Neste trabalho foram investigadas a atividade antialérgica de extratos, ou substâncias isoladas, obtidos de fontes naturais. Para isso foi utilizado o sistema biossensor baseado em mastócitos os quais liberam a enzima beta-hexosaminidase usada como marcador da degranulação. Para algumas substâncias naturais da classe dos flavonóides (quercetina-Qc e rutina-Rt) e ácidos polifenólicos (ácido dimetoxicinâmico-Dm e ácido cafeico-Cf), os ensaios biológicos foram conduzidos na presença de beta-ciclodextrina (beta-CD) a fim de estudar a eficiência do ensaio biológico e o efeito de complexação na atividade antialérgica. Inicialmente, foram investigadas, as propriedades espectroscópicas destes flavonóides e ácidos polifenólicos, na ausência, e presença de beta-CD. As mudanças nos espectros de absorção e fluorescência, em presença de beta-CD, mostraram que ocorre a associação dos fármacos com a beta-CD. Assim, as constantes de incorporação (Kc) foram determinadas pelo método de Higuchi e Connors e os resultados mostraram maior incorporação da Qc (Kc = 172 /M) na cavidade da beta-CD quando comparada a Rt (Kc = 139 /M). No caso dos polifenóis, Dm mostrou incorporação maior em relação ao Cf, com valores de Kc iguais a 718 e 278 /M, respectivamente. Os valores de Kc foram considerados apropriados para a aplicação de compostos de inclusão como agentes terapêuticos. Assim, os complexos de inclusão sólidos, foram preparados por uma adaptação do método da co-evaporação e caracterizados por Análise Termogravimétrica (TGA), Análise Térmica Diferencial (DTA), Calorimetria Diferencial de Varredura (DSC), espectroscopia na região do Infravermelho (FTIR) e Ressonância Magnética Nuclear de Prótons (1H-RMN). O parâmetro físico-químico para interação hidrofóbica (log P) foi determinado para os flavonóides e acidos polifenólicos e os resultados indicaram que a hidrofobicidade seguiu a seguinte ordem: Dm > Cf > Qc > Rt. Os complexos de inclusão foram mais eficazes para inibir a liberação da beta-hexosaminidase do que os fármacos na forma livre. A atividade anti-alérgica da Qc livre (IC50= 5,1 µM) mostrou um aumento de oito vêzes quando complexada com a beta-CD (IC50= 0,62 µM). Um aumento da atividade foi observado, também, para os complexos Rt/CD, Cf/CD e Dm/CD. Este efeito foi mais forte para os compostos com maior hidrofobicidade. A atividade antialérgica das substâncais naturais livres provenientes de várias classes de plantas tais como flavonóides, ácidos polifenólicos, terpenos, alcalóides e iridóides foi, também, investigada. Os flavonóides tais como quercetina (IC50= 5,1 µM), 7-metil quercetina (IC50= 6,2 µM), caempferol-3-glicosideo (IC50= 6,7 µM) and 4-O-(6-trans-p-coumaroil)--D-glicopyranosil okanina (IC50= 5,8 µM) mostraram a maior atividade antialérgica comparados ao fumarato de cetotifeno (IC50= 15,1 µM). Os extratos provenientes de diversas espécies de plantas tais como Bidens sulphurea, Bidens gardneri, Bidens graveolens, Mikania parodii Cabrera e Mikania pilosa Baker foram, também, investigados. Os resultados mostraram maior atividade para o extrato de Bidens obtido de acetato de etila. Este extrato é rico em derivados metilados de quercetina os quais exibiram forte atividade antialérgica quando utilizados no ensaio biológico como substância isolada. / Anti-allergic activity of extracts and isolated compounds obtained from natural sources was investigated using the mast-cell based biosensor system. Mast cells release beta-hexosaminidase enzyme which is used as a marker of degranulation. Flavonoids (quercetin-Qc and rutin-Rt) and polyphenolic acids (caffeic acid-Cf and dimethoxy cinnamic acid-Dm) were used as inclusion compounds with beta-cyclodextrin (beta-CD) in order to compare the efficiency of the biological assay and the anti-allergic activity of the drugs free or associated with beta-CD. Spectroscopic properties of the flavonoids and polyphenolic acids were monitored in the absence or presence of beta-CD. The absorbance and fluorescence spectra showed drug association with beta-CD; subsequently the stability constants (kc) of the drugs were obtained in accordance with the method of the Higuchi-Connors. The results showed higher association of Qc with beta-CD (Kc= 172 /M) compared to Rt (Kc= 139 /M). For the polyphenolic acids, Dm exhibited the higher association with beta-CD compared to Cf (718 and 278 /M respectively). The Kc values felt within the range considered adequate for the formation of inclusion complex, and they can be used to improve the bioavailability of the flavonoids and polyphenolic acids. The solid inclusion compounds were obtained by an adaptation of the co-evaporation method and characterized by Thermo Gravimetric Analysis (TG), Differential Thermal Analysis (DTA), Differential Scanning Calorimetry (DSC), Fourier Transform Infrared Spectroscopy (FTIR) and Proton Nuclear Magnetic Resonance Spectroscopy (1H NMR). Physico-chemical parameter for hydrophobic interaction (log P) was determined for the flavonoids and polyphenolic acids and the results indicated that the hydrophobicity followed the order: Dm > Cf > Qc > Rt. The inclusion complexes were more effective at inhibiting beta-hexosaminidase release than plain drugs. The anti-allergic activity of plain Qc (IC50= 5.05 M) showed eightfold improvement when included inside the beta-CD cavity (IC50= 0.62 M). Higher biological activity on the part of the complex was also observed for the complexes Rt/CD, Cf/CD and Dm/CD. This effect was stronger for the compounds with higher hydrophobicity. The anti-allergic activity of plain natural compounds from several classes of plants such as flavonoids, polyphenolic acids, terpenes, alkaloids and iridoids was investigated. Flavonoids such as quercetin (IC50= 5.1 µM), 7-methyl quercetin (IC50= 6.2 µM), kaempferol-3-glycoside (IC50= 6.7 µM) and 4-O-(6-trans-p-coumaroil)--D-glucopyranosyl okanin (IC50= 5.8 µM) showed the stronger anti-allergic activity compared with ketotifen fumarate, a reference drug (IC50= 15.1 µM). Extracts proceeding from different species of plants such as Bidens sulphurea, Bidens gardneri, Bidens graveolens, Mikania parodii Cabrera and Mikania pilosa Baker were also investigated. The results showed stronger anti-allergic activity for ethyl acetate extracts obtained from Bidens specie. This extracts are rich in methylated quercetin derivatives which showed strong anti-allergic activity when assayed as isolated substances

