Introdução: A importância do parênquima pulmonar na fisiopatologia da asma tem sido recentemente enfatizada, particularmente nos pacientes com asma grave e de difícil controle. O óxido nítrico (NO) é um importante modulador da resposta contrátil, inflamatória e de remodelamento pulmonar que ocorrem na asma. Embora seu papel na modulação em vias aéreas proximais e distais já tenha sido estabelecido, seus efeitos no parênquima pulmonar foram pouco investigados. Objetivos: Avaliar se a inibição da arginase 2, por intermédio do tratamento com N-hydroxy-nor-L-arginine (nor-NOHA), e da iNOS, por intermédio da administração de 1400W, pode modular a resposta constritora de parênquima distal, o estresse oxidativo, a expressão de iNOS, assim como a atividade da arginase, em modelo de inflamação alérgica crônica pulmonar em cobaias. Métodos: Os animais foram expostos a sete inalações com soro fisiológico ou com ovoalbumina em doses crescentes (1~5mg/ml- 4 semanas) e tratados com 1400W (2mg/Kg ip. diariamente) iniciando após a 7ª inalação e nor- NOHA (10M - infusão no banho durante a avaliação da mecânica oscilatória), ou associação de 1400W + nor-NOHA (administrado conforme descrito anteriormente). Setenta e duas horas após a sétima inalação os animais foram anestesiados, exsanguinados e a foi realizada a avaliação da mecânica oscilatória do parênquima pulmonar, sendo obtidos os valores de resistência e elastância tecidual na condição basal e após desafio (0,1% de ovoalbumina). Em seguida, os fragmentos de tecido pulmonar periférico foram fixados em solução de formolaldeído a 4%, por 48 horas. Depois de terminada a fixação, o material foi submetido às técnicas histológicas habituais com parafina, para obtenção de cortes de 4m de espessura. Os cortes foram corados para Hematoxilina e Eosina e utilizando técnica de imunohistoquímica foram avaliados o número de células iNOS positivas, a expressão de PGF2, do fator de transcrição NF-kB e de arginase 2 no septo alveolar, por intermédio de avaliação morfométrica. A expressão de arginase 2, NF-kB e PGF2 nos fragmentos de tecido pulmonar periférico foram avaliados em aumento 400X utilizando o programa Image-Pro Plus 4.5v Image Analysis System e os resultados foram expressos em porcentagem. Em relação ao número de células iNOS positivas nos fragmentos pulmonares, foi utilizada a técnica de contagem de pontos, determinada pelo número de pontos que coincidiam em células positivas em cada campo dividido pelo número de pontos que incidiam no tecido pulmonar, no aumento de 1000x. Foram analisados 10 campos por corte, selecionados de forma randômica. Os resultados foram expressos como células por unidade de área (104m2). Para a avaliação da atividade da arginase 2, foi utilizado o método descrito no kit da BioAssay Systems que utiliza um cromogênio, que forma um complexo corado com uréia, produzido na reação de arginase. A determinação da atividade da arginase 2 envolveu a medida da velocidade de reação, expressa em termos de atividade por miligrama de proteína (U/mg). Resultados: Não houve alteração no percentual de estruturas (vasos, vias aéreas e tecido alveolar) no tecido pulmonar nos quatro grupos experimentais, sendo que mais de 90% de tecido do fragmento pulmonar foi constituído de alvéolos. Os animais que foram expostos à ovoalbumina apresentaram um aumento da resistência e da elastância tecidual (após desafio antigênico), no número de células iNOS positivas, na percentagem de expressão de PGF2, arginase 2 e NF-kB no septo alveolar, assim como da atividade de arginase 2 comparativamente aos grupos controles (p<0,001). O tratamento com nor-NOHA ou 1400W isoladamente em animais expostos à ovoalbumina atenuou a resposta de elastância e resistência teciduais após desafio antigênico (p<0,001), o número de células iNOS positivas (p<0,001), na expressão de PGF2, de arginase 2 e NF-kB no septo alveolar (p<0,001). Porém a atividade da arginase 2 só foi reduzida nos grupos que foram tratados com nor-NOHA e na associação de 1400W e nor-NOHA (p<0,05). A associação de 1400W e nor-NOHA em animais expostos à ovoalbumina potencializou a atenuação da expressão PGF2 no septo alveolar (p<0,001). Conclusões: O presente estudo sugere que, neste modelo experimental de inflamação crônica pulmonar, a inibição da arginase tem papel importante na modulação das repostas constritoras e de estresse oxidativo no parênquima pulmonar distal. A associação da inibição da iNOS à da arginase 2 potencializa o controle da resposta de estresse oxidativo. Estas respostas são, pelo menos em parte, moduladas pela ativação de NF-kB. A inibição destas vias enzimáticas pode representar uma estratégia futura para o tratamento de pacientes com asma grave e de difícil controle / Introduction: The importance of the lung parenchyma in the pathophysiology of asthma has recently been emphasized, particularly in patients with severe asthma and difficult to control. Nitric oxide (NO) is an important modulator of contractile response, lung inflammation and remodeling occurring in asthma. Although its role in the modulation proximal and distal airways tone control has already been established, its effects on the lung parenchyma were rarely investigated. Aims: To evaluate the inhibition of arginase 2, through treatment with N-hydroxy-nor-L-arginine (nor-NOHA) and / or iNOS, through the administration of 1400W, can modulate the constrictor response of distal parenchyma, oxidative stress, the expression of iNOS and the activity of arginase, in a model of chronic allergic lung inflammation in guinea pigs. Methods: The seven animals were exposed to inhaled saline or ovalbumin with increasing doses (1~5mg/ml-4 weeks) and treated with 1400W (2mg/kg ip daily) beginning after the 7th inhalation and nor-NOHA (10M - infusion in the bath during the evaluation of mechanical oscillation), or combination of 1400W + nor- NOHA (administered as formerly). Seventy-two hours after the seventh inhalation the animals were anesthetized, and exsanguinated and it was performed evaluation was made of oscillatory mechanics of lung parenchyma, and obtained the values of tissue resistance and elastance at baseline and after challenge (0.1% ovalbumin). Then the peripheral lung tissue fragments were fixed in formolaldeíde 