevalence in Western countries, including Canada, and increased exacerbations have been associated with ambient air pollution. The maintenance of airways tone is critically dependent on the endogenous bronchodilator, nitric oxide (NO). The nitric oxide synthase (NOS) isoenzymes produce NO from the amino acid, L-arginine, and competition for substrate with the arginase isoenzymes can limit NO production. Imbalances between these pathways have been implicated in the airways hyperresponsiveness (AHR) of asthma. The overall objective of this work was to determine whether arginase and downstream polyamine metabolites are functionally involved in airways responsiveness in animal models of asthma and the adverse responses of allergic animals to air pollution. To this purpose, the expression profiles of proteins involved in L-arginine metabolism were determined in lung tissues from human asthmatics and murine models of ovalbumin (OVA)-induced airways inflammation. Expression of arginase 1 was increased in human asthma and animal models. Competitive inhibition of arginase attenuated AHR in vivo. The roles of the downstream metabolites of arginase, the polyamines (putrescine, spermidine and spermine) were examined by administering them via inhalation to anaesthetized mice. It was demonstrated that spermine increases methacholine responsiveness in normal and allergic mice. Additionally, inhibition of polyamine synthesis improved AHR in a murine model. Thus, arginase and downstream polyamine metabolites contribute to AHR in asthma. Finally, the potential role of arginase in the exacerbation of asthma by air pollution was investigated. For this purpose, murine sub-acute and chronic murine models of allergic airways inflammation were employed, which exhibit inflammatory cell influx and remodeling/AHR, respectively, to determine the role of arginase in the response to concentrated ambient fine particles plus ozone. Allergic mice that were exposed to air pollution exhibited increased arginase activity and expression, compared to filtered air-exposed controls. Furthermore, inhibition of arginase attenuated the air pollution-induced AHR. Thus, the studies of the arginase pathway and downstream metabolites described in this thesis indicate that arginase inhibition may be a therapeutic target in asthma and may also protect susceptible populations against the adverse health effects of air pollution.

Asthma is a chronic respiratory disease with a high prevalence in Western countries, including Canada, and increased exacerbations have been associated with ambient air pollution. The maintenance of airways tone is critically dependent on the endogenous bronchodilator, nitric oxide (NO). The nitric oxide synthase (NOS) isoenzymes produce NO from the amino acid, L-arginine, and competition for substrate with the arginase isoenzymes can limit NO production. Imbalances between these pathways have been implicated in the airways hyperresponsiveness (AHR) of asthma. The overall objective of this work was to determine whether arginase and downstream polyamine metabolites are functionally involved in airways responsiveness in animal models of asthma and the adverse responses of allergic animals to air pollution. To this purpose, the expression profiles of proteins involved in L-arginine metabolism were determined in lung tissues from human asthmatics and murine models of ovalbumin (OVA)-induced airways inflammation. Expression of arginase 1 was increased in human asthma and animal models. Competitive inhibition of arginase attenuated AHR in vivo. The roles of the downstream metabolites of arginase, the polyamines (putrescine, spermidine and spermine) were examined by administering them via inhalation to anaesthetized mice. It was demonstrated that spermine increases methacholine responsiveness in normal and allergic mice. Additionally, inhibition of polyamine synthesis improved AHR in a murine model. Thus, arginase and downstream polyamine metabolites contribute to AHR in asthma. Finally, the potential role of arginase in the exacerbation of asthma by air pollution was investigated. For this purpose, murine sub-acute and chronic murine models of allergic airways inflammation were employed, which exhibit inflammatory cell influx and remodeling/AHR, respectively, to determine the role of arginase in the response to concentrated ambient fine particles plus ozone. Allergic mice that were exposed to air pollution exhibited increased arginase activity and expression, compared to filtered air-exposed controls. Furthermore, inhibition of arginase attenuated the air pollution-induced AHR. Thus, the studies of the arginase pathway and downstream metabolites described in this thesis indicate that arginase inhibition may be a therapeutic target in asthma and may also protect susceptible populations against the adverse health effects of air pollution.

