Submitted by Karina Calciolari null (karinaveterinaria@hotmail.com) on 2016-04-18T19:09:55Z No. of bitstreams: 1 Dissertação_Karina Calciolari.pdf: 3537872 bytes, checksum: 25bdb76342a16861139075d0c9e32394 (MD5) / Approved for entry into archive by Felipe Augusto Arakaki (arakaki@reitoria.unesp.br) on 2016-04-19T17:12:27Z (GMT) No. of bitstreams: 1 calciolari_k_me_jabo.pdf: 3537872 bytes, checksum: 25bdb76342a16861139075d0c9e32394 (MD5) / Made available in DSpace on 2016-04-19T17:12:27Z (GMT). No. of bitstreams: 1 calciolari_k_me_jabo.pdf: 3537872 bytes, checksum: 25bdb76342a16861139075d0c9e32394 (MD5) Previous issue date: 2016-02-22 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / As afecções do sistema respiratório são a segunda causa de queda de desempenho e afastamento dos equinos do esporte ou trabalho, as quais estão atrás somente das desordens musculoesqueléticas. A estabulação e alimentação a base de fenos predispõe os equinos a inalação de grande número de agentes irritantes as vias aéreas. No intuito de mensurar a influência dos tipos de cama utilizado em baias de equinos nas respostas alérgicas das vias aéreas estudou-se 3 grupos com 5 equinos cada sendo dois mantidos em cocheiras com diferentes tipos de forração à base de madeira (maravalha de pinus autoclavada-MA e serragem-SE) e por último um grupo controle à pasto por 45 dias com avaliações quinzenais (D0, D15, D30 e D45). Foi realizado nestes momentos o exame físico, coleta de amostras de sangue venoso e arterial para realização de hemograma e hemogasometria respectivamente, lavado broncoalveolar com análise citológica e mensuração de fosfatase alcalina, além de amostras das camas (limpa e suja) e feno pré-secado para análise micológica. No exame físico foi notado tosse e espirros em maior intensidade e número nos equinos do grupo SE. A análise hematológica permaneceu dentro dos padrões de normalidade. Já nos achados hemogasométricos os maiores valores de pH foram observados nos equinos à pasto (7,47 ± 0,02). Nos grupos embaiados foi observado valores de pressão parcial de dióxido, de D15 à D45, inferiores (valores entre 38,3 a 43,7 mmHg) ao grupo a pasto (valores entre 43,7 a 46,5 mmHg), sendo os valores de pressão parcial de oxigênio inversamente proporcionais (MA e SE: valores entre 103,6 a 127,78 mmHg e à pasto: 88,2 a 103,6 mmHg). Os principais achados no lavado broncoalveolar basearam-se na contagem de células nucleadas totais no que apresentou maiores valores em todos os momentos nos animais à pasto (251,0 a 336,8 x 103/ µL), seguido do grupo SE (197,0 a 248,6 x 103/ µL) e por último o MA (140,6 à 270,2 x 103/ µL). Dentre as células nucleadas os macrófagos representaram maior porcentagem seguidos dos linfócitos (51,2 a 80,8% e 6,6 a 33,2% respectivamente). Um padrão inesperado foi observado na população de neutrófilos, os quais com os passar dos momentos houve decréscimo, com excessão do grupo MA que apresentou um pico no D30 (35%), relacionado ao pico de fosfatase alcalina (27,6 ± 14,98 UI/L) neste mesmo momento. Foram identificados cerca de 12 gêneros fúngicos em ambas camas e 17 no alimento volumoso, sendo neste último encontrado gêneros patogênicos como Aspergillus (0,85 ± 2,2%), Fusarium (5,35 ± 9,23%) e Penicílium (4,81 ± 9,53%), porém em baixas porcentagens. A principal diferença entre as camas baseou-se na capacidade da produção e dispersão de partículas finas passíveis de inalação, a qual foi maior para a serragem (MA: 15,29 % e SE: 18,28%). Concluiu-se que a cama de maravalha além de ter manejo mais fácil, apresentou menor capacidade irritativa das vias aéreas superiores do que a serragem. As condições de manejo e estabulações utilizadas foram ideais, servindo de modelo quanto a ventilação e higienização das baias. / Horse respiratory diseases is second most commom cause of poor performance and premature retirement, which the one are musculoskeletal disorders. The stabling and poor quality feedings may the favor predispose of diseases in airways. In order we measure the influence of the types of bedding used in the horse stalls about responses allergic airway. Three groups of five horses each are formated, two groups kept in stables but each group with a different type of bedding (autoclaved wood- MA shaving and sawdust-SE) . The third group is the control and stays at pasture. The groups remained in this condition for 45 days with biweekly reviews (D0, D15, D30 and D45). It was realize the physical examination, venous and arterial blood samples collection to realize hemogram and hemogasometry analysis, the bronchoalveolar lavage samples was colected to realize the cytological analysis and to measure alkaline phosphatase. In addition, samples of the beds (clean and dirty) and haylage was collected to realize mycological analysis. In the physical examination was noticed higher intensity of coughing and sneezing at SE group. Hematological analysis remained within the normal standards. Findings of hemogasometric analysis were observed the biggest pH values at horses to pasture (7.47 ± 0.02). At stables horses was observed smaller values of partial pressure dioxide (D15 to D45), (values between 38.3 to 43.7 mmHg) to horses to pasture (values between 43.7 to 46.5 mmHg). The values oxygen partial pressure it was inversely proportional to partial pressure dioxide (MA and SE: values between 103.6 to 127.78 mmHg and pasture: 88.2 to 103.6 mmHg). It was observate higher values of nucleated cells of bronchoalveolar lavage, at all times, in animals to pasture (251.0 to 336.8 x 103 / mL), followed by the SE group (197.0 to 248.6 x 103 / mL) and finally MA group (140.6 to 270.2 x 103 / mL). The macrophages cells represented the