4% for 48 hours. After completion of fixation, the material was subjected to routine histological techniques with paraffin to obtain sections of 4m thickness. The sections were stained with Hematoxylin and Eosin and by using immunohistochemical technique, we assessed the number of iNOS positive cells and expression of PGF2, of the transcription factor NF-kB and arginase 2 in the alveolar septum, through morphometric analysis. The expression of arginase 2, NF-kB and PGF2 in peripheral lung tissue fragments were evaluated at 400X increase by Image - Pro Plus Image Analysis System 4.5v and the results were obtained as a ratio between the amount of expression of arginase 2, NF-kB and PGF2 and are expressed as percentage. Regarding the content of iNOS in lung fragments, we used the technique of counting points, determined by the number of points coinciding in the positive cells in each field divided by the number of points that focused on lung tissue, using the increase of 1000x. Were increase of 10 fields analyzed per section, selected randomly. The results were expressed as cells per unit area (104m2). To assess the activity of arginase 2 we used the method bioassay described in the kit Systems using a chromogen which forms a colored complex with urea produced in the reaction of arginase. The determination of the activity of arginase 2 involved the measurement of reaction rate, expressed in terms of activity per milligram of protein (U/mg). Results: There were no changes in the percentages of structures (vessels, airways and alveolar tissue) lung tissue in four experimental groups, with more than 90% of the tissue fragment consisted of pulmonary alveoli. The animals that were exposed to ovalbumin, showed an increase in tissue resistance and elastance after antigen challenge, the number of iNOS positive cells, the of expression of PGF2, of NF-kB and arginase 2 in the alveolar septum well as in the activity of arginase 2, compared to control groups (p <0.001). The treatment with nor-NOHA or 1400W alone in animals exposed to ovalbumin attenuated the tissue elastance and resistance after antigenic challenge (p <0.001), the number of iNOS positive cells (p <0.001), the expression of PGF2, arginase 2 and NFkB in the alveolar septum (p<0.001). However the activity of arginase 2 was reduced in only in the groups that were treated with nor-NOHA and the association of 1400W and nor-NOHA (p <0.05). The combination of 1400W and nor-NOHA, in animals exposed to ovalbumin enhanced the attenuation of the expression of PGF2 in the alveolar septum (p <0.001). Conclusions: The present study suggests that in this experimental model of chronic pulmonary inflammation, inhibition of arginase 2 plays an important role in the modulation of constrictor responses and oxidative stress in the distal lung parenchyma. The association of iNOS inhibition to the arginase 2 enhances the control of oxidative stress response. These responses are, at least in part, modulated by the activation of NF-kB. The inhibition of these enzyme pathways may represent a future strategy for the treatment of patients with severe asthma and difficult to control

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

L'huile d'olive est la principale source d’apports lipidique dans le régime méditerranéen. Elle est reconnue comme une source de nutriments et est à l’origine de plusieurs effets biologiques bénéfiques pour la santé. Plusieurs études montrent notamment que la consommation d'huile d'olive peut être efficace dans le traitement de maladies cardiovasculaires. Cependant, peu d'études mettent en évidence un effet antiinflammatoire, antiallergique et anti-Alzheimer de l’huile d’olive. De même, aucune étude n’a déterminé un lien entre ces effets et les variétés d’olives dont sont issues les huiles ainsi que les composés présents responsables de ces effets. Le but de ce travail est donc d'évaluer l'effet biologique, et plus particulièrement le potentiel antiinflammatoire et antiallergique des composés hydrophiles obtenus après extraction en phase solide (SPE-Diol), à partir de plusieurs variétés d’huiles d'olive extra vierge (EVOO) issues de trois années de récolte différentes. L'effet neuroprotecteur de la variété Picholine, contre la maladie d'Alzheimer a également été évalué. Les résultats montrent que l'effet anti-inflammatoire et l’effet anti-allergique varies de façon concentration dépendante et en fonction de la variété d’EVOO. De plus, nous avons mis en évidence que la variété de Picholine a un effet neuroprotecteur. L’ensemble de ce travail a établi une corrélation entre des activités anti-inflammatoire, anti-allergique et anti-Alzheimer des huiles d’olives issues de plusieurs variétés et les différents composés présents, ce qui a permis de mettre en avant la variété Picholine. / Olive oil is the primary source of fat in the Mediterranean diet. It is not only known as a distinctive source of nutrients but also as an origin of several biological effects that represent a very important factor for the health. Several studies demonstrate that olive oil consumption has to be effective against heart and cardiovascular diseases. Few studies show up the anti-inflammatory, the anti-allergic and the anti-Alzheimer effects of olive oil but there is no study about the cultivar effect and the interference that could occur between the different olive oil compounds. The aim of this work is to evaluate the biological effect and more particularly the potential anti-inflammatory and anti-allergic of hydrophilic components obtained by solid phase extraction (SPE-Diol) of different extra virgin olive oil (EVOO) cultivars and from three different harvest years. Moreover the neuroprotective effect of Picholine cultivar against Alzheimer’s disease was also studied. The results show that the anti-inflammatory and the anti-allergic activities differ depending on the EVOO cultivar and they are concentration-dependent. The results show also that Picholine cultivar has an anti-Alzheimer activity. All of these studies have established a correlation between the anti-inflammatory, the anti-allergic and the anti-Alzheimer effects of different EVOO cultivars and different compounds present, which enabled us to highlight the Picholine cultivar.