Background: According to recent studies in children, IgE sensitization not only against perennial allergens, but also against food allergens, is related to asthma risk and increased airway inflammation. During the last decade, a new technique for IgE determination based on allergen components has become available, but its use in epidemiological studies has been limited. Aims: To investigate the relationship between the pattern of IgE sensitization to allergen components and the prevalence of asthma, airway inflammation and hyperresponsiveness in a population-based setting. To examine the relationship of IgE sensitization to allergen extract, and airway inflammation, airway hyperresponsiveness and blood eosinophilia in asthmatics. To examine the natural history of IgE sensitization to food allergens in adults. To compare extract-based and component-based IgE measurements in relation with new-onset respiratory disease and airway inflammation and hyperresponsiveness. Methods: The present thesis is based on cross-sectional and longitudinal analyses of the adult, the population-based study ECRHS (European Community Health Survey) and a cross-sectional, observational study of young subjects with asthma. IgE sensitization was examined by means of both extract-based and component-based tests. Airway inflammation was assessed by exhaled NO and airway hyperresponsiveness with methacholine test. Results: IgE sensitization to food allergens independently related to increased airway inflammation in both a population-based study and a study of asthmatics. Furthermore, a relation was found with increased blood eosinophils in asthmatics. The decrease in prevalence of IgE sensitization against food allergens during a 9-year follow-up was larger than the decrease of aeroallergens. Subjects with IgE sensitization to both cat extract and components showed more frequent airway inflammation, greater bronchial responsiveness and higher likelihood of developing asthma and rhinitis than subjects with IgE sensitization only to cat extract. Conclusions: The presence of IgE antibodies against food allergens was independently associated with airway and systemic inflammation. Both aeroallergens and food allergens should be examined in order to understand the signaling of local and systemic inflammation in asthma. Prevalence of IgE sensitization to food decreased in adults to a larger extent than IgE sensitization against aeroallergens. Measurement of IgE sensitization to cat allergen components appears to have a higher clinical value than extract-based measurement

Aims. The overall aim was to examine the severity, treatment and level of control in patients with asthma in primary care in Sweden. The specific aims were to assess what matters to asthma patients, evaluate symptoms, medication and identify factors related to asthma severity, compare the extent of asthma control in 2001 and 2005, and investigate the development of asthma and degree of asthma control in adolescents and young adults who had reported asthma six years earlier. Methods. The first study was a telephone interview of a representative sample of Swedish asthmatics. In the second study a random sample of 1,136 patients answered two questionnaires. A classification of the asthma severity similar to that in the GINA guidelines was made. In the third study two surveys were performed, in 2001 and in 2005, with a random sample of 1,012 and 224 asthma patients, respectively, and a classification of asthma control similar to the recent GINA guidelines was made. In the fourth study 71 individuals who reported physician-diagnosed asthma in a population-based survey in 1997 and were defined as current asthmatics, were reinvestigated in 2003 with a skin prick test, methacholine challenge test, eucapnic voluntary hyperventilation test and measurement of exhaled nitric oxide. Results. Common situations causing symptoms of asthma were physical exertion and contact with pets. Nocturnal symptoms were frequent. In primary care 35% of the women and 24% of the men were classified as having severe asthma. Female sex, increasing age, not filling the asthma prescription owing to cost, daily smoking, and pollen allergy increased the odds of having severe asthma. In 2001, 37% had achieved asthma control, as compared with 40% in 2005. Uncontrolled asthma was more common in women and smokers. In the 2003 study of adolescents and young adults with asthma six years earlier, the definition of current asthma was fulfilled by 50 of the 71 subjects and one third had achieved asthma control. Conclusions. The majority of the asthmatics reported a large number of symptoms and limitations in their daily living. Many asthma patients in primary care have insufficient asthma control. One reason for lack of control might be undertreatment with inhaled corticosteroids.

<p>Asthma is a disease characterized by transient airway smooth muscle contraction leading to episodes of reversible airway narrowing. It affects over 300 million people worldwide and is implicated in over 250 000 deaths annually. The primary clinical features of asthma include airway inflammation, hyperresponsiveness, and remodeling. Generally, asthmatic patients experience exacerbations between periods of diminished symptoms. Interestingly, in addition to these above mentioned hallmarks, asthmatics have also been shown to react differently to ventilatory mechanical strain. This is most evident when assessing the effect of a deep inspiration (DI), clinically measured as a breath taken from functional residual capacity to total lung capacity, in healthy individuals <em>versus</em> asthmatics. These deep inspiratory efforts have been shown to produce a bronchodilatory response in healthy individuals, whereas in asthmatics, DIs are less effective in producing bronchodilation, can cause more rapid airway re-narrowing, and even bronchoconstriction in moderate to severe asthmatics. The mechanism by which a DI is able to cause bronchoconstriction remains ambiguous. Previous theories suggest that this phenomenon is intrinsic to airway smooth muscle (ASM) itself. However, the airway inflammation present in asthmatic airways may also add to the increased ASM contractility following stretch, by the release of mediators that can prime the contractile apparatus to react excessively in the presence of stretch.</p> <p>Thus, collectively, the studies contained in this thesis are linked to the general theme of greater characterization of the signalling mechanisms that regulate airway stretch-activated contractions using a pharmacological approach in intact bovine bronchial segments, with the hope of providing novel insights into the mechanisms that regulate the DI-induced bronchoconstriction seen in asthmatics.</p> / Doctor of Philosophy (Medical Science)