Doença alérgica ocular acomete cerca de ¼ da população mundial. Dentro do espectro da doença alérgica ocular, a cérato-conjuntivite vernal, que afeta principalmente crianças, pode apresentar-se sob forma severa e persistente, levando a dano do tecido corneal e comprometimento da função visual. Traumatismo epitelial crônico, induzido pelo ato de coçar os olhos, associado ao intenso prurido ocular tem sido apontado como fator de risco importante na patogênese do ceratocone. Pode estimular a apoptose prematura dos ceratócitos, provocando mudanças estruturais do estroma da córnea. A associação de cérato-conjuntivite vernal e ceratocone tem sido apontada como freqüente na literatura oftalmológica através de estudos descritivos e qualitativos, que, entretanto, não auxiliam na detecção precoce da doença ectásica corneal, prejudicando sua análise epidemiológica, seu estudo genético e a definição de sua patogênese. Propôs-se estudo clínico caso-controle de pacientes com cérato-conjuntivite vernal do Ambulatório de Alergia Ocular do Departamento de Oftalmologia da Santa Casa de São Paulo, com finalidade de obter-se, por meio da análise topográfica computadorizada de córnea utilizando-se de descritor quantitativo da superfície anterior da córnea (sumário diagnóstico de Holladay), informações sobre as alterações topográficas da superfície anterior da córnea, que pudessem determinar a freqüência da associação entre cérato-conjuntivite vernal e ceratocone, além de seus efeitos sobre o desempenho da visão destes pacientes. Os resultados obtidos neste estudo mostram alta freqüência de ceratocone em pacientes com cérato-conjuntivite vernal. A performance visual destes pacientes é influenciada pelas aberrações provocadas por alterações da asfericidade corneal e de outras variáveis topográficas. / Allergic ocular disease affects ¼ of the world population. Inside the spectrum of the allergic ocular disease, vernal keratoconjunctivitis, that affects mostly children, may present as severe and persistent form, leading to corneal tissue damage and disturbing visual function. Chronic epithelial trauma, provoked by eye rubbing due to intense ocular itching, has been postulated as an important risk factor in the pathogenesis of keratoconus. It may induce early keratocyte apoptosis that results in structural changes to the corneal stroma. The association of keratoconus with vernal keratoconjunctivitis has been observed to be frequent in the ophthalmological literature by descriptive and qualitative studies, unable to detect earlier forms of this ectatic corneal disease, weakening epidemiological analysis, genetic studies and the definition of its pathogenesis. We proposed a case-control clinical study of patients with vernal keratoconjunctivitis from the Ambulatory of Ocular Allergy of the Department of Ophthalmology of Santa Casa of São Paulo, aiming for information on the anterior corneal curvature and visual performance, using a quantitative descriptor analyzer (Holladay Diagnostic Summary). The results suggest high frequency of the keratoconus in patients with vernal keratoconjunctivitis. The visual performance is affected by the induced aberration caused by changed corneal asphericity and other topographic variables.

A dermatite de contato alérgica (DCA) ao íon níquel (Ni2+) é uma dermatose inflamatória frequente nos países industrializados. Envolve a ativação de células T específicas ao Ni2+, seguida da proliferação e indução de um perfil misto de citocinas, tanto pró-inflamatórias quanto reguladoras, sugerindo que vários subtipos de células T (helper - Th e citotóxica - Tc) estão envolvidos na resposta imune. Este estudo teve como objetivo a análise das citocinas TNF-alfa, INF-y, IL-2, IL-4, IL-10, IL-13, IL-17 e IL-23 pela técnica de imuno-histoquímica, para tentar identificar a prevalência de um ou mais subtipos de células T (Th/Tc), nos eczemas crônico e agudo de pacientes com DCA ao Ni2+. Avaliamos 20 pacientes (17 mulheres e 3 homens, com idade mediana de 46 anos) apresentando eczema crônico, pelo contato cotidiano do paciente com o Ni2+. Foram coletadas duas biópsias de pele em cada um dos 20 pacientes, a primeira no local do eczema crônico ao Ni2+, antes da aplicação do teste de contato (TC); e a segunda no local do eczema agudo, provocado pelo TC com o sulfato de níquel, 48 horas após sua fixação, nas leituras positivo forte (++) ou positivo muito forte (+++). Foram 160 amostras de eczema agudo e 160 de eczema crônico, perfazendo um total de 320 amostras. Apenas três amostras foram excluídas devido a algum tipo de falha técnica, como, por exemplo, o descolamento dos cortes de pele da lâmina. Para a análise dos dados utilizou-se o software estatístico STATA versão 13. As amostras coradas revelaram resultados positivos para as oito citocinas estudadas, e estas apresentaram valores heterogêneos. Esta heterogeneidade foi medida pelo coeficiente de variação, indicando a variabilidade do conjunto dos dados obtidos. O TNF-alfa, IFN-y, IL-4, IL-13 e IL-17 tiveram prevalência maior no eczema crônico do que no eczema agudo, a IL-2 e IL-23 apresentaram maior prevalência no eczema agudo, em comparação com o eczema crônico e a IL-10 apresentou prevalência similar tanto no eczema agudo quanto no crônico, porém, estas prevalências foram muito baixas, em ambos os eczemas. O TNF-alfa foi a citocina que mais prevaleceu no eczema crônico e a IL-2 foi a mais prevalente no eczema agudo. Porém, estas prevalências foram estatisticamente significantes apenas para a IL-4 e IL-13. Verificamos, nos eczemas crônico e agudo, a presença de um perfil misto de citocinas dos subtipos de células T (Th/Tc), sugerindo que as respostas imunes são expressas ao mesmo tempo. Entretanto, são necessários mais estudos para uma compreensão mais ampla sobre o perfil das citocinas na DCA ao Ni2+, o que poderia levar a novas abordagens terapêuticas / Allergic contact dermatitis (ACD) to nickel (Ni+2) is a inflammatory dermatosis, common in industrialized countries. It involves the activation of nickel-specific T cells, followed by the proliferation and induction of a mixed profile of both proinflammatory and regulatory cytokines, suggesting that several T cell subtypes (helper - Th and cytotoxic - Tc) are involved in the immune response. This study aimed to analyze the cytokines TNF-alfa, INF-y, IL-2, IL-4, IL-10, IL-13, IL-17 and IL-23 using the immunohistochemistry technique in order to try to identify the prevalence of one or more T cell subtypes (Th/Tc) in the chronic and acute eczema of patients with ACD to Ni+2. We evaluated 20 patients (17 women and 3 men, median age of 46 years) with chronic eczema, by the patient\'s daily contact with Ni+2. Two skin biopsies were collected in each of the 20 patients, the first at the site of the chronic eczema to Ni+2, prior to the application of the contact test (CT); and the second at the site of acute eczema caused by CT with nickel sulphate, 48 hours after its fixation in the strong positive (++) or very strong positive (+++) readings. There were 160 samples of acute eczema and 160 of chronic eczema, a