Introdução: A importância do parênquima pulmonar na fisiopatologia da asma tem sido recentemente enfatizada, particularmente nos pacientes com asma grave e de difícil controle. O óxido nítrico (NO) é um importante modulador da resposta contrátil, inflamatória e de remodelamento pulmonar que ocorrem na asma. Embora seu papel na modulação em vias aéreas proximais e distais já tenha sido estabelecido, seus efeitos no parênquima pulmonar foram pouco investigados. Objetivos: Avaliar se a inibição da arginase 2, por intermédio do tratamento com N-hydroxy-nor-L-arginine (nor-NOHA), e da iNOS, por intermédio da administração de 1400W, pode modular a resposta constritora de parênquima distal, o estresse oxidativo, a expressão de iNOS, assim como a atividade da arginase, em modelo de inflamação alérgica crônica pulmonar em cobaias. Métodos: Os animais foram expostos a sete inalações com soro fisiológico ou com ovoalbumina em doses crescentes (1~5mg/ml- 4 semanas) e tratados com 1400W (2mg/Kg ip. diariamente) iniciando após a 7ª inalação e nor- NOHA (10M - infusão no banho durante a avaliação da mecânica oscilatória), ou associação de 1400W + nor-NOHA (administrado conforme descrito anteriormente). Setenta e duas horas após a sétima inalação os animais foram anestesiados, exsanguinados e a foi realizada a avaliação da mecânica oscilatória do parênquima pulmonar, sendo obtidos os valores de resistência e elastância tecidual na condição basal e após desafio (0,1% de ovoalbumina). Em seguida, os fragmentos de tecido pulmonar periférico foram fixados em solução de formolaldeído a 4%, por 48 horas. Depois de terminada a fixação, o material foi submetido às técnicas histológicas habituais com parafina, para obtenção de cortes de 4m de espessura. Os cortes foram corados para Hematoxilina e Eosina e utilizando técnica de imunohistoquímica foram avaliados o número de células iNOS positivas, a expressão de PGF2, do fator de transcrição NF-kB e de arginase 2 no septo alveolar, por intermédio de avaliação morfométrica. A expressão de arginase 2, NF-kB e PGF2 nos fragmentos de tecido pulmonar periférico foram avaliados em aumento 400X utilizando o programa Image-Pro Plus 4.5v Image Analysis System e os resultados foram expressos em porcentagem. Em relação ao número de células iNOS positivas nos fragmentos pulmonares, foi utilizada a técnica de contagem de pontos, determinada pelo número de pontos que coincidiam em células positivas em cada campo dividido pelo número de pontos que incidiam no tecido pulmonar, no aumento de 1000x. Foram analisados 10 campos por corte, selecionados de forma randômica. Os resultados foram expressos como células por unidade de área (104m2). Para a avaliação da atividade da arginase 2, foi utilizado o método descrito no kit da BioAssay Systems que utiliza um cromogênio, que forma um complexo corado com uréia, produzido na reação de arginase. A determinação da atividade da arginase 2 envolveu a medida da velocidade de reação, expressa em termos de atividade por miligrama de proteína (U/mg). Resultados: Não houve alteração no percentual de estruturas (vasos, vias aéreas e tecido alveolar) no tecido pulmonar nos quatro grupos experimentais, sendo que mais de 90% de tecido do fragmento pulmonar foi constituído de alvéolos. Os animais que foram expostos à ovoalbumina apresentaram um aumento da resistência e da elastância tecidual (após desafio antigênico), no número de células iNOS positivas, na percentagem de expressão de PGF2, arginase 2 e NF-kB no septo alveolar, assim como da atividade de arginase 2 comparativamente aos grupos controles (p<0,001). O tratamento com nor-NOHA ou 1400W isoladamente em animais expostos à ovoalbumina atenuou a resposta de elastância e resistência teciduais após desafio antigênico (p<0,001), o número de células iNOS positivas (p<0,001), na expressão de PGF2, de arginase 2 e NF-kB no septo alveolar (p<0,001). Porém a atividade da arginase 2 só foi reduzida nos grupos que foram tratados com nor-NOHA e na associação de 1400W e nor-NOHA (p<0,05). A associação de 1400W e nor-NOHA em animais expostos à ovoalbumina potencializou a atenuação da expressão PGF2 no septo alveolar (p<0,001). Conclusões: O presente estudo sugere que, neste modelo experimental de inflamação crônica pulmonar, a inibição da arginase tem papel importante na modulação das repostas constritoras e de estresse oxidativo no parênquima pulmonar distal. A associação da inibição da iNOS à da arginase 2 potencializa o controle da resposta de estresse oxidativo. Estas respostas são, pelo menos em parte, moduladas pela ativação de NF-kB. A inibição destas vias enzimáticas pode representar uma estratégia futura para o tratamento de pacientes com asma grave e de difícil controle / Introduction: The importance of the lung parenchyma in the pathophysiology of asthma has recently been emphasized, particularly in patients with severe asthma and difficult to control. Nitric oxide (NO) is an important modulator of contractile response, lung inflammation and remodeling occurring in asthma. Although its role in the modulation proximal and distal airways tone control has already been established, its effects on the lung parenchyma were rarely investigated. Aims: To evaluate the inhibition of arginase 2, through treatment with N-hydroxy-nor-L-arginine (nor-NOHA) and / or iNOS, through the administration of 1400W, can