biggest percentage of nucleates cells on bronchoalveolar lavage followed by lymphocytes (51.2 to 80.8% and 6.6 to 33.2% respectively). An unexpected pattern was observed in percentange of neutrophils cells, that it was a decrease over the time. One exception it was with MA group that had the biggest percentage values in D30 (35%), it was related to alkaline phosphatase peak at same time (27.6 ± 14.98 IU / L). Were identified about 12 fungi genres in both beds and 17 fungi genres at haylage. We indentified pathogenic genres in haylage as Aspergillus (0.85 ± 2.2%), Fusarium (5.35 ± 9.23%) and Penicillium (4 , 81 ± 9.53%), however in lower percentages. It was observate a difference production capacity and fine particle dispersion between the bed, that the biggest it was notice at sawdust (MA: 15.29% and SE: 18.28%). It was concluded that wood shavings bed is easier to use and showed less irritative capacity upper airway to que sawdust. As management and stabling conditions used were ideals and it was a ideal model of stables.

Surfactant proteins, SP-A and SP-D, are collagen-containing calcium-dependent (C-type) lectins, called, collectins. Their primary structure has four regions: a cysteine-linked N- terminal region involved in multimerization, a collagen region composed of Gly-X-Y repeats, coiled-coil neck region, and the C-terminal carbohydrate recognition domains (CRD) or C-type lectin domain. SP-A looks like a bouquet, while SP-D is a cruciform- like structure, with four arms of equal length. SP-A and SP-D have been shown to act as innate immune molecules at pulmonary as well as extra-pulmonary sites by binding to pathogens, allergens and apoptotic/necrotic cells via their CRD region. SP-A and SP-D can induce pathogen neutralization and enhanced phagocytosis. In addition, SP-A and SP-D can interact via CRDs with allergens and dampen allergic reaction in vitro and in vivo. This thesis examines in vitro interaction of a recombinant fragment of human SP-D containing neck and CRD regions (rhSP-D) with IAV and Aspergillus fumigatus, in addition to characterizing a dichotomy of the effects of SP-A and SP-D on dendritic cells in an attempt to explain how SP-A and SP-D modulate DC functions differentially. Experiments involving interaction of rhSP-D with IAV pandemic strain show that it can be a restrictive factor against the virus, in addition to modulating immune response by a macrophage cell line. The rhSP-D can have anti-A. fumigatus effect directly and indirectly in the context of pathogen as well as allergen. A comparison has been made between two recombinant fragments of SP-D that have been expressed with and without 8 Gly-X-Y repeats for their fungistatic properties. The effects of SP-A and SP-D on cultured DC maturation, and effector cytokine and proliferative response of co-cultured cells have also been examined in vitro.

Made available in DSpace on 2015-05-14T13:00:11Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 2644910 bytes, checksum: 94bc3ca3dd5252435423f525d32a2ea9 (MD5) Previous issue date: 2009-04-01 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Asthma is a chronic inflammatory disease characterized by cell migration and bronchial hyperreactivity (HRB). One strategy to treat allergic diseases is the development of new drugs with good efficacy and few side effects. Flavonoids are plant compounds known for its antioxidant, antiallergic and anti-inflammatory activities. Based on this premise, the study aimed to select flavonoids by the immunopharmacological screening and to evaluate prophylactic and therapeutic treatments in the experimental asthma model. For this purpose the flavonoids tested were myricetin, isoramnetin and glycosylated kampherol known as 3-O-[β-D-glycopiranosil-(1→6)-α-L-rhamnopyranosyl]-7-O-α-L-rhamnopyranosyl-kamferol (GRRK). The experimental model was BALB/c mice sensitized and challenged with ovalbumin (OVA) and the treatments were before (prophylactic) or after (therapeutic) to establishment of the experimental asthma model. The three flavonoids inhibited the animal death during the anaphylactic chock reaction induced by OVA and the inflammatory cell migration to the lung; however only the GRRK was used in the following experiments because of its best yield in the purification process. Both prophylactic and therapeutic treatments with GRRK (30 mg/kg) decreased significantly the total number of inflammatory cells (P< 0.05), eosinophils cells (P< 0.05), IL-5 (P< 0.05) and IL-13 (P< 0.05) production from bronchoalveolar lavage (BAL), IgE OVA-specific title (P< 0.01) in the sera, the bronchial hyperreactivity (HRB) (P< 0.05) induced by methacholine and mucus production (P< 0.001) by the globbet cells from the lung as compared with non-treated GRRK animals. The results obtained in the different treatments with GRRK were statistically comparable with that observed in the OVA-sensitized group treated with dexametasone. Moreover, treatment with GRRK was able to reduce the number of CD4+ T cells (p <0.001), B cells (p <0.001), expression of major complex of compatibility II class (MHC II class) and CD40 molecules in the antigen presenting cells (CD11b+ CD11c+) from BAL. Although the GRRK induced a suppressive effect in the Th2 immune response it was not able to exacerbate the Th1 immune response by IFN- production. The production of this cytokine was not modified when compared with non treated animals. The GRRK treatment also was not able to modify the regulatory immune response because there was no significant change in the production of TGF- and the number of TCD4+Foxp3+, a marker of LT regulatory (TReg). These results demonstrated that GRRK treatment before (prophylactic) or