Orientador: Renée Laufer Amorim / Banca: Noeme Sousa Rocha / Banca: Luiz Henrique de Araújo Machado / Banca: Mirela Tinucci. Costa / Banca: Sara Maria Carvalho e Suzano / Resumo: O mastocitoma canino (MCT) é uma neoplasia maligna de grande importância na clínica oncológica devido ao seu comportamento biológico agressivo e alta freqüência. A COX-2 e a PGE2 têm sido associadas à promoção e progressão tumoral e seus principais mecanismos envolvem estímulos da angiogênese tumoral e a inibição da morte celular programada. O VEGF é um potente indutor da angiogênese e a caspase-3 tem um importante papel na via efetora da apoptose. Compreender o mecanismo pela qual a COX-2 pode estimular a progressão tumoral no mastocitoma, permite ampliar o conhecimento do comportamento biológico desta neoplasia e direcionar tratamentos mais eficazes. O presente trabalho fez um estudo retrospectivo em 24 casos de mastocitoma canino (MCT). As neoplasias foram classificadas de acordo com Patnaik et al. (1984) e a expressão da COX-2, PGE2, VEGF e caspase-3 foram avaliadas usando a técnica de imunoistoquímica. A expressão da COX-2 foi correlacionada à expressão do VEGF, PGE2 e caspase-3 nos diferentes graus histopatológicos. A imunomarcação da caspase-3 foi menor nos tumores indiferenciados comparados com os bem diferenciados. Comparando os dados da expressão da COX-2 com os demais marcadores foi observado a correlação positiva entre COX-2 e PGE2, COX-2 e VEGF nas graduações II e III. A correlação entre COX-2 e caspase-3 foi somente detectada no grau III. / Abstract: The canine mast cell tumor (MCT) is a malignant neoplasia with great importance on the clinical practice due to its aggressive behavior and high frequency. The COX-2 and the PGE2 have been associated to the tumor initiation, promotion and progression, and its main mechanisms involve the stimuli of tumor angiogenesis and the inhibition of apoptosis. The VEGF is a powerful inductor of angiogenesis and the caspase-3 is responsible for most part of the apoptotic effects. The understanding of the mechanism by which the COX-2 stimulates the tumor progression in the mast tumor cells provides an extension through the biological behavior of this neoplasia and leads to a better and effective treatment. The present work was a retrospective study in 24 cases of MCT. The neoplasias were classified according to Patnaik et al. (1984) and the expression of COX-2, PGE2, VEGF and caspase-3 were evaluated using the immunohistochemistry technique. The expression of COX-2 was correlated to the expression of VEGF, PGE2 and caspase-3 in the different histopathologic grades. Caspase-3 immunolabeling was lower in the undifferentiated tumors compared to the more differentiated ones. Comparing the COX-2 expression data to the other markers it was observed a positive correlation between COX-2 and PGE2, COX-2 and VEGF in grade II and III. Correlation between COX-2 and caspase-3 was detected only on grade III. Keywords: COX-2, PGE2, VEGF, caspase-3, mast cell tumor. / Doutor

As doenças periodontais afetam os tecidos de suporte dos dentes e são desencadeadas por microrganismos que possuem a capacidade de invadir os tecidos periodontais. A evolução desta doença é influenciada pela resposta inflamatória e imunológica do hospedeiro e envolve a participação de diversos tipos celulares. Atualmente, existem evidências de que os mastócitos, além de outras funções, possuem a capacidade de eliminar bactérias, através da fagocitose. Assim sendo, este estudo teve por objetivo avaliar a capacidade fagocítica dos mastócitos frente ao periodontopatógeno A. actinomycetemcomitans, além de comparar sua capacidade fagocítica com a dos macrófagos, considerados fagócitos profissionais. Para este fim, foram realizados ensaios fagocíticos in vitro utilizando mastócitos e macrófagos murinos, desafiados ora com A. actinomycetemcomitans ora com Escherichia coli, opsonizados ou não, sob diferentes proporções célula: bactérias. Após 1 hora de desafio, as células foram coradas com laranja de acridina e avaliadas qualitativamente utilizando-se microscópio de varredura confocal a laser e quantitativamente através do microscópio de fluorescência convencional. Nossos resultados demonstraram que os mastócitos murinos se mostraram eficientes quanto a sua capacidade fagocítica frente a A. actinomycetemcomitans. Os valores percentuais de mastócitos com A. actinomycetemcomitans internalizados, na ausência de opsonização com complemento, foram maiores que aqueles na presença da opsonização, sugerindo a participação de receptores opsoninas-independentes no reconhecimento deste patógeno pelos mastócitos, além do receptor de complemento tipo 3 (CR3). Comparando os dois tipos celulares, verificou-se que ambas as células apresentaram importante atividade fagocítica contra A. actinomycetemcomitans, porém os valores