total of 320 samples. Only three samples were excluded due to some kind of technical failure, such as detachment of the skin cuts from the microscope slide. Statistical software STATA version 13 was used to analyze the data. The stained samples showed positive results for the eight cytokines studied, and these presented heterogeneous values. This heterogeneity was measured by the coefficient of variation, indicating the variability of the data set obtained. TNF-alfa, IFN-y, IL-4, IL-13 and IL-17 had a higher prevalence in chronic eczema than in acute eczema, IL-2 and IL-23 were more prevalent in acute eczema compared to chronic eczema and IL-10 presented similar prevalence in both acute and chronic eczema, however, a very low prevalence in both eczema. TNF-alfa was the most prevalent cytokine in chronic eczema and IL-2 was the most prevalent in acute eczema. However, these prevalences were statistically significant only for IL-4 and IL-13. In chronic and acute eczema, we observed the presence of a mixed cytokine profile of the T cell subtypes (Th/Tc), suggesting that immune responses are expressed at the same time. However, further studies are needed for a broader understanding of the cytokine profile in ACD to Ni+2, which could lead to new therapeutic approaches

INTRODUÇÃO: A conjuntivite alérgica é a forma mais comum de doença alérgica que afeta o olho. Neste trabalho, desenvolvemos um modelo murino reprodutível e simular a doença humana, para possibilitar o estudo dos mecanismos fisiopatológicos da conjuntivite alérgica crônica. MÉTODOS: Imunizamos os camundongos BALB/c e C57Bl/6 com extrato do ácaro Dermatophagoides pteronyssinus (Dpt). Foi realizada a dissecção dos linfonodos ilíacos e para-aórticos, e a enucleação dos olhos. O plasma obtido pela punção cardíaca foi utilizado para a dosagem de IgE e IgG totais e específicas para Dpt. Os olhos enucleados foram enviados para estudo anátomo-patológico da conjuntiva e córnea. RESULTADOS: 1) Houve uma diferença estatisticamente significante entre as duas linhagens (BALB/c e C57Bl/6) para os grupos imunizados com 5 ?g e 500 ?g na gradação clínica e histopatológica, dosagens de IgE Total e Específica, proliferação de linfócitos específica para Dpt e IgG Específica, e na dosagem das IL-5, IL-8 e IL-13; 2) Os níveis de IgG Total não se mostraram significantes para as duas linhagens nos grupos imunizados com 5 ?g e 500 ?g; 3) Os níveis de IL-4 e IL-10 tiveram uma diferença significante nos animais da linhagem BALB/c imunizados com 5 ?g e 500 ?g, mas não nos camundongos da linhagem C57BI/6; 4) Os níveis de IFN-? foram maiores nos camundongos C57BI/6 que receberam as menores quantidades de antígeno. Porém nos camundongos BALB/c o fenômeno foi o inverso; 5) O exame histológico revelou afilamento corneano, infiltrado linfocítico corneano e conjuntival, degeneração da conjuntiva e úlceras de córnea nos animais que obtiveram as maiores gradações clínicas da doença (camundongos BALB/c imunizados com 500 ?g de Dpt e camundongos C57Bl/6 imunizados com 5 ?g. CONCLUSÃO: Desenvolveu-se um modelo simples e reprodutível de conjuntivite alérgica crônica do Dermatophagoides pteronyssinus depois de repetidas exposições ao antígeno, o qual apresenta manifestações clínicas similares à doença humana, e serve como modelo de estudo dos mecanismos imunológicos envolvidos no desenvolvimento da doença / INTRODUCTION: Allergic conjunctivitis is the most common form of allergic disease that affects the eye. In this study we developed a reproducible mouse model and simulated human disease to enable the study of physiopathologic mechanisms of chronic allergic conjunctivitis. METHODS: We immunized BALB/c and C57B1/6 mice with Dermatophagoides Pteronyssinus (Dpt) dust mite extract. The iliac and paraaortic lymph nodes were dissected and the eyes were enucleated. The plasma obtained by cardiac puncture was used to measure Total and Specific IgE and IgG and Dpt-specific. Lymph node cells were used to measure Dpt specific proliferation cytokine detection in the culture supernatant. Eyes were enucleated for histopathological analysis of the conjunctiva and cornea. RESULTS: 1) There was a statistically significant difference between the 2 strains (BALB/c and C57B1/6) for the 2 groups immunized with 5?g and 500?g in the clinical and histopathological score, Total and Specific IgE dosages, proliferation Dpt-specific lymphocytes, dust mite Specific IgG, and in the levels of IL-5, IL-8 and IL-13; 2) The level of Total IgG was not significantly different between the 2 lineages in the groups immunized with 5?g and 500?g; 3) The levels of IL-4 and IL-10 showed a significant difference in BALB/c mice sensitized with 5?g and 500?g, but not in C57B1/6 mice; 4) The IFN-? levels were higher in C57B1/6 mice that received the smallest quantity of antigen. But among BALB/c mice the phenomenon was inversed; 5) The histological examination revealed that there was a tapering of the cornea, lymphocytic infiltration of the cornea and conjunctiva, conjunctival degeneration and corneal ulcers in the animals that developed the highest clinical scores of disease (BALB/c immunized with 500 ug of Dpt and C57Bl/6 immunized with 5 ?g of Dpt). Conclusion: A simple and reproducible model of chronic allergic conjunctivitis to Dermatophagoide pteronyssinus was developed after repeated exposure to the allergen, which exhibit similar clinical manifestations as human disease, therefore serving as a template to study the immunological mechanisms involved in the development of disease

全球過敏性疾病的患病率逐漸增加。大約30 - 40的世界人口患有一個或多個過敏性疾病，它絶對是一個國際性的公共健康問題。在過去三十年，過敏性皮膚炎的發病率增加了2-3 倍，當中患病率最高的是嬰兒和兒童，而且現時並沒有明確的治療方法。然而，對過敏性疾病有效的治療方法仍然缺乏，大多數傳統的治療涉及臨床改善，但不針對促進過敏性炎症發病機制中的主要因素。這些傳統的治療方法都有不良副作用。因此，發展一個更安全和非類固醇的治療方式成為了新的趨勢。 / 從過往的臨床試驗中，患有中度至嚴重過敏性皮膚炎的兒童服用由五種中藥制成的Pentaherbs（PHF）膠囊藥丸後， 顯著地改善他們的生活質數，降低了過敏性皮膚炎指數（SCORAD）及減少使用傳統藥物類固醇的份量，更沒有出現任何不良藥物作用。實驗結果指出PHF 有潛力替代類固醇，成為治療過敏性皮膚炎的取代品。在本研究中，我們使用炎症相關的細胞因子IL-33，激活在有或沒有與皮膚成纖維細胞一起培植下的嗜鹼性細胞系KU812 細胞， 來探討了PHF，牡丹皮（DP，PHF 的五種草藥之一）和沒食子酸（GA，牡丹皮的主要成分之一）的抗炎和抗過敏特性。 / 在過敏性炎症中，嗜鹼性粒細胞是一個重要的效應細胞。我們利用細胞因子IL-33 激活嗜鹼性粒細胞系KU812 細胞，並從研究結果發現出PHF，DP 和GA 能有效及顯著地抑制細胞間粘附分子ICAM-1 的表達，炎症相關趨化因子CCL2，CCL5，CXCL8 和促炎細胞因子IL-6 的釋放。這證實出PHF, DP 和GA有抗炎和抗過敏的特性。在進一步的研究中，我們加入一種常用醫治過敏性炎症藥物的合成類固醇地塞米松, 與PHF, DP 和GA 結合使用。從各種組合的不同濃度地塞米松與PHF，DP 和GA 中，我們發現聯合使用低濃度為0.01 微克/毫升的地塞米松和10 微克/毫升GA 可進一步抑制ICAM-1 在KU812 的表達，趨化因子CCL2 和CCL5 釋放。此外，沒食子酸可顯著抑制細胞內信號分子p38 絲裂原活化蛋白激酶 (MAPK)，IκB-α和JNK 的表達。這表明了黏附分子的表達，趨化因子和細胞因子的釋放的抑製是經由p38 MAPK，IκB-α和JNK 訊息傳遞路徑所調節。實驗證實PHF，DP 和GA 具有抗炎和抗過敏的特性，與過往的臨床試驗結果一致。 / 為了更進一步研究沒食子酸和地塞米松在過敏性炎症的發病機制中扮演的角色， 我們建立了一個體外的模仿患者皮膚皮炎症的模型，共同培養嗜鹼性細系KU812 細胞和皮膚成纖維細胞 。我們發現，單沒食子酸的應用已經可以顯著地抑制在KU812 細胞和成纖維細胞表面粘附分子的表達，並減低釋放過敏性炎症相關的趨化因子CCL2，CCL5，CXCL8 和促炎細胞因子IL-6。此外，地塞米松和沒食子酸的結合使用能增強抑制KU812 細胞面上ICAM-1 的表達，和皮膚成纖維細胞面上ICAM-1 和VCAM-1 的表達，與及趨化因子CCL2 和CXCL8，促炎性細胞因子IL-6 的釋放。 / 上述調查結果表明，天然植物產品PHF，DP 和GA 是具有消炎和抗過敏的作用，抑制嗜鹼性粒細胞趨化遷移至發炎處和隨後釋放的過敏性炎症介質，如炎症相關的趨化因子和促炎細胞因子。結果表明，沒食子酸天然植物產品可能是一個潛在的過敏性皮膚炎的治療劑，而沒食子酸和地塞米松的結合使用，可以有效降低在患者治療皮膚炎症時使用地塞米松的劑量。總結，這項研究結果揭示使用天然植物衍生產品具有更安全,高效力和副作用少的一種新治療方式。 / The worldwide prevalence of allergic diseases has been increasing gradually. Around 30 - 40% of the world population suffers from one or more allergic conditions, it is definitely a national public health issue. The incidence of Atopic Dermatitis (AD) has increased by 2-3 folds in the past 3 decades with no definitive cure, where the case is the highest during the early infancy and childhood. However, effective treatments on allergic diseases are still lacking, with most of the traditional treatment involves clinical improvement but not targeting the primary factors promoting the pathogenesis of allergic inflammation. These traditional treatments have undesirable side effects. Therefore, there has been a rising interest in the development of a safer and nonsteroid immunomodulation formula to cure the disease. / From previous clinical trails, it is revealed that children with moderate-to-severe AD treated with traditional Chinese Medicine, Pentaherbs formula (PHF), have significantly improved their quality of life, lowered the Scoring of Atopic Dermatitis (SCORAD) index and the use of topical steroids without any adverse drug effect, suggesting that PHF can be an alternative potential adjunct therapy for AD. In this present studies, we elucidated the in vitro anti‐inflammatory and anti‐allergic activities of PHF, Cortex Moutan / Danpi (DP, one of the five herbs in PHF) and gallic acid (GA, one of the main ingredients in Danpi) using human basophilic KU812 cells, with or without human dermal fibroblast, upon the activation with alarmin inflammation-related cytokine IL-33. / Human basophilic KU812 cells activated by alarmin cytokine IL-33 were used as basophil cell model for study since basophils are a crucial effector cells in allergic inflammation. Our results showed that PHF, DP and GA exhibited the anti-inflammatory and anti‐allergic activities indicated by significant suppressive effects on the intercellular adhesion molecule (ICAM)‐1 expression, the release of inflammation‐related chemokines CCL2, CCL5, CXCL8 and proinflammatory cytokine IL-6 from IL-33‐activated KU812 basophilic cells. The studies were further investigated with the combined use of synthetic steroid dexamethasone which is a common drug for AD. Among various combinations with different concentrations of dexamethasone with PHF, DP and GA, we demonstrated that the combined use of a concentration as low as 0.01 μg/ml dexamethasone and 10 μg/ml GA could further suppress ICAM‐1 expression, chemokines CCL2 and CCL5 release in IL-33 activated KU812 cells. Furthermore, gallic acid could significantly suppress the intracellular signaling molecules p38 MAPK, IκB-α and JNK in KU812 cells, thereby suggesting the underlying mechanisms for the suppressive effect on adhesion molecules expression, and the chemokines and cytokines release. Both in vivo and in vitro experiments show that PHF, DP and GA exhibit the anti-inflammatory and anti‐allergic activities in concordance to the previous clinical trials using PHF on AD children. / In order to further study the involvement of gallic acid and dexamethasone in the pathogenesis of AD, we then established an in vitro skin inflammatory cell model by co‐culturing human basophilic KU812 cells and human dermal fibroblasts mimicking the skin lesions of the AD patients. We revealed that the application of gallic acid alone could already significantly suppress the adhesion molecules expression on KU812 cell and fibroblasts, and the release of AD-related chemokines CCL2, CCL5, CXCL8 and pro-inflammatory cytokine IL-6 from the co‐culture. In addition, the combined use of dexamethasone and gallic acid showed an enhanced suppressive effect on ICAM-1 on KU812, and ICAM-1 and VCAM-1 on fibroblasts, AD‐releated chemokines CCL2 and CXCL8, and pro-inflammatory cytokine IL-6. / The above findings suggest that PHF, DP and GA are anti-inflammatory and anti-allergic natural plant products by suppressing the transmigration of basophils into the inflamed sites and the subsequent release of allergic inflammation mediators e.g. inflammation-related chemokines and proinflammatory cytokines.The results suggest that natural plant product gallic acid could be a potential therapeutic agent in treating skin inflammation in AD, and the combined use of gallic acid with dexamethasone could lower the dosage of dexamethasone used in AD patients. Together, of the results of this study shed light for a novel therapeutic modality of AD using a safer natural plant derived product with high potency and less side effects to treat AD. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Liu, Yan Ping Kelly. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 107-122). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.I / ABSTRACT --- p.III / 摘要 --- p.VI / PUBLICATIONS --- p.IX / ABBREVIATIONS --- p.XI / TABLES OF CONTENTS --- p.XIII / Chapter CHAPTER 1: --- General Introduction / Chapter 1.1 --- Allergy --- p.1 / Chapter 1.1.1 --- Definition of Allergy --- p.1 / Chapter 1.1.2 --- Allergic diseases and their prevalence --- p.2 / Chapter 1.1.3 --- Allergic inflammation and its characteristics --- p.2 / Chapter 1.1.4 --- Treatment of allergy --- p.4 / Chapter 1.1.5 --- Atopic Dermatitis --- p.7 / Chapter 1.2 --- Biology of basophils --- p.8 / Chapter 1.2.1 --- Development of basophils --- p.8 / Chapter 1.2.2 --- Morphology and phenotype --- p.9 / Chapter 1.2.3 --- Mast cells and basophils --- p.11 / Chapter 1.2.4 --- Basophils and allergic inflammation --- p.12 / Chapter 1.2.5 --- Human basophilic KU812 cell line --- p.13 / Chapter 1.3 --- Adhesion molecules in allergic inflammation --- p.14 / Chapter 1.3.1 --- Selectins --- p.14 / Chapter 1.3.2 --- Integrins --- p.16 / Chapter 1.3.3 --- Immunoglobulin gene super family --- p.16 / Chapter 1.4 --- Chemokines in allergic inflammation --- p.18 / Chapter 1.4.1 --- C chemokines --- p.18 / Chapter 1.4.2 --- CC chemokines --- p.18 / Chapter 1.4.3 --- CXC chemokines --- p.19 / Chapter 1.4.4 --- CX3C chemokines --- p.19 / Chapter 1.5 --- Cytokines in allergic inflammation --- p.20 / Chapter 1.5.1 --- Proinflammatory cytokines --- p.20 / Chapter 1.5.2 --- Anti-inflammatory cytokines --- p.23 / Chapter 1.6 --- Signal Transduction in allergic inflammation --- p.25 / Chapter 1.6.1 --- Intracellular signaling mechanisms --- p.25 / Chapter 1.6.2 --- RAS-RAF-MAPK pathway --- p.27 / Chapter 1.6.3 --- JAK/STAT pathway --- p.28 / Chapter 1.6.4 --- PI3K-Akt pathway --- p.28 / Chapter 1.6.5 --- NF-κB pathway --- p.28 / Chapter 1.7 --- Aim of Study --- p.29 / Chapter Chapter 2: --- Materials and Methods / Chapter 2.1 --- Materials --- p.32 / Chapter 2.1.1 --- Cell Culture --- p.32 / Chapter 2.1.2 --- Serum Supplements --- p.33 / Chapter 2.1.3 --- Recombinant human cytokine --- p.33 / Chapter 2.1.4 --- Dexamethasone --- p.33 / Chapter 2.1.5 --- Phosphate-buffered saline --- p.34 / Chapter 2.1.6 --- Dimethyl sulfoxide --- p.34 / Chapter 2.1.7 --- Nucleotide-binding oligomerization domain ligands --- p.34 / Chapter 2.1.8 --- BAY 117082 --- p.34 / Chapter 2.1.9 --- Cell surface and intracellular immunofluorescence staining --- p.35 / Chapter 2.1.10 --- In vitro XTT based toxicology assay kit --- p.38 / Chapter 2.1.11 --- Quantitative analysis of inflammatory mediators release --- p.38 / Chapter 2.1.12 --- Natural Products --- p.39 / Chapter 2.1.13 --- Animal Experiment --- p.40 / Chapter 2.2 --- Methods --- p.41 / Chapter 2.2.1 --- Cell Culture --- p.41 / Chapter 2.2.2 --- Preparation of plant extracts --- p.42 / Chapter 2.2.3 --- Cell toxicity of the natural products --- p.42 / Chapter 2.2.4 --- Flow cytometric analysis of cell surface expression of molecules --- p.43 / Chapter 2.2.5 --- CBA assay --- p.43 / Chapter 2.2.6 --- Flow cytometric analysis of activated intracellular molecules --- p.44 / Chapter 2.2.7 --- Allergic asthmatic mice model --- p.45 / Chapter 2.2.8 --- Statistical analysis --- p.45 / Chapter Chapter 3: --- Anti-inflammatory and anti-allergic properties of Pentaherbs formula, Danpi and Gallic acid / Chapter 3.1 --- Introduction --- p.46 / Chapter 3.1.1 --- Basophils in inflammation --- p.46 / Chapter 3.1.2 --- IL-33 --- p.47 / Chapter 3.1.3 --- Natural plant products --- p.48 / Chapter 3.1.4 --- Dexamethasone --- p.51 / Chapter 3.1.5 --- Hypothesis and aim of study --- p.52 / Chapter 3.2 --- Results --- p.54 / Chapter 3.2.1 --- Cell cytotoxicity of PHF, DP and GA on human basophilic KU812 cells --- p.54 / Chapter 3.2.2 --- Effect of adhesion molecules expression on IL-33-activated KU812 cells treated with PHF, DP and GA --- p.56 / Chapter 3.2.3 --- Effect of PHF, DP and GA on inflammation-related chemokines CCL2,CCL5, CXCL-8 production from IL-33-activated KU812 cells --- p.59 / Chapter 3.2.4 --- Effect of PHF, DP and GA on pro-inflammatory cytokine IL-6 production from IL-33-activated KU812 cells --- p.64 / Chapter 3.2.5 --- Intracellular signaling pathways involved in GA treatment on IL33-activated KU812 cells --- p.67 / Chapter 3.2.6 --- Effect on the adhesion molecules expression, chemokines and cytokines release of IL-33-activated human basophilic KU812 cells upon the combined treatment of PHF/DP/GA with dexamethasone --- p.73 / Chapter 3.2.7 --- In vivo effect of PHF and DP on Th2 and inflammatory cytokines concentration in serum or BALF in allergic inflammatory mice models --- p.76 / Chapter 3.3 --- Discussion --- p.79 / Chapter Chapter 4: --- Gallic acid and Dexamethasone in Atopic Dermatitis / Chapter 4.1 --- Introductions --- p.84 / Chapter 4.1.1 --- Atopic Dermatitis --- p.84 / Chapter 4.1.2 --- Basophils in AD --- p.86 / Chapter 4.1.3 --- Dermal fibroblasts in AD --- p.86 / Chapter 4.1.4 --- Hypothesis --- p.87 / Chapter 4.2 --- Results --- p.88 / Chapter 4.2.1 --- Effect of the combined use of GA and dexamethasone on ICAM-1 expression on KU812 cells co-cultured with fibroblasts --- p.88 / Chapter 4.2.2 --- Effect of the combined use of GA and dexamethasone on ICAM-1 and VCAM-1 expression on fibroblasts co-cultured with KU812 cells --- p.90 / Chapter 4.2.3 --- Effect on chemokines release from the co-culture upon the treatment with GA and dexamethasone --- p.93 / Chapter 4.2.4 --- Effect on cytokine release from the co-culture treated with GA and dexamethasone --- p.96 / Chapter 4.3 --- Discussions --- p.98 / Chapter Chapter 5: --- Concluding Remarks and Future Prospective / Chapter 5.1 --- Concluding remarks --- p.100 / Chapter 5.2 --- Future prospective --- p.101 / References --- p.107

Une cellule dendritique (CD) qui exprime le facteur de transcription GILZ durant l’apprêtement de l’antigène et sa présentation aux cellules effectrices, génère des lymphocytes T régulateurs (LTregs) CD25high Foxp3+ sécréteurs d’IL-10. La production de GILZ est dépendante de l’action des glucocorticoïdes, de l’IL-10 et du TGF-.Nous avons mis en évidence chez l’homme qu’une corticothérapie orale de 48h induit l’expression de GILZ dans les cellules présentatrices de l’antigène circulantes (CPAs) de sujets allergiques. Les CPAs isolées après la corticothérapie génèrent des LTregs CD25high Foxp3+ IL-10+ spécifiques de l’allergène.Nous également constaté in vitro que les mastocytes participent à l’activation des CDs au cours des réactions allergiques en régulant l’expression de GILZ. Les médiateurs d’origine mastocytaire, dont l’histamine, diminuent l’expression de GILZ dans les CDs et altèrent ainsi leur capacité à activer des LTregs. Nous avons identifié le mécanisme par lequel l’histamine diminue l’expression de GILZ dans les CDs humaines. L’histamine inhibe l’activité transcriptionnelle de Foxo3, un facteur de transcription régulant l’expression de GILZ. Enfin, nous avons démontré que des souris transgéniques qui surexpriment GILZ constitutivement dans les CDs sont protégées contre le développement de l’asthme allergique. L’ensemble de ces résultats permet d’envisager de nouvelles stratégies d’immunomodulation dans l’allergie, centrée sur la régulation de l’expression de GILZ dans les CDs. / We previously showed in vitro that DCs with a high level of GILZ activate regulatory T cells (Tregs) whereas DCs with low level of GILZ trigger effector T lymphocytes. Glucocorticoids (GCs), IL-10 and TGF- are potent inducers of GILZ expression. The aim of this thesis is to extend the above findings to induction of tolerance to allergens. Modulation of GILZ expression by DCs should induce allergen-specific Tregs able to inhibit the activation and proliferation of allergen specific T cell clones. In order to validate this concept we demonstrated that:- allergen-specific tolerance can be achieved in allergic patients treated with oral GC through the induction of GILZ expression in their antigen-presenting cells, and the role of allergen-specific Tregs in this effect,- mast cells play a role in the activation of DCs by inhibiting their expression of GILZ and thus their ability to stimulate Tregs against harmless environmental allergens,- GILZ-expressing DCs protect against allergic asthma in a model of transgenic mice over-expressing GILZ in their DCs.The present study supports the concept of an immune regulation of allergic responses through the modulation of GILZ expression by DCs and opens new perspectives in the development of innovative immunotherapies in the treatment of allergic diseases.