after (therapeutic) the establishment of allergic asthma, restored the morpho-functional changes in the airways by Th2-dependent immunossupression, although independent of Th1 and TReg. / A asma é uma doença inflamatória crônica caracterizada por migração celular para os pulmões e hiperreatividade brônquica (HRB). Uma estratégia para o tratamento de doenças alérgicas é o desenvolvimento de novos medicamentos com boa eficácia e poucos efeitos colaterais. Flavonóides são compostos derivados de plantas conhecidos por suas atividades antioxidante, antialérgica e antiinflamatória. Partindo desta premissa, o trabalho teve como objetivo selecionar flavonóides a partir de análises imunofarmacológicas e avaliar os tratamentos profiláticos ou terapêuticos no modelo de asma alérgica experimental. Para tal, os flavonóides testados foram: myricetina, isoramnetina e kanferol glicosilado conhecido por 3-O-[β-D-glycopiranosil-(1→6)-α-L-ramnopiranosil]-7-O-α-L-ramnopiranosil-kanferol (GRRK). O modelo experimental foi realizado em camundongos BALB/c sensibilizados e desafiados com ovalbumina (OVA) e os tratamentos foram antes (profilático) ou após (terapêutico) estabelecer o modelo de asma alérgica experimental. Os três flavonóides inibiram a morte dos animais durante o choque anafilático induzido por OVA e a migração de células da inflamação para os pulmões, entretanto, apenas o GRRK foi usado nos demais experimentos, devido ao seu melhor rendimento no processo de purificação. Os tratamentos profilático e terapêutico com GRRK (30 mg/kg) diminuíram, de forma significativa, o número total de células inflamatórias (P< 0,05), de eosinófilos (P< 0,05) a produção de IL-5 (P< 0,05) e IL-13 (P< 0,05) no lavado broncoalveolar (LBA), o título de IgE-OVA-específica (P< 0,01) no soro, a hiperreatividade brônquica (HRB) (P< 0,05) induzida com metacolina e a produção de muco (P< 0,001) pelas células caliciformes de pulmão quando comparados com os animais não tratados com GRRK e sensibilizado com OVA. Os resultados obtidos nos diferentes tratamentos com GRRK foram comparáveis estatisticamente aos efeitos observados no grupo de animais tratados com a droga padrão dexametasona. Além disso, o tratamento com GRRK foi capaz de diminuir: o número de linfócitos T CD4+ (P< 0,001) e de linfócitos B (P< 0,001), a expressão de moléculas de classe II do complexo principal de histocompatibilidade (MHC II) e da molécula CD40 em células apresentadoras de antígeno (CD11b+CD11c+) do LBA. Embora o tratamento com GRRK tenha induzido um efeito supressor na resposta imune de perfil Th2, não foi capaz de exacerbar a resposta imune Th1 com a produção de IFN- A produção desta citocina se manteve inalterada quando comparada ao grupo de animais não tratados. O tratamento também não foi capaz de alterar a reposta imune reguladora, pois não houve mudança significativa na produção de TGF- e no número de células TCD4+Foxp3+, marcador de LT regulador (LTReg). Esses resultados demonstram que o tratamento com GRRK antes (profilático) ou depois (terapêutico) de estabelecer a asma alérgica experimental, restabeleceu as alterações morfofuncionais das vias aéreas, por um mecanismo modulador das células Th2, embora independente de Th1 e de Treg

L’allergie est une pathologie touchant un très grand nombre de personnes à travers le monde en particulier dans les pays industrialisés. Actuellement seule l’éviction totale de l’allergène évite toute nouvelle réaction allergique. Jusqu’à présent différents types de tests ont été développés afin de déterminer le pouvoir allergisant d’un grand nombre de composés. Parmi ces tests certains font appel à l’utilisation de la voie animale. Cependant, dans le cadre de la législation sur les cosmétiques ces tests sont amenés à disparaître. Dans ce contexte il est nécessaire de développer des stratégies intégrées combinant à la fois des tests in silico, in chemico et in vitro. Ce travail de thèse propose d’analyser des données de réactivité issues de tests « in chemico » réalisés au laboratoire, et de les comparer avec des données biologiques issues de tests « in vitro » sur l’activation de la voie Keap1/Nrf2/ARE chez les lignées de cellules dendritiques de type THP-1 et MoDCs (INSERM UMR-996). Ainsi, la réactivité de 10 composés connus pour leur implication dans des réactions allergiques a été étudiée au cours de ce travail permettant d’apporter des éléments de réponse sur l’existence d’une corrélation entre la capacité d’une molécule à réagir avec une protéine, et donc à être sensibilisante, et le stress oxydant induisant la maturation des cellules dendritiques. / Contact dermatitis is one of the most common health problems and is highly prevalent in industrialized countries. Currently, only the total eviction of the allergen can prevent further allergic reactions. Since several years, different tests have been developed to determine the sensitization potential of a large number of compounds. Some of them are based on animal models. However, the cosmetic legislation now bans this kind of tests. In this context, the development of integrated strategies based on in silico, in chemico and on in vitro assays seems to be necessary. The aim of this PhD work is to compare reactivity data from in chemico tests realized at the laboratory, with biological data obtained from in vitro tests concerning the activation of the Keap1/Nrf2/ARE pathway in THP-1 and MoDCs dendritic cells (INSERM UMR-996). The reactivity of 10 compounds known for their implication in allergic contact dermatitis has been studied to show if a correlation exists between the potential of sensitization of allergens and the oxidative stress inducing dendritic cells maturation.