percentuais de mastócitos com bactérias internalizadas sem complemento foram maiores que aqueles de macrófagos com bactérias internalizadas com complemento, em uma das proporções (1:10). Os resultados deste trabalho sugerem o papel dos mastócitos como fagócitos profissionais na patogênese da doença periodontal induzida por placa dentobacteriana. / Periodontal diseases affect the supporting tissues of the teeth and are triggered by microorganisms which are capable of invading periodontal tissues. The evolution of this disease is influenced by inflammatory and immune response of the host and involves the participation of different cell types. Currently, there is evidence that mast cells, among other functions, have the ability to eliminate bacteria by phagocytosis. Thus, this study aimed to evaluate the phagocytic ability of mast cells against the periodontopathogens A. actinomycetemcomitans, and compare with the phagocytic capacity of macrophages, which are considered professional phagocytes. Therefore, in vitro phagocytic assays were conducted using murine mast cells and macrophages, challenged with A. actinomycetemcomitans or Escherichia coli, at the same time, opsonized or not, under different proportions cell: bacteria. After 1 hour of challenge, cells were stained with acridine orange and qualitatively assessed by using a confocal laser scanning electron microscope and quantitatively by the conventional scanning fluorescence microscope. The results demonstrated that phagocytic ability of murine mast cells was effective against A. actinomycetemcomitans. The percentages of mast cells with A. actinomycetemcomitans internalized in the absence of opsonization with complement, were higher than those in the presence of opsonization, suggesting the involvement of opsonin-independent receptors in recognition of this pathogen by mast cells, as well as complement receptor type 3 (CR3). Comparing the two cell types, it was observed that both cells showed significant phagocytic activity against A. actinomycetemcomitans, however, the percentages of mast cells with internalized bacteria without complement were higher than those of macrophages with internalized bacteria with complement, in one of the proportions (1:10). The results suggest the role of mast cells as professional phagocytes in the pathogenesis of periodontal disease induced by dental plaque.

Mastocitoma é uma das principais neoplasias cutâneas em cães, caracterizada por uma multiplicação anormal de mastócitos, com comportamento biológico muito variável. Os principais fatores prognósticos incluem grau histopatológico, marcadores de proliferação, como índice mitótico, Ki67 e AgNOR, além de estadiamento clínico. Diversos estudos concentram-se na avaliação da relação da apoptose com a oncogênese e seu papel no prognóstico. Em condições fisiológicas, a apoptose ocorre na maturação e senescência celular, mantendo a homeostasia dos diferentes tecidos, removendo do organismo células que tenham sofrido alguma mutação. A genética da apoptose pode ser interrompida frente à ocorrência de mutações, levando à perda do controle na proliferação celular, o que resulta no desenvolvimento de uma neoplasia. O presente estudo avaliou a ocorrência de apoptose por meio de ensaio TUNEL em mastocitomas cutâneos caninos, com o objetivo de testar sua relação com as graduações histopatológicas e o valor prognóstico quanto à sobrevida pós-cirúrgica, assim como compará-lo à expressão imuno-histoquímica de caspase 3 e Ki67. Quarenta e quatro mastocitomas cutâneos caninos, provenientes de 36 cães, foram submetidos à avaliação histopatológica para graduação quanto à diferenciação tumoral, à análise imuno-histoquímica para avaliação das expressões de Ki67 e caspase 3. A marcação positiva para TUNEL não mostrou relação com grau histopatológico, nem foi um bom indicador para sobrevida ou mortalidade em função da doença. Apesar disso, houve correlação positiva entre os índices apoptóticos. / Mast cell tumor is a very common neoplasm in dogs and characterized by an abnormal proliferation of mast cells, with variable biological behavior. The main prognostic factors include histological grade, proliferation markers, such as mitotic index, Ki67 and AgNOR, and clinical staging. Several studies focus in the relation of apoptosis and oncogenesis and its role in prognostication. In physiological conditions, apoptosis occurs due to aging and cell senescence, maintaining the homeostasis of different tissues by removing mutated cells. The genetics of apoptosis can be interrupted by mutations, leading to loss of control in cellular proliferation, and resulting in cancer development. This study evaluated the occurrence of apoptosis by TUNEL assay in canine cutaneous mast cell tumors, compared it with histopathological grading and the immunohistochemical expressions of caspase-3