A dermatite de contato alérgica (DCA) ao íon níquel (Ni2+) é uma dermatose inflamatória frequente nos países industrializados. Envolve a ativação de células T específicas ao Ni2+, seguida da proliferação e indução de um perfil misto de citocinas, tanto pró-inflamatórias quanto reguladoras, sugerindo que vários subtipos de células T (helper - Th e citotóxica - Tc) estão envolvidos na resposta imune. Este estudo teve como objetivo a análise das citocinas TNF-alfa, INF-y, IL-2, IL-4, IL-10, IL-13, IL-17 e IL-23 pela técnica de imuno-histoquímica, para tentar identificar a prevalência de um ou mais subtipos de células T (Th/Tc), nos eczemas crônico e agudo de pacientes com DCA ao Ni2+. Avaliamos 20 pacientes (17 mulheres e 3 homens, com idade mediana de 46 anos) apresentando eczema crônico, pelo contato cotidiano do paciente com o Ni2+. Foram coletadas duas biópsias de pele em cada um dos 20 pacientes, a primeira no local do eczema crônico ao Ni2+, antes da aplicação do teste de contato (TC); e a segunda no local do eczema agudo, provocado pelo TC com o sulfato de níquel, 48 horas após sua fixação, nas leituras positivo forte (++) ou positivo muito forte (+++). Foram 160 amostras de eczema agudo e 160 de eczema crônico, perfazendo um total de 320 amostras. Apenas três amostras foram excluídas devido a algum tipo de falha técnica, como, por exemplo, o descolamento dos cortes de pele da lâmina. Para a análise dos dados utilizou-se o software estatístico STATA versão 13. As amostras coradas revelaram resultados positivos para as oito citocinas estudadas, e estas apresentaram valores heterogêneos. Esta heterogeneidade foi medida pelo coeficiente de variação, indicando a variabilidade do conjunto dos dados obtidos. O TNF-alfa, IFN-y, IL-4, IL-13 e IL-17 tiveram prevalência maior no eczema crônico do que no eczema agudo, a IL-2 e IL-23 apresentaram maior prevalência no eczema agudo, em comparação com o eczema crônico e a IL-10 apresentou prevalência similar tanto no eczema agudo quanto no crônico, porém, estas prevalências foram muito baixas, em ambos os eczemas. O TNF-alfa foi a citocina que mais prevaleceu no eczema crônico e a IL-2 foi a mais prevalente no eczema agudo. Porém, estas prevalências foram estatisticamente significantes apenas para a IL-4 e IL-13. Verificamos, nos eczemas crônico e agudo, a presença de um perfil misto de citocinas dos subtipos de células T (Th/Tc), sugerindo que as respostas imunes são expressas ao mesmo tempo. Entretanto, são necessários mais estudos para uma compreensão mais ampla sobre o perfil das citocinas na DCA ao Ni2+, o que poderia levar a novas abordagens terapêuticas / Allergic contact dermatitis (ACD) to nickel (Ni+2) is a inflammatory dermatosis, common in industrialized countries. It involves the activation of nickel-specific T cells, followed by the proliferation and induction of a mixed profile of both proinflammatory and regulatory cytokines, suggesting that several T cell subtypes (helper - Th and cytotoxic - Tc) are involved in the immune response. This study aimed to analyze the cytokines TNF-alfa, INF-y, IL-2, IL-4, IL-10, IL-13, IL-17 and IL-23 using the immunohistochemistry technique in order to try to identify the prevalence of one or more T cell subtypes (Th/Tc) in the chronic and acute eczema of patients with ACD to Ni+2. We evaluated 20 patients (17 women and 3 men, median age of 46 years) with chronic eczema, by the patient\'s daily contact with Ni+2. Two skin biopsies were collected in each of the 20 patients, the first at the site of the chronic eczema to Ni+2, prior to the application of the contact test (CT); and the second at the site of acute eczema caused by CT with nickel sulphate, 48 hours after its fixation in the strong positive (++) or very strong positive (+++) readings. There were 160 samples of acute eczema and 160 of chronic eczema, a total of 320 samples. Only three samples were excluded due to some kind of technical failure, such as detachment of the skin cuts from the microscope slide. Statistical software STATA version 13 was used to analyze the data. The stained samples showed positive results for the eight cytokines studied, and these presented heterogeneous values. This heterogeneity was measured by the coefficient of variation, indicating the variability of the data set obtained. TNF-alfa, IFN-y, IL-4, IL-13 and IL-17 had a higher prevalence in chronic eczema than in acute eczema, IL-2 and IL-23 were more prevalent in acute eczema compared to chronic eczema and IL-10 presented similar prevalence in both acute and chronic eczema, however, a very low prevalence in both eczema. TNF-alfa was the most prevalent cytokine in chronic eczema and IL-2 was the most prevalent in acute eczema. However, these prevalences were statistically significant only for IL-4 and IL-13. In chronic and acute eczema, we observed the presence of a mixed cytokine profile of the T cell subtypes (Th/Tc), suggesting that immune responses are expressed at the same time. However, further studies are needed for a broader understanding of the cytokine profile in ACD to Ni+2, which could lead to new therapeutic approaches

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No. of bitstreams: 2 Dissertação - Barbara Hess Rodrigues Gonçalves - 2016.pdf: 1742093 bytes, checksum: 80bd9955d0b421c59b1e6faf6d41f773 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-12-13 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Canine atopic dermatitis (CAD) is the second most frequent disease in dermatological clinical routine of dogs. It is defined as a pruritic allergic skin disease, with genetic predisposition and clinical features, being related to the response to environmental allergens. Interleukin 31 (IL) 31 is a cytokine that participates in inflammatory processes and it is associated with pruritic diseases, especially those involving chronic inflammation such as allergic dermatitis. Produced by mononuclear cells, IL-31 is described to play an important role in the pathogenesis of atopic dermatitis. Two studies were performed in order to correlate clinical features, histopathological changes and the presence of IL-31 in the skin of dogs with atopic dermatitis. 34 dogs were selected from clinical routine, which 31 animals were diagnosed with CAD and three were healthy. The animals were evaluated for pruritus level by the owners' report and by clinical examination according to the CAD extent and severity index (CADESI-4). The dogs were grouped in discreetly, moderately and markedly compromised by CAD following the sum of scores’ values assigned in the clinical examination. Cutaneous samples from the axillary and interdigital regions of each dog were collected and submitted to histopathological (HE and toluidine blue) and immunohistochemical analyzes (IL-31). There was a correlation between the clinical score and the microscopic changes. As well, there was correlation among all the microscopic changes, but not between the degree of pruritus and the clinical score of CAD. Also, the inflammatory infiltrate was more intense in the axillary region in relation to interdigital skin. An increased numbers of cells immunostained for IL-31 was observed in dogs severely compromised by CAD. There was a correlation between the clinical score and the amount of interdigital mast cells, with an amount of cells immunostained for IL-31 in the axilla. It was also verified correlation between the amount of mast cells and cells immunostained for IL-31 in the axilla, as well as between acanthosis and all other histopathological alterations of the skin in the axillary region. We concluded that there is a correlation between severity of dermathological lesions, IL-31 immunostaining, mast cell count and histopathological changes in dogs with atopic dermatitis, but there is no correlation between the degree of pruritus reported by owners and severity of cutaneous lesions in animals with CAD. Moreover, the intensity of the inflammatory process may vary depending on the anatomical site of the lesion. / A dermatite atópica canina (DAC) é a segunda doença mais incidente na rotina de atendimento clínico