O N-metil- 3-4, metilenodioximetanfetamina (MDMA) ou ecstasy tem sido freqüentemente usado por jovens. Analisamos neste trabalho, dentro de uma perspectiva neuroimune, os efeitos da administração aguda de MDMA em parâmetros comportamentais, neuroendócrinos, hematatológicos e imunes de camundongos Balb/C, usando para esta última finalidade um modelo de asma experimental. O MDMA produziu: 1- aumento diferencial da atividade locomotora nas diferentes zonas do campo aberto; aumento na locomoção total e diminuição da atividade exploratória no hole-board; aumento da porcentagem de entradas e da taxa de permanência nos braços abertos do LCE; aumento no tempo gasto na caixa de saída e diminuição do número de acessos de risco em uma caixa de exposição a um predador; 2- aumento dos níveis séricos de corticoterona; 3- aumento dos níveis de noradrenalina no estriado e córtex frontal, aumento nos níveis de dopamina e de DOPAC no estriado, diminuição dos níveis de DOPAC corticais, aumento de 5-HT e 5-HIAA no estriado, diminuição dos níveis de 5- HIAA e do turnover de serotonina no hipotálamo e diminuição do \"turnover\" de dopamina no estriado e córtex frontal; 4- alteração na migração de leucócitos em camundongos alérgicos com diminuição da porcentagem de linfócitos e monócitos circulantes, diminuição do número de granulócitos no lavado bronco alveolar (LBA), efeitos estes que foram revertidos pelo pré-tratamento com RU-486; 5 redução da expressão de L-selectinas por monócitos e tendência de redução da expressão de L -selectinas por neutrófilos no pulmão; 6- diminuição das produções espontâneas de IL-4, IL-5 e IL-10 e de IL-4 em cultura estimulada com LPS; 7- redução da contração da traquéia isolada de animais alérgicos e 8- redução da desgranulação dos mastócitos em brônquios intrapulmonares. Sugeriu-se que o estresse/ansiedade induzidos pelo MDMA tenham ativado o eixo HHA e/ou do sistema nervoso autonômico simpático dos camundongos, alterando a resposta imune dos mesmos na vigência de um modelo de asma. A inflamação alérgica pulmonar desponta, assim, como importante ferramenta para o entendimento da ação de drogas de abuso em processos neuroimunológicos. / The N-metil- 3-4, metilenodioximetanfetamina (MDMA) or ecstasy is a drug widely used amongst young people. This study was undertaken to analyze, under a neuroimmune perspective, the effects of acute MDMA administration on behavioral, neuroendocrine, hematological and immune parameters on Balb/C mice, using for the latter purpose the allergic lung inflammatory response model. It was observed that MDMA produced in mice: 1- a differential increase on total locomotion in the different open-field zones; an increase on total locomotion and a decrease on exploratory activity in the hole-board; an increase on both percentage of entrances and time spent on plus-maze open arms; an increase on time spent in the starting box and a decrease of risk assessments in a predator exposition box; 2- an increase in corticosterone serum levels; 3- an increase in striatal and frontal cortex noradrenaline levels, an increase in striatal dopamine and DOPAC levels, a decrease in cortical DOPAC levels, an increase in striatal 5-HT and 5-HIAA levels, a decrease in both 5-HIAA levels and 5-HT turnover rates in hypothalamus and a decrease in striatal and cortical dopamine \"turnover\" rates; 4- an alteration on leukocyte migration in allergic mice, i.e., decreased percentage of peripheral blood lymphocytes and monocytes, decreased number of granulocytes on bronchoalveolar lavage fluid ( LBA); these effects were reverted by previous RU-486 treatment; 5- a decrease in L-selectin expression by monocytes and a tendency towards a decrease in L-selectin expression by lung neutrophils; 6- a decrease on expontaneous production of IL-4, IL-5 e IL-10 and IL-4 in LPS-stimulated cultures; 7- a decrease in the contraction of allergic mice isolated trachea; and, 8- a decrease in bronchial mastocytes degranulation. It was suggested that MDMA-induced anxiety/stress symptoms increasing HHA-axis and/or the autonomic nervous system activities this leading to the immune changes observed presently in the allergic lung inflammation model of asthma used. This model, thus, emerges as a useful tool for the understanding of neuroimmune effects of drugs of abuse.