<p><strong>Introduction</strong></p> <p>Asthma is a chronic respiratory disease that is becoming more prevalent. Airway hyperresponsivness, a key feature of asthma, involves increased narrowing of the airways in response to bronchoconstricting agents. Airway smooth muscle (ASM) functioning is largely responsible for hyperresponsiveness yet the mechanisms behind excitation-contraction coupling are not fully understood. Src tyrosine kinase contributes to contraction in other smooth muscle types. Furthermore, STIM1, Orai1, IPLA₂b and RyRs play a role in ASM excitation-contraction coupling.</p> <p><strong>Aim</strong></p> <p>We sought to determine whether Src activity is involved in serotonin (5-HT)- and acetylcholine (ACh)-induced ASM contraction. We also examined whether the gene expression of molecules involved in sarcoplasmic reticulum emptying and refilling is altered during airway hyperresponsiveness.</p> <p><strong>Methods</strong></p> <p>Bovine tracheal ASM strips were pre-treated with the non-specific tyrosine kinase inhibitor genistein (10^-4M), src kinase family inhibitors PP1 (10^-5M) and PP2 (10^-5M) or vehicle and challenged with either 5-HT or ACh to determine the involvment of Src in contraction. Western blotting was used to examine Src activity following 5-HT or ACh treatment. Female BALB/c mice were exposed to an intranasal injection of [1.7mg/ml] HDM extract or saline. Real time, reverse-transcriptase polymerase chain reaction was used to examine gene expression.</p> <p><strong> </strong></p> <p><strong>Results</strong></p> <p>Genistein, PP1 and PP2 significantly reduced 5-HT-induced ASM contractions and Src activity was significantly increased in response to 5-HT. ACh-induced contractions were significantly reduced by genistein, but not PP1 and PP2. However, Src activity was significantly increased by ACh. RyR3 mRNA expression was significantly increased, Orai1 was significantly decreased, and STIM1, IPLA₂b, RyR1 and RyR2 were unchanged by the house dust mite treatment.</p> <p><strong>Conclusion</strong></p> <p>These data suggets 5-HT-induced ASM contraction involves Src activity. However, ACh-induced ASM contractions might not require Src. The changes in RyR3 and Orai1 expression might alter Ca²⁺-handling in such a way as to potentiate airway hyperresponsiveness but further investigation is required.</p> / Master of Science (MSc)

Doctor of Philosophy (PhD) / Asthma is a chronic inflammatory disease of the airways. Corticosteroid medication is the most effective currently available treatment. Complications of corticosteroid therapy are dose-dependent, however, the clinical efficacy of varying doses of inhaled corticosteroids has been studied with mixed results. A randomized, double-blind, parallel group study was used to evaluate the inhaled corticosteroid dose-response relationship for clinical endpoints and in vitro parameters of underlying airway inflammation and remodelling. The mannitol provocation test with Forced Oscillation Technique (FOT) was used to derive potential dose-differentiating endpoints. In vitro inflammatory markers were measured in alveolar macrophages from bronchoalveolar lavage. Basement membrane thickness was measured from bronchial biopsies. Eleven nonasthmatic subjects were enrolled for comparison. This thesis addresses the null hypothesis that there is no significant difference in clinical and biological effects between low dose (200mcg/day, n=11) and high dose (1000mcg/day, n=11) treatment (for 6-7 weeks) with inhaled fluticasone propionate (FP) for a range of clinical outcomes and in vitro markers of airway inflammation and remodelling. Significant changes after FP included increased FEV1, reduced airway hyperresponsiveness (AHR) (by FOT and FEV1), exhaled nitric oxide and Juniper symptom score. In addition, significant reductions occurred in expression of GM-CSF, TNF-alpha and IL-1ra in macrophages. A lower baseline FOT-derived respiratory system conductance was predictive of a greater degree of improvement in symptoms. No statistically significant differences in the changes after treatment between low and high dose FP were found in spirometry, exhaled nitric oxide, symptom scores, AHR, alveolar macrophage cytokine levels (GM-CSF, TNF-alpha, IL-1ra, IL-10) and basement membrane thickness, although there were trends towards greater improvements in many of the parameters after high dose FP. Basement membrane thickness appeared to be reduced by high dose FP, although this reduction was not statistically significant. There was a weak, but statistically significant, negative correlation between basement membrane thickness and FOT-derived conductance (r2=0.135, p=0.042). With the recognition of the limitations in the interpretation of these data, the results suggest that, in previously steroid naïve mild to moderate asthmatics, there may be only minimal benefit derived from an additional 800µg/day of inhaled fluticasone above the low dose of 200µg/day.