dermatológico de cães. É definida como doença cutânea alérgica pruriginosa, de predisposição genética e características clínicas definidas, estando relacionada a resposta a alérgenos ambientais. A interleucina 31 (IL) 31 é uma citocina que participa de processos inflamatórios e está associada a doenças pruriginosas, principalmente as que envolvem inflamação crônica como as dermatites alérgicas. Produzida por células mononucleares, a IL-31 é descrita por desempenhar papel importante na patogênese da dermatite atópica.Com o objetivo de correlacionar as alterações clínicas, histopatológicas e a presença da IL-31 na pele de cães com dermatite atópica foram realizados dois estudos. Para isso, foram selecionados 34 cães da rotina de atendimento clínico, sendo 31 com diagnóstico de DAC e três hígidos. Os animais foram avaliados quanto ao nível de prurido a partir do relato de seus proprietários e quanto ao índice de extensão e severidade da DAC (CADESI-4) a partir do exame clínico. Os cães foram agrupados em discretamente, moderadamente e acentuadamente comprometidos pela DAC após somatório dos valores de escores atribuídos no exame clínico. Amostras cutâneas da região axilar e interdigital de cada cão foram colhidas e submetidas às análises histopatológica (HE e azul de toluidina) e imunoistoquímica (IL-31). Houve correlação entre o escore clínico e as alterações microscópicas, assim como entre as alterações microscópicas, mas não entre o grau de prurido e o escore clínico da DAC. Também o infiltrado inflamatório foi mais intenso na região axilar em relação a interdigital. Maior número de células imunomarcadas para IL-31 foi observado nos cães acentuadamente comprometidos pela DAC. Houve correlação entre o escore clínico e a quantidade de mastócitos no interdígito, do mesmo modo com a quantidade de células imunomarcadas para IL31 na axila. Também foi verificada correlação entre a quantidade de mastócitos e células imunomarcadas para IL-31 na axila, assim como entre acantose e todas as demais alterações histopatológicas da pele na região axilar. Conclui-se que há correlação entre a gravidade das lesões, a imunomarcação de IL-31, a contagem de mastócitos e as alterações histopatológicas em cães com dermatite atópica, mas não há correlação entre o grau de prurido relatado pelos proprietários e a gravidade das lesões cutâneas em animais com DAC. Ainda, a intensidade do processo inflamatório pode variar em função do sítio anatômico da lesão.

As relações bidirecionais entre o Sistema Nervoso e o Sistema Imune são relevantes para a manutenção da homeostase do organismo. Estudos realizados em nosso laboratório mostraram que 14 dias de coabitação com um conspecífico doente (injetado com células do tumor de Ehrlich-TAE) produziu mudanças comportamentais, endócrinas e imunológicas. Este estudo analisa os efeitos da convivência com um animal portador de tumor de Ehrlich em camundongos OVA sensibilizados e desafiados sobre a resposta alérgica pulmonar. Pares de camundongos machos foram separados em três grupos: naïve, controle e experimental. Os animais do grupo naïve não foram manipulados sendo utilizados para a avaliação de parâmetros basais. Um animal de cada par dos grupos experimental e controle foi imunizado com OVA. No dia D(0), os animais imunizados receberam uma dose reforço de OVA. No dia D(0) os camundongos do grupo experimental que não foram manipulados foram inoculados com 5x106 células de tumor de Ehrlich; seus companheiros de gaiola moradia foram designados CAD (companheiro do animal doente). Os camundongos não perturbados de cada par do grupo controle foram tratados (i.p.) em D(0) com 0,9% de NaCl, sendo designados CAS (companheiro do animal saudável). O desafio intranasal com OVA foi realizado nos camundongos CAS e CAD nos dias D(12) e D(13); colheram-se o sangue e os tecidos no dia D(14). Em comparação com o grupo CAS, os camundongos do grupo CAD apresentaram 14 dias após a coabitação: (1) aumento do número de eosinófilos e neutrófilos no LBA, (2) diminuição na contagem de células da medula óssea, (3) aumento do níveis de IL-4 e IL-5 e diminuição de IL-10 e INF-ϒ no sobrenadante do LBA, (4) aumento dos níveis de IgG1-OVA, diminuição dos níveis de IgG2a-OVA e nenhuma alteração na IgE-OVA no sangue periférico, (5) aumento na expressão de ICAM-1, VCAM-1 e L-selectina em granulócitos do LBA, (6) diminuição da reatividade da traquéia à metacolina in vitro, (7) aumento da desgranulação de mastócitos, (8) nenhuma alteração nos níveis plasmáticos de corticosterona, (9) aumento dos níveis de adrenalina e noradrenalina plasmáticas, (10) diminuição no tempo de permanência e entradas nos braços abertos do labirinto em cruz elevado, (11) diminuição da expressão de IL-6 no PVN e (12) diminuição da expressão de C-fos no PFC. Estes resultados mostram que a convivência forçada com um animal portador de um tumor ascitico de Ehrlich exacerba a inflamação alérgica pulmonar de camundongos. Eles foram discutidos como decorrentes da estimulação do Sistema Nervoso Autônomo Simpático (SNS) pelo estresse psicológico gerado pela coabitação com o parceiro doente, via liberação de adrenalina e noradrenalina e consequente mudança no perfil de citocinas Th1/Th2 para uma resposta do tipo Th2. Esta alteração seria, provavelmente, um dos mecanismos responsáveis pelo aumento do recrutamento celular para as vias aéreas dos camundongos do grupo CAD. / The bidirectional relationship between the nervous system and the imune system is relevant for homeostatic organism maintenance. Studies from our laboratory showed that 14 days of cohabitation with a sick conspecific (injected with Ehrlich tumor cells-TAE) produced behavioral, endocrinological and immunological changes. This study analyzes the effects of cohabitation with an Ehrlich tumor-bearing animal on ovalbumin (OVA)-induced lung inflammatory response in mice. Pairs of male mice were separate into three groups: naïve, control and experimental. Animals of the naïve group were kept undisturbed being used for assessment of basal parameters. One animal of each experimental and control pair of mice was immunized with OVA. On D(0), these OVA-immunized animals received an OVA booster. At this day (D(0)) the experimental mice that were kept undisturbed were inoculated with 5x106 Ehrlich tumor cells; their immunized cage-mates were then referred as to CSP(companion of sick partner). The undisturbed mice of each control pair were i.p. treated on D(0) with 0.9% NaCl; their sensitized cage-mate were subsequently referred as CHP (companion of health partner). The intranasal OVA challenge was performed on CSP and CHP mice on D(12) and D(13); blood and tissue collection were performed on D (14). Fourteen days after cohabitation, in comparison to the CHP mice, the CSP mice displayed the following: (1) an increased number of eosinophils and neutrophils in the BAL, (2) a decreased bone marrow cell count, (3) increased levels of IL-4 and IL-5 and decreased levels of IL-10 and INF-ϒ in the BAL supernatant, (4) increased levels of IgG1-OVA, decreased levels of IgG2a-OVA and no changes in OVA-specific IgE in the peripheral blood, (5) increased expression of ICAM-1, VCAM-1 and L-selectin in the BAL granulocytes, (6) decreased tracheal reactivity to metacholine measured in vitro , (7) increased mast cell degranulation, (8) no changes in plasma corticosterone levels (9) increased levels of plasmatic adrenaline and noradrenaline, (10) decreased time and % of entries on open arms of elevated plus maze, (11) decreased expression of IL-6 on PVN and (12) decreased expression of C-fos on PFC. These results suggest that cohabitation with an Ehrlich tumor bearing mice exacerbates allergic lung inflammatory response in mice. Most probably, the changes observed in CSP mice are a consequence of the psychological stress induced by forced cohabitation with the sick partner. Strong involvement of the sympathetic nervous system through adrenaline and noradrenaline release and a shift of the Th1/Th2 cytokine profile toward a Th2 response were considered to be the mechanisms underlying the cell recruitment to the animal´s airways.