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2016-08-31T14:54:02Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) AVALIAÇÃO DO BRONCOESPASMO INDUZIDO POR EXERCÍCIO EM RINÍTICOS NÃO ASMÁTICOS - EDIL DE ALBUQUERQU.pdf: 3820113 bytes, checksum: c21e0ff0d89c8a2255c71a63f439e88b (MD5) / Made available in DSpace on 2016-08-31T14:54:02Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) AVALIAÇÃO DO BRONCOESPASMO INDUZIDO POR EXERCÍCIO EM RINÍTICOS NÃO ASMÁTICOS - EDIL DE ALBUQUERQU.pdf: 3820113 bytes, checksum: c21e0ff0d89c8a2255c71a63f439e88b (MD5) Previous issue date: 2016-02-29 / CAPEs / As queixas respiratórias após exercício físico são comuns em riniticos, asmáticos e em indivíduos sem estas doenças. Uma das causa é o broncoespasmo induzido pelo exercício (BIE), definido como o estreitamento dos brônquios após exercício físico vigoroso. Estes sintomas podem limitar as atividades destes adolescentes e acarretar em consequências negativas nas atividades diárias e na qualidade de vida. O objetivo principal desse estudo foi avaliar a prevalência do BIE em riníticos não asmáticos e comparar as respostas no volume expiratório forçado no 1º segundo (VEF1) aos testes de Hiperventilação Eucápnica Voluntária (HEV) e Corrida em esteira (CE). Métodos: Foi realizado um estudo descritivo, analítico, inferencial, transversal, para avaliação de prevalência, com amostras por conveniência de indivíduos dos 10 aos 20 anos de idade, realizado no Serviço de Pneumologia do Hospital das Clínicas - UFPE. Foram mensuradas variáveis antropométricas (peso e estatura), e aplicados questionários para avaliação da gravidade da rinite, asma no passado e de queixas respiratórias aos exercícios. O VEF1 foi determinado em duplicata através da espirometria, antes e aos 3, 5, 7, 10, 15 e 30 minutos após cada teste diagnóstico. Para o teste da CE, os indivíduos realizaram o exercício por um período de oito a nove minutos, sendo que nos seis minutos finais deveria manter a frequência cardíaca alvo (estimada em 80% a 90% da FC máxima : 220- idade). Para a realização da HEV, os indivíduos realizaram uma manobra de hiperventilação durante seis minutos, respirando ar seco adicionado de 5% de CO2, com uma ventilação minuto alvo de 21 vezes o seu VEF1 basal. O BIE foi diagnosticado quando houve uma redução > 10% do valor basal detectado em dois momentos consecutivos após a provocação. Resultados: Foram estudados 35 indivíduos testados com média da idade de 16,3 + 3,6 anos. O BIE foi diagnosticado em 13 sujeitos (37%) por um dos dois métodos ou ambos. Em 9 indivíduos após a CE e em 10 após a HEV e em 6 por ambos, mostrando uma concordância moderada entre os métodos (Kappa = 0,489). O VEF1 basal expresso como percentual do predito foi maior naqueles indivíduos com testes negativos que nos positivos (101% e 85%, p<0,02 para a CE e 101% versus 85%, p< 0,001 para a HEV) e não foram verificadas diferenças na frequência cardíaca máxima alcançada entre os sujeitos com teste positivo e negativo na corrida em esteira e nem na ventilação atingida quando os pacientes foram submetidos à HEV. Conclusão: A prevalência do BIE em pacientes com rinite em nossa região é comparável àquelas descritas na literatura mais recente. Houve uma concordância moderada entre os testes empregados para o diagnóstico do BIE, o que indica cautela quando se pretende comparar os dois métodos ou mesmo considerar seus resultados de forma intercambiável. / Respiratory complaints after exercise are common in rhinitis, asthma, and in individuals without these diseases. One cause is exercise-induced bronchospasm (EIB), defined as the narrowing of the bronchi after strenuous exercise. These symptoms can limit the activities of these adolescents and result in negative consequences in daily activities and quality of life. The main objective was to evaluate the prevalence of EIB and compare the answers in FEV1 to eucapnic voluntary hyperventilation testing (EVH) and Treadmill running (TR) in rhinitis but non-asthmatic patients. Methods: We conducted a descriptive, analytical, inferential, cross-sectional study, to assess EIB prevalence with a convenience samples of 10 to 20 year- old patients, held at the Pulmonology Department of Hospital das Clínicas – UFPE – Recife/Brazil. Anthropometric variables were measured (height and weight), and questionnaires to evaluate the severity of rhinitis, past asthma history and respiratory complaints on exercise. Forced expiratory volume in 1 second (FEV1) was determined in duplicate before and at 3, 5, 7, 10, 15 and 30 minutes after each diagnostic test. For TR test, subjects performed the exercise for a period of eight to nine minutes, in the final six minutes should maintain the target heart rate (estimated at 80% to 90% of maximum heart rate: 220- age). To perform the EVH, individuals sustained a hyperventilation maneuver breathing dry air enriched with 5% CO2 for six minutes with minute ventilation target at 21 times their baseline FEV1. EIB was diagnosed when there was a reduction > 10% from baseline FEV1 value detected at two consecutive times after challenges. Results: Of the 35 included