modulate the constrictor response of distal parenchyma, oxidative stress, the expression of iNOS and the activity of arginase, in a model of chronic allergic lung inflammation in guinea pigs. Methods: The seven animals were exposed to inhaled saline or ovalbumin with increasing doses (1~5mg/ml-4 weeks) and treated with 1400W (2mg/kg ip daily) beginning after the 7th inhalation and nor-NOHA (10M - infusion in the bath during the evaluation of mechanical oscillation), or combination of 1400W + nor- NOHA (administered as formerly). Seventy-two hours after the seventh inhalation the animals were anesthetized, and exsanguinated and it was performed evaluation was made of oscillatory mechanics of lung parenchyma, and obtained the values of tissue resistance and elastance at baseline and after challenge (0.1% ovalbumin). Then the peripheral lung tissue fragments were fixed in formolaldeíde 4% for 48 hours. After completion of fixation, the material was subjected to routine histological techniques with paraffin to obtain sections of 4m thickness. The sections were stained with Hematoxylin and Eosin and by using immunohistochemical technique, we assessed the number of iNOS positive cells and expression of PGF2, of the transcription factor NF-kB and arginase 2 in the alveolar septum, through morphometric analysis. The expression of arginase 2, NF-kB and PGF2 in peripheral lung tissue fragments were evaluated at 400X increase by Image - Pro Plus Image Analysis System 4.5v and the results were obtained as a ratio between the amount of expression of arginase 2, NF-kB and PGF2 and are expressed as percentage. Regarding the content of iNOS in lung fragments, we used the technique of counting points, determined by the number of points coinciding in the positive cells in each field divided by the number of points that focused on lung tissue, using the increase of 1000x. Were increase of 10 fields analyzed per section, selected randomly. The results were expressed as cells per unit area (104m2). To assess the activity of arginase 2 we used the method bioassay described in the kit Systems using a chromogen which forms a colored complex with urea produced in the reaction of arginase. The determination of the activity of arginase 2 involved the measurement of reaction rate, expressed in terms of activity per milligram of protein (U/mg). Results: There were no changes in the percentages of structures (vessels, airways and alveolar tissue) lung tissue in four experimental groups, with more than 90% of the tissue fragment consisted of pulmonary alveoli. The animals that were exposed to ovalbumin, showed an increase in tissue resistance and elastance after antigen challenge, the number of iNOS positive cells, the of expression of PGF2, of NF-kB and arginase 2 in the alveolar septum well as in the activity of arginase 2, compared to control groups (p <0.001). The treatment with nor-NOHA or 1400W alone in animals exposed to ovalbumin attenuated the tissue elastance and resistance after antigenic challenge (p <0.001), the number of iNOS positive cells (p <0.001), the expression of PGF2, arginase 2 and NFkB in the alveolar septum (p<0.001). However the activity of arginase 2 was reduced in only in the groups that were treated with nor-NOHA and the association of 1400W and nor-NOHA (p <0.05). The combination of 1400W and nor-NOHA, in animals exposed to ovalbumin enhanced the attenuation of the expression of PGF2 in the alveolar septum (p <0.001). Conclusions: The present study suggests that in this experimental model of chronic pulmonary inflammation, inhibition of arginase 2 plays an important role in the modulation of constrictor responses and oxidative stress in the distal lung parenchyma. The association of iNOS inhibition to the arginase 2 enhances the control of oxidative stress response. These responses are, at least in part, modulated by the activation of NF-kB. The inhibition of these enzyme pathways may represent a future strategy for the treatment of patients with severe asthma and difficult to control

Introdução: Estima-se que 10% dos doentes com asma t€m sintomas e limita‚es importantes, como exacerba‚es freqƒentes ou redu„o persistente da fun„o respirat…ria As altera‚es do par€nquima pulmonar distal tem sido recentemente abordadas na fisiopatologia da asma. Apesar do uso de corticoster…ides, pacientes com asma refrat†ria t€m mais estresse oxidativo, assim como apresentam ativaao da iNOS. Al‡m disso, muitos dos dispositivos utilizados para administra„o de ester…ides inalat…rios geram partˆculas que n„o chegam efetivamente ‰s vias a‡reas distais e ao par€nquima pulmonar. Objetivos: Avaliamos os efeitos do tratamento com montelucaste ou dexametasona tratamentos associados ou n„o a um inibidor especˆfio da iNOS (1400W) na resposta eosinofˆlica, remodelamento da matriz extracelular, estresse oxidativo, conteŠdo de actina, c‡lulas positivas para IL4, IL5, MMP9, TIMP1, IFN, TGF do par€nquima em cobaias com inflama„o crnica pulmonar. Métodos: As cobaias foram inaladas com ovalbumina (grupo OVA) 2X/semana por 4semanas. Ap…s a 4Ž inala„o, as cobaias foram tratadas diariamente com montelucaste (grupo OVAM 10mg/Kg/PO/dia) ou dexametasona (grupo OVAD 5mg/Kg/IP/dia). O inibidor da iNOS, 1400W (grupo OVAW 1mg/kg/dia) foi administrado intraperitonealmente nos Šltimos 4 dias (OVAW, OVADW e grupos OVAMW). Ap…s 72 horas da 