Introdução: Asma e rinite alérgicas são doenças inflamatórias crônicas das vias aéreas cuja principal etiopatogenia é a reação de hipersensibilidade ao ácaro da poeira. O único tratamento específico que modifica a história natural da doença é a imunoterapia alérgeno-específica (ITAE). Porém, as reações adversas ainda são uma dificuldade na consolidação da ITAE como forma de tratamento. Um meio de minimizar as reações e maximizar os efeitos terapêuticos é o uso de adjuvantes, entre eles vacinas e lipopolissacárides bacterianos, que objetivam aumentar o desvio da resposta imune de perfil T-helper (TH) 2 para TH1. A vacina de Bordetella pertussis de célula inteira (Pw) mostrou ter um papel protetor em modelos experimentais de asma induzida por ovalbumina. Porém, seu papel na alergia respiratória induzida por ácaros ainda não é conhecido. Nosso objetivo foi avaliar os efeitos da vacina tríplice bacteriana (difteria-tétano-coqueluche - DTPw) em um modelo murino de alergia respiratória crônica induzida pelo ácaro Dermatophagoides pteronyssinus (Derp). Métodos: O protocolo teve duração de 30 dias. Camundongos BALB/c foram divididos em 6 grupos, os quais foram sensibilizados por via subcutânea com solução salina ou Derp 50mcg, em três injeções. Três grupos foram imunizados com Derp, apenas com o ácaro ou associado as vacinas de difteria-tétano (DT) e DTPw, respectivamente. Os outros três receberam soro fisiológico, com ou sem vacinas DT e DTPw. Os animais, posteriormente, sofreram desafio intranasal com soro fisiológico ou Derp por 7 dias e foram sacrificados 24 horas após o último desafio. Medimos IgE, IgG1 e IgG2a séricas específicas anti-Derp; resistência, elastância e hiperresponsividade das vias aéreas; densidade de leucócitos polimorfonucleares (PMN) e área de muco ácido no epitélio nasal; celularidade no lavado bronco-alveolar (BAL); e remodelamento das vias aéreas inferiores. Resultados: Os animais sensibilizados com Derp produziram altos níveis de imunoglobulinas específicas, apresentaram aumento da densidade de PMN e da área de muco ácido nasal, elevação das células inflamatórias no BAL às custas de macrófagos e remodelamento significativo das vias aéreas. As vacinas levaram à redução dos níveis de IgE específica e o grupo Derp-DTPw apresentou os menores níveis, similares aos grupos salina. A vacina DTPw não alterou os níveis de IgG1 ou IgG2a em relação ao grupo Derp. Não houve diferença entre todos os 6 grupos quanto aos parâmetros ventilatórios e quanto à contagem de eosinófilos no BAL. As vacinas DT e DTPw inibiram o infiltrado PMN nasal e DTPw modulou a produção do muco ácido. Os grupos vacinados tiveram redução da celularidade no BAL e do remodelamento em comparação com os controles, com resultados mais expressivos no grupo Derp-DTPw em relação ao grupo Derp-DT. Conclusões: Neste modelo murino de alergia respiratória crônica induzida por ácaro, a vacina DTPw diminuiu a IgE específica sérica, as células inflamatórias no nariz e no BAL, o muco ácido nasal e o remodelamento das vias respiratórias inferiores. / Background: Asthma and allergic rhinitis are chronic inflammatory diseases of the airways whose primary etiology is hypersensitivity reaction to house dust mite (HDM). The only specific treatment which modifies the natural history of the disease is the allergen specific immunotherapy (ASIT). However, adverse reactions are still a difficulty in consolidating ASIT as a good treatment option. One way to minimize adverse reactions and maximize therapeutic effects is the use of adjuvants, including bacterial lipopolysaccharide and vaccines that aim to shift immune response profile from T helper (TH) 2 to TH1. Bordetella pertussis whole-cell vaccine (Pw) has shown to have a protective role in experimental models of ovalbumin-induced asthma. Nevertheless, its role in respiratory allergy induced by HDM is unknown. Our objective was to evaluate the effects of diphtheria-tetanus-pertussis (DTPw) vaccine in a murine model of respiratory allergy induced by the HDM Dermatophagoides pteronyssinus (Derp). Methods: The protocol lasted 30 days. BALB/c mice were divided into 6 groups, which were sensitized subcutaneously (s.c.) with saline solution or Derp 50mcg, in three injections. Three groups were submitted to Derp alone or associated with diphtheria-tetanus (DT) vaccine and DTPw, respectively. The other three received saline solution with or without DT and DTPw vaccines. Subsequently mice underwent intranasal challenge with saline or Derp for 7 days and were sacrificed 24h after the last challenge. We measured serum specific IgE, IgG1 and IgG2a anti-Derp, airway resistance, elastance and hyperresponsiveness, density of polymorphonuclear (PMN) leukocytes infiltration in nasal mucosa, acidic nasal mucus content, cellularity in bronchoalveolar lavage (BAL) and lower airways remodeling. Results: HDM sensitized mice produced high levels of specific immunoglobulins, increased density of PMN leukocytes and acidic mucus in nasal mucosa, inflammatory cells in BAL due to macrophages and developed significant airway remodeling. Vaccines have led to a progressive reduction in specific IgE levels and Derp-DTPw group had lower levels, similar to groups sensitized with saline solution. DTPw vaccine did not alter the levels of IgG1 and IgG2a compared to Derp group. There was no difference between all six groups on behalf of ventilatory parameters and eosinophil counts in BAL. Both DT and DTPw vaccines inhibited PMN infiltrate in the nose and DTPw modulated acidic nasal mucus. Vaccinated groups had reductions in BAL cellularity and remodeling compared to controls, and Derp-DTPw group had better results than Derp-DT group. Conclusions: In this murine model of chronic respiratory allergy induced by HDM, DTPw vaccine decreased specific IgE, inflammatory cells in the nose and BAL, acidic nasal mucus and lower airways remodeling