subjects the mean age was 16.3 + 3,6 years. EIB was diagnosed in 13 patients (37%) by one of the two methods or both. In nine individuals after TR and 10 after EVH and in 6 by both methods, showing a moderate agreement between the methods (Kappa = 0.489). Baseline FEV1 expressed as a percentage of predicted was higher in those with negative tests com testes negativos (101% and 85%, p<0,02 for TR and 101% versus 85%, p< 0,001 for EVH) but no differences were observed in maximum heart rate achieved among patients with positive and negative treadmill tests nor the ventilation achieved when patients underwent EVH. Conclusion: The prevalence of EIB in patients with rhinitis in our region is comparable to those described in the most recent literature. There was a moderate agreement between the tests, which suggests that we have to be cautious when trying to compare them or when considering its results interchangeably.

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

Les sensibilisants de contact sont des molécules réactives électrophiles qui ont la capacité de modifier des protéines de la peau pour former un antigène. Au delà de ce mécanisme d'hapténisation, le signal de danger induit par les sensibilisants conduisant à l'activation des cellules dendritiques (DC) est un élément déterminant dans l'induction de cellules T spécifiques de l'haptène. Dans le contexte du 7ième amendement à la directive cosmétique européenne, la mise en place d'une batterie de tests in vitro permettant de prédire le potentiel sensibilisant de molécules est indispensable pour l'industrie cosmétique. Tandis que la plupart des études in vitro étudient les signaux de danger induits par les sensibilisants dans des modèles homéostasiques, nous nous sommes intéressés à l'effet des sensibilisants sur la mise en place d'une réponse inflammatoire. Lorsque la lignée U937 est différenciée avec du PMA et stimulée avec du LPS, les facteurs de transcription NF-κB et Nrf2 sont activés et l'acide arachidonique (AA) est métabolisé au travers de la cascade cPLA2 / COX-2. L'ensemble de ces voies activées conduit à la production par les U937 d'un grand nombre de médiateurs inflammatoires (IL-1β, TNF-α, IL-6, IL-10, IL-8, PGE2, PGD2, TxB2). Dans ce modèle, nous avons analysé l'effet de 6 sensibilisants de potentiels variés (DNCB, PPD, HQ, PG, CIN, EUG) et montré que de façon inattendue, tous les sensibilisants étudiés diminuent significativement et de façon spécifique la production de tous les prostanoïdes et en particulier de PGE2 induite par PMA/LPS. Nous avons de plus démontré que selon les sensibilisants, les cibles de cette inhibition au sein de la cascade métabolique de l'AA diffèrent, même si elles se focalisent la plupart du temps (sauf pour le DNCB) sur l'enzyme COX-2 (inhibition de son expression et/ou de son activité). Pour le DNCB, le mécanisme d'inhibition semble plutôt impliquer sa capacité à réagir fortement avec les groupements résidus thiols, ce qui se traduit en particulier par la déplétion du GSH intracellulaire et engendrerait l'inhibition des synthases dépendantes du GSH pour leurs activités. En parallèle de cette étude mécanistique, nous avons appréhendé la problématique du point de vue statistique et vérifié sur un set plus important et diversifié de molécules (160 molécules) que le paramètre « inhibition de PGE2 » pouvait être un bon test de prédiction de l'HSRC. L'étude statistique a permis de déterminer le modèle prédictif du test PGE2 et de mettre en évidence de bonnes performances (78%) par rapport aux prédictions du LLNA. Au-delà, une certaine complémentarité du test PGE2 avec d'autres tests in vitro (MUSST, Nrf2-HTS) a pu être mise en évidence. En conclusion, au travers de cette étude, nous avons pu mettre en évidence de nouvelles propriétés biochimiques des sensibilisants. Même si la signification biologique de la diminution de PGE2 par les sensibilisants de contact demeure complexe d'interprétation, ce paramètre a permis le développement d'un test qui prédit avec de bonnes performances le caractère sensibilisant de molécules et dont la position au sein d'une batterie prédictive d'évaluation de l'allergie de contact reste à être précisée. / Contact sensitizers are defined as reactive molecules (electrophilic) which have the ability to modify skin proteins to form an antigen (hapten). In addition to the haptenation mechanism, danger signals, leading to the activation of dendritic cells, are described to be crucial for the effective induction of an hapten-specific T cell immune response. In the context of the 7th amendment to the Cosmetic Directive, the cosmetic industry is concerned by the challenge of finding non-animal approaches to assess the sensitizing potential of chemicals. While danger signals induced by sensitizers in steady-state conditions have already been analyzed, we chose to investigate the impact of sensitizers on the course of an inflammatory response. For this purpose we used the U937 cell line differentiated with PMA and activated with LPS. In these conditions, cells produce a large amount of inflammatory mediators (IL-β, TNF-α, IL-6, IL-10, IL-8, PGE2, PGD2, TxB2) through