7Ž inala„o, as cobaias foram anestesiadas, e os fragmentos de tecido pulmonar distal foram submetidos ‰ avalia„o histopatol…gica. Resultados: Houve um aumento no infiltrado eosinofˆlco, nas c‡lulas positivas para IL4, IL5, TIMP1, MMP9, iNOS, IFN TGF, conteŠdo de actina, isoprostano PGF2 alfa, fibras col†genas e el†sticas nos animais OVA em compara„o com animais SAL (p<0,05). Houve uma diminui„o no nŠmero de eosin…filos, c‡lulas positivas para IL4, IL5, MMP9, TIMP1, IFN, TGF, conteŠdo de actina, col†geno e isoprostano PGF2 alfa em todos os grupos tratados em compara„o com animais OVA (p<0,05). O conteŠdo de fibras el†sticas foram reduzidas somente nos grupos OVAMW, OVADW e OVAW em compara„o com animais OVA (p<0,05). A associa„o de 1400W e o tratamento com montelucaste (grupo OVAMW) potencializou a redu„o do conteŠdo de actina, fibras el†sticas, isoprostano PGF2 alfa de c‡lulas positivas para IL4, IL5, TIMP1, IFN TGF e iNOS em rela„o ao grupo montelucaste (OVAM) (p<0,05). Os tratamentos com 1400W e dexametasona (grupo OVADW) contribuˆram para uma maior redu„o do conteŠdo das fibras el†sticas, actina e isoprostanoPGF2 alfa e o nŠmero de c‡lulas positivas para IL4, IL5, IFN e TIMP1 em rela„o ao grupo dexametasona (OVAD) (p<0,05). Conclusões: O tratamento com corticoster…ides associados ‰ inibi„o da iNOS contribuiu para uma maior redu„o da remodela„o da matriz extracelular, diminuiu o estresse oxidativo, e tamb‡m foi eficiente para atenuar a resposta inflamat…ria Th2 no par€nquima pulmonar distal. Por outro lado, o tratamento com montelucaste associado à inibição da iNOS mostrou uma maior eficácia para reduzir o teor de fibras elásticas, a ativação do estresse oxidativo, conteúdo de actina e expressão das células positivas para IL4, IL5 no parênquima pulmonar distal. Estas associações podem representar futuras ferramentas farmacológicas para o controle das alterações histopatológicas pulmonares distais induzidas pela inflamação crônica / Introduction: It is estimated that 10% of asthma patients have symptoms and important limitations such as frequent exacerbations or persistent reduction of resiratory function, despite the use of corticosteroids. The alterations of distal lung parenchyma have been recently evaluated on asthma pathophysiology, particulary in patients with refractory asthma and difficcult to control. These patients have increased oxidative stress responses, mainly with significant activation of iNOS. Aims: We evaluated the effects of montelukast or dexamethasone treatments associated or not to an iNOS inhibitor (1400W) on eosinophilic response, extracellular matrix remodeling, oxidative stress, actin content, IL4, IL5, MMP9, TIMP1, IFN gama, TGF beta positive cells of distal lung parenchyma in guinea pigs with chronic alergic inflammation. Methods: Guinea Pigs were inhaled with ovalbumin (OVA group) twice a week for four weeks. After 4th inhalation, GP were treated with montelukast (OVAM group-10mg/Kg/PO/day) or dexamethasone (OVAD group-5mg/Kg/IP/day). The treatment with iNOS inhibitor 1400W (OVAW group-1mg/kg/day) was given daily in the last 4 days (OVAW, OVADW and OVAMW groups). After 72 hours of 7th inhalation, GP were anesthetized, lung strips were retired and submitted to histopathological evaluation. Results: There was an increase in eosinophilic infiltrate, in the number of positive cells for IL4, IL5, TIMP1, MMP9, iNOS, IFN gama TGF beta, actin, isoprostane PGF2 alpha, elastic and collagen fiber contents in OVA animals comparing to SAL group (p<0,05). There was a decrease in the number of eosinophils, IL4, IL5, MMP9, TIMP1, IFN gama, TGF beta positive cells, collagen, actin and isoprostane PGF2 alpha content in all treated groups compared to OVA animals (p<0.05), but the treatment with montelukast did not reduce the positive cells for IFN gama, compared to OVA (p>0.05). Elastic fiber content were reduced only in OVAMW, OVADW and OVAW groups compared to OVA animals (p<0.05). The association of 1400W and montelukast treatments potentiated the reduction of actin, elastic fibres and isoprostane PGF2 alpha contents and the number of IL4, IL5, TIMP1, IFN gama, TGF beta and iNOS positive cells compared to montelukast group (p<0.05). The treatments with 1400W and dexamethasone contributed to a greater reduction of elastic fibers, actin and isoprostane PGF2 alpha contents and the number of IL4, IL5, IFNgama and TIMP1 positive cells compared to dexamethasone group (p<0.05). Conclusions: Corticosteroid treatment associated to iNOS inhibition contributes to a greater reduction of extracellular matrix remodeling, decreases the oxidative stress, and also is efficient to attenuate the Th2 inflammatory response in distal lung parenchyma. On the other hand, montelukast treatment associated to iNOS inhibition showed a higher efficacy to reduce elastic fibres content, oxidative stress activation, actin content and IL4 and IL5 expression in distal lung parenchyma. These associations may represent future pharmacological tools for controlling distal pulmonary histopathological alterations induced by chronic inflammation

The eosinophil is a leukocyte whose intracellular mediators are considered to play a central role in the pathogenesis of allergic diseases, including allergic asthma, allergic rhinitis and atopic dermatitis, and which is also involved in immunological responses to parasites. Eosinophil differentiation and maturation from bone marrow progenitors is regulated by interleukin-5 (IL-5), which may be secreted by T helper 2 (Th2) T lymphocytes, and is consistently upregulated in allergic conditions. Eotaxin is a potent chemoattractant for circulating and tissue eosinophils, and the production of this chemokine promotes eosinophil infiltration and accumulation within sites of allergic inflammation.¶ Eosinophils obtained from inflammatory tissues and secretions display an altered phenotype in comparison to peripheral blood eosinophils, with increased surface expression of major histocompatibility complex (MHC) proteins and adhesion molecules (Hansel et al., 1991), and migration across the microvascular endothelium may also increase their capacity to generate an oxidative burst (Walker et al., 1993; Yamamoto et al., 2000). Eosinophils are phagocytic cells, and have been shown to present simple (no requirement for intracellular processing) and complex antigens to MHC-restricted, antigen-specific T lymphocytes (Del Pozo et al., 1992; Weller et al., 1993). Furthermore, eosinophils express the costimulatory molecules required for effective antigen presentation (Tamura et al., 1996), and ligation of costimulatory molecules on the eosinophil cell surface can induce the release of eosinophil derived cytokines (Woerly et al., 1999; Woerly et al., 2002). Therefore the eosinophil may also regulate immune responses.¶ To date, no studies have demonstrated the ability of eosinophils to modulate activated T lymphocyte function via presentation of relevant antigen in the context of MHC class II (MHC-II), concomitant with Th2 cytokine release. In the experiments described in this thesis, murine eosinophils have been observed to rapidly migrate to sites of antigen deposition within the airways mucosa of naïve mice, suggesting a potential role for this granulocyte in the primary response to inhaled antigen. However, human allergic diseases are often diagnosed after the establishment of allergic responses, and symptom development. Therefore, a murine model of allergic airways disease (AAD) was used to investigate the ability for eosinophils to participate as antigen presenting cells (APCs), and thereby modulate activated T lymphocyte function both in vitro and in vivo. Detailed histological analysis of the pulmonary draining lymph nodes following antigen challenge in sensitised mice revealed a rapid infiltration of eosinophils into this tissue, which preceded the accumulation of eosinophils in bronchoalveolar lavage fluid (BALF). This suggested that eosinophils were preferentially translocating to the draining lymph nodes following antigen challenge, and that the subsequent accumulation of these cells in the BALF was a consequence of continued antigen delivery to the lower airways.¶ Eosinophil trafficking to lymphoid tissue via the afferent lymphatics was substantiated using electron microscopy of lymph node sections and the intravenous (i.v.) transfer of fluorescently labeled eosinophils, which did not traffic to lymph nodes via the blood. During the resolution of AAD, eosinophils were noted for their persistence in the pulmonary draining lymph nodes. These observations suggested a continued modulation of T cell function by lymph node dwelling eosinophils during AAD resolution, particularly in light of recent observations for draining lymph node T cell proliferation following instillation of antigen-pulsed eosinophils into the allergic mouse lung (Shi et al., 2000).¶ To further investigate the antigen presenting capacity, eosinophils were obtained from the BALF of mice with AAD, and their surface expression of MHC class II (MHC-II) proteins and costimulatory molecules confirmed using flow cytometric analysis. The ability to acquire and process complex antigen both in vitro and in vivo was also confirmed using naturally quenching fluorescenated ovalbumin (OVA), which is degraded into fluorescent peptides by the action of intracellular proteases. Thus, eosinophil expression of the surface molecules necessary for effective antigen presentation was confirmed, as was their ability to process complex antigen. Further investigations revealed that eosinophils can present complex OVA antigen to CD4+ T lymphocytes obtained from the allergic mouse, and to in vitro derived OVA-specific Th2 cells. In the presence of exogenous antigen, eosinophils co-cultured with T lymphocytes were able to induce Th2 cytokine production, and demonstrated an ability for eosinophils to modulate T lymphocyte function in vitro.¶ The ability for eosinophils to act as antigen presenting cells in vivo was also investigated. Eosinophils obtained from the antigen-saturated lungs of OVA sensitised and challenged mice were transferred to the peritoneal cavities of naïve host mice. When subsequently challenged with aerosolised OVA, eosinophil recipients developed a pulmonary eosinophilia similar to that of OVA sensitised and challenged mice. To validate this finding, the experimental procedure was altered to accommodate the use of non-allergy derived eosinophils, which were pulsed with OVA in vitro, prior to transfer into naïve recipients. When subsequently challenged with aerosolised OVA, eosinophil recipients developed a peripheral blood and pulmonary eosinophilia, and stimulation with OVA induced IL-5 and IL-13 cytokine production from pulmonary draining lymph node cells. Notably, the AAD induced by transfer of antigen pulsed eosinophils did not induce detectable OVA-specific IgG1, which may be attributed to the lack of soluble antigen required for B cell antibody production.¶ During the course of these investigations, an OVA T cell receptor (TCR) transgenic mouse (OT-II) was procured with a view to defining the interaction between eosinophils and activated T lymphocytes (Barnden et al., 1998). Despite having specificity for the OVA323-339 peptide, an immunodominant epitope that skews naïve T cell responses towards Th2 cytokine release (Janssen et al., 2000), T lymphocytes from the OT-II mouse preferentially secreted IFN-γ in response to stimulation with either OVA peptide or OVA. These mice were further characterised in a mouse model of AAD, and found to be refractory to disease induction and progression, which may be attributed to significant IFN-γ secretion by transgenic CD4+ T lymphocytes during antigen sensitisation. Indeed, these cells were noted for their ability to attenuate pulmonary eosinophilia when transferred to OVA sensitised and challenged wild type mice, although serum OVA-specific IgG1, peripheral blood eosinophilia levels and airways response to methacholine challenge remained intact.¶ Knowledge of the biased Th1 phenotype in naïve OT-II provided a unique opportunity to investigate the fate of T lymphocytes bearing high affinity OVA-specific TCRs following neonatal antigen exposure to soluble OVA. In a previous study, subcutaneous (s.c.) administration of soluble OVA to wild type neonatal mice was suspected to have deleted OVA-specific T cells from the T cell repertoire (Hogan et al., 1998a). Using flow cytometry and TCR specific antibody, the delivery of s.c. OVA to OT-II neonates did not alter transgenic T cell populations in adult mice. Instead, it was surprising to find a skewing towards the Th2 phenotype and loss of IFN-γ secretion following OVA sensitisation and challenge in adult mice. A mechanism for this reprogramming of the transgenic T cell from the Th1 to a Th2 phenotype following OT-II neonatal exposure to soluble OVA is proposed, and further experimentation may validate this hypothesis.¶ In conclusion, eosinophils residing in the allergic lung have the capacity to interact with activated T cells, both within this tissue and the draining lymph nodes. Despite their relative inefficiency as antigen presenting cells (Mawhorter et al., 1994), eosinophils may participate en masse in the serial triggering of activated TCRs, and provide appropriate costimulatory signals that modulate T lymphocyte function. Through the elaboration of Th2 cytokines and stimulation of T cell proliferation, antigen presenting eosinophils may transiently prolong or exacerbate the symptoms of allergic diseases. Alternatively, eosinophils presenting relevant antigens may inhibit T cell activity via degranulation, and such activity has recently been observed in a parasite model (Shinkai et al., 2002). Finally, experiments in the OT-II mouse have provided valuable information to suggest that therapies designed to modulate eosinophil numbers in allergic tissues through the secretion of opposing cytokines such as IFN-γ, may be of limited benefit. The results shown here suggest that airways dysfunction remains intact despite significantly reduced pulmonary eosinophilia

The aim of this thesis was to identify risk factors for the onset of adult asthma. Other objectives were to study determinants of smoking habits and the association between sensitization and outcome of asthma. In 1990, a questionnaire was distributed to 12,732 individuals from three age groups (16, 30-39 and 60-69 years) in two counties of Sweden. In a second phase, 2538 subjects who had reported respiratory symptoms and 600 controls were invited to clinical investigations, 81% participated. At the follow-up in 2003 subjects of the remaining cohort (11,282) were re-invited. Analyses are based on the 67% (n=7563) who responded to both questionnaires 1990 and 2003. In 2003, 17.2% of the young adults, 11.4% of the middle-aged and 10.3% of the elderly reported having, or having had, asthma. A total of 791 subjects reported onset of asthma during the 13-year study period. Lifestyle factors such as smoking, obesity, hard physical training and a low consumption of fruit and fish were constant risk factors for onset of asthma after adjusting for socioeconomic group. A smoker’s risk of asthma onset was increased by 37%. The impact of risk factors differed between the age-groups. BMI had a significantly higher impact in the middle-aged and elderly. In subjects participating in the clinical investigations in 1990, sensitization to pets, were determinants of both persistent asthma and onset of asthma in 2003. The risk for persistent asthma was threefold. The risk for onset of asthma was more than doubled. Smoking at baseline in 1990 was the strongest determinant of being a smoker in 2003. Allergic sensitization and clinically verified asthma were not associated with smoking habits in 2003. No differences in changing smoking habits could be identified between smokers with or without asthma. In conclusion, modifiable lifestyle factors are important risk factors for adult onset asthma. The co-occurrence and interplay between asthma and cigarette smoking is still puzzling.

Background It is debated whether a low total diversity of the gut microbiota in early childhood is more important than an altered prevalence of particular bacterial species for the increasing incidence of allergic disease. The advent of powerful, cultivation-free molecular methods makes it possible to characterize the total microbiome down to the genus level in large cohorts. Objective We sought to assess microbial diversity and characterize the dominant bacteria in stool during the first year of life in relation to atopic eczema development. Methods Microbial diversity and composition were analyzed with barcoded 16S rDNA 454-pyrosequencing in stool samples at 1 week, 1 month, and 12 months of age in 20 infants with IgE-associated eczema and 20 infants without any allergic manifestation until 2 years of age (ClinicalTrials.gov ID NCT01285830). Results Infants with IgE-associated eczema had a lower diversity of the total microbiota at 1 month (P = .004) and a lower diversity of the bacterial phylum Bacteroidetes and the genus Bacteroides at 1 month (P = .02 and P = .01) and the phylum Proteobacteria at 12 months of age (P = .02). The microbiota was less uniform at 1 month than at 12 months of age, with a high interindividual variability. At 12 months, when the microbiota had stabilized, Proteobacteria, comprising gram-negative organisms, were more abundant in infants without allergic manifestation (Empirical Analysis of Digital Gene Expression in R [edgeR] test: P = .008, q = 0.02). Conclusion Low intestinal microbial diversity during the first month of life was associated with subsequent atopic eczema. / <p>Funding Agencies|BioGaia AB, Stockholm, Sweden||Ekhaga Foundation, the Heart and Lung foundation||Research Council for the South-East Sweden|F2000-106|Olle Engqvist Foundation||Swedish Asthma and Allergy Association||Swedish Research Council||University Hospital of Linkoping||Soderberg Foundation||Vardal Foundation for Health Care Science and Allergy Research, Sweden||BioGaia AB||</p>

Background & Objectives: There has been few investigation of the association between the farming related activities or specific characteristics and atopic disease in rural Canadian children. In population-based studies, assuring the quality of information from questionnaires is of concern. We conducted this study in order to: first, identify the prevalence and risk factors of atopy and allergic conditions among school-age children in a rural region of Canada. Also, we sought to evaluate the validity and reliability of a questionnaire report of allergy to assess in this population. Methods: As part of a longitudinal study of lung health in rural residents, we conducted a cross-sectional baseline study in rural Saskatchewan, Canada. This included an initial survey phase followed by a clinical testing phase. A sub-sample of 584 children (grades 1-8) completed skin prick testing to assess atopic status. Of these, 480 children completed a questionnaire report of allergy and atopic outcomes and participated in skin prick testing (SPT). Atopy was defined as a positive reaction to any of 6 allergens (local grasses, wheat dust, cat dander, house dust mite, Alternaria, Clasdosporium)≥3mm compared to the negative control. Agreement between questionnaire report and objective measures of atopy was considered overall and between the specific allergens tested on SPT and those assessed on questionnaire. We considered percent concordance, Kappa, sensitivity, specificity, and the positive and negative predictive values of reported allergies or allergic conditions in comparison to SPT as the gold standard. Results: The prevalence of atopy as well as allergen-specific sensitizations was similar between farm and non-farm children but supported the notion that livestock farming is protective against atopy. Also, we found that 25.0% of children reported a history of allergic conditions by questionnaire and 19.4% were atopic detected by skin pick test. In our study, the agreement between questionnaire report of specific allergic triggers and atopy measured by SPT was high (83.0% - 89.5%). Conclusion: In children, livestock exposure has a protective effect on SPT positivity.The agreement between questionnaire report of allergic symptoms and atopy measured by SPT was high and the agreement between atopy and report of allergic conditions was moderate.

Diss. (sammanfattning) Stockholm : Karol inst., 2004. / Härtill 5 uppsatser.