the activation of pathways leading to the activation of the transcription factors NF-κB and Nrf2 and through AA metabolism by the cPLA2/COX-2 cascade. Interestingly, we showed that 6 contact sensitizers with various potential (DNCB, PPD, HQ, PG, CIN, EUG) significally and specifically decrease the production of prostanoïds and in particular of PGE2 induced by PMA/LPS. We further demonstrated that there is no unique inhibition profile of the sensitizers even if the majority (except for DNCB) of the effects applies on COX-2 (i.e. inhibition of the expression and/or activity). For DNCB, inhibition mechanism appears to be dependant of its capacity to react with thiols residues and in particular to deplete intracellular glutathione possibly leading to the inactivation of the PG-synthases. In parallel, we assess a statistical analysis on 160 molecules that allow us to define the test parameters (a molecule is a sensitizer if the PGE2 inhibition at 24h is more than 60%) and to calculate the test performance toward LLNA (78%). Moreover we demonstrated that the PGE2 test could be complementary to other already existing in vitro tests like MUSST or Nrf2-HTS. In summary, we add here a new insight into the multiple biochemical effects described so far for sensitizers. Even if the underlying biological relevance remains unclear, the parameter “PGE2 inhibition” is good test for skin sensitization evaluation. Further studies will precise how this parameter could be implemented into an alternative testing strategy for the evaluation of skin sensitization.

Introduction: Le traitement de la rhinite allergique (RA) est maintenant bien établi et la plupart des patients atteints par cette maladie y répondent. Néanmoins, il existe un pourcentage de patients qui ne sont pas contrôlés, malgré un traitement maximal, amenant au concept de SCUAD (Severe Chronic Upper Airway Disease), acronyme anglais pour "atteinte sévère et chronique des voies aériennes supérieures". En Chine, les données concernant la RA non contrôlée et le SCUAD sont insuffisantes. Le test de contrôle de la rhinite allergique (Allergic Rhinitis Control Test, ARCT) est un outil validé pour évaluer le contrôle de la RA et identifier la RA sévère. Néanmoins, l'ARIA (Allergic Rhinitis and its Impact on Asthma, acronyme anglais pour Rhinite Allergique et son Impact sur l'Asthme) n'offre pas de définition claire du contrôle de la RA et, du fait de l'absence de critères uniformes, le choix de la pharmacothérapie varie dans différentes régions et populations. Objectif: La première étude a eu pour objectif d'évaluer la prévalence et les caractéristiques des patients avec RA non contrôlée et SCUAD à Wuhan. A partir de cette étude préliminaire, une autre étude a été mise en oeuvre, pour évaluer le rôle de l'ARCT en tant qu'outil pour guider une pharmacothérapie par paliers, dans le but d'atteindre le contrôle de la RA. Méthode : Dans la première étude, tous les patients consultant pour une RA ont été évalues en prospectif par EVA et ARCT, et mis sous traitement selon le guide ARIA. Au bout de 15 jours (J15), une interview téléphonique a permis de ré-évaluer la RA au moyen d'une échelle numérique et de l'ARCT. La RA non contrôlée était définie par un score ARCT<20. Les patients souffrant de SCUAD étaient définis par un score ≥5 à J15. Dans la 2ème étude prospective, un traitement pharmacologique standard a été proposé aux patients souffrant de RA. Les paliers allaient du palier 1 (antihistaminique H1 à la demande) jusqu'au palier 5 (corticoïde oral). La RA était traitée et évaluée tous les 15 jours par ARCT. Si le score ARCT était ≥20, maintenu pendant 15 jours, le patient terminait l'étude. Si l'ARCT était <20, le patient recevait le prochain palier de traitement, selon une démarché prédéfinie, progressive, jusqu'au palier 5. Résultats: Au total, 252 patients ont été inclus dans la 1ère étude. La RA modérée/sévère était présente en 82,9% des patients, avec un impact sur le sommeil, travail, activités sociales et physique. Les patients avec RA non contrôlée à J15 étaient ceux avec un poids plus important (P=0,042), antécédents d'infections ORL ou de prise d'antibiotiques pour infections respiratoires dans les derniers 12 mois (62,3 vs. 45,6%, P=0,018), de tabagisme (P=0,024) et de dysosmie (P=0,005). Les patients avec SCUAD avaient plus fréquemment des antécédents d'infections ORL ou de prise d'antibiotiques pour infections respiratoires (P=0,014) et de dysosmie (P=0,003) et moins fréquemment de dermatite atopique (P=0,017). 255 patients ont été inclus dans la 2ème étude. Seulement 3,2% sont restés non contrôlés à la fin de l'étude. Les patients avec une RA modérée à sévère selon ARIA, RA persistante, impact modéré à sévère sur la qualité de vie, antécédents d'asthme, rhinorrhée et toux avaient toujours besoin d'un traitement associé, ainsi qu'un traitement prolongé pour atteindre le contrôle. Après ajustement sur chacune des variables, le seul facteur de risque restant significatif était la présence d'un asthme (il était moins probable que ces patients soient contrôlés par les premiers paliers de traitement). Conclusion: Les patients ayant une RA non-contrôlée ou atteints de SCUAD sont nombreux. L'EVA et l'ARCT sont des outils simples qui peuvent être utilisés dans l'évaluation globale de la sévérité et du contrôle de la RA. L'ARCT offre un critère objectif pour guider le traitement par paliers... / Background : The treatment of Allergic rhinitis (AR) is now well established and most patients respond well to the treatment. However, there are still some patients with uncontrolled AR despite optimal maximum treatment, leading to the concept of severe chronic upper airway disease (SCUAD). In China, there are insufficient epidemiological data regarding uncontrolled AR and SCUAD. Allergic Rhinitis Control Test (ARCT) has been validated for assessing AR control and to identify severe AR. However, Allergic Rhinitis and its Impact on Asthma (ARIA) still has no clear definition of AR control, and due to the absence of uniformed criteria, pharmacotherapy adjustment regimens varies in different areas and populations. ObjectiveThe first study aimed to assess the prevalence and the characteristics of patients with uncontrolled AR and SCUAD in Wuhan. On the basis of the preliminary study, a further study is designed to assess ARCT as a questionnaire driven stepwise pharmacological treatment to achieve AR control. Methods : In the first epidemiology study, all patients consulting for AR were prospectively assessed using VAS and ARCT and put on standardized treatment based on ARIA guidelines. After 15 days, they were reevaluated by a telephone interview using a numerical scale and ARCT. A score of ARCT <0 defined uncontrolled AR and a score ≥5 at day-15 defined SCUAD patients.In the second study, a standard pharmacotherapy regimen from step1 (oral second generation H1 antihistamine as needed) to step 5 (oral corticosteroid) was applied prospectively in a Chinese AR population. The AR patients were initiated with ARIA appropriate step treatment and assessed with ARCT every 15 days. If ARCT score was equal or above 20 and maintained for 15 days, the patient would finish the study; if ARCT score was strictly less than 20, the patient would receive higher step treatment according to a predefined open design up to step 5. Results: A total of 252 patients were included in the first study. Moderate/severe AR was diagnosed in 82.9% of the patients and they had an impact on sleep, work life, social activities and physical activities. Patients with uncontrolled ARat day-15 more frequently presented a higher weight (P=0.042), past history of ENT infection or antibiotics intake for respiratory infection in the last 12 months (P=0.018), smoking (P=0.024) and smell disturbance (P=0.005). Patients with SCUAD more frequently presented a past history of ENT infection or antibiotics intake for respiratory infection (P=0.014) and smell disturbance (P=0.003), while less commonly had atopic dermatitis (P=0.017). 255 patients were enrolled in the second study. Only 3.2% patients remained uncontrolled at the endpoint of the study. Patients with ARIA moderate/severe or persistent symptoms, moderate/severe impaired quality of life, asthma history, rhinorrhea and cough symptoms always needed up to step 4 and prolonged treatments to achieve disease control. After adjustment on each of the variables, the only factor that remained significant was asthma (less likely to be in a group controlled by the first steps’ therapies) .Conclusion: Uncontrolled AR and SCUAD patients are numerous. VAS and ARCT are simple and quantitative methods and self-completion questionnaire that can be used for a global evaluation of the severity and control of AR. ARCT offers an objective criterion for the stepwise pharmacotherapy of AR. Risk factor analysis did not reveal strong clinical characteristics that would help the physician to control AR better.

Charles University Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Sciences Author: Bc. Petra Porubová Supervisor: prof. RNDr. Ctirad Andrýs, Ph.D. Title of diploma thesis: Allergic phenotypes in patients with respiratory allergy evaluated by microchip ISAC technique Key words: allergic inflammation, allergen specific IgE, component resolved diagnostics, microchip, ISAC Background: The aim of this diploma thesis was to evaluate the sensitization profile in the group of pacients with allergic rhinitis and/or bronhial asthma and to identify possible differences between patients with different disease severity. Methods: ImmunoCAP ISAC was used to determine specific IgE antibodies against 112 allergen components. The evaluated group included 152 patients (57.9 % women) from the allergology outpatient office of the Department of Clinical Immunology and Allergology, University Hospital Hradec Králové. Results: The most common allergens in this group of patients were the components Phl p 1 (68,4 %) and Bet v 1 (65,1 %). Differences in the number of positive and negative reactions were demonstrated between patients with different severity of allergic rhinitis. Correlations were found between the number of positive/negative reactions and the severity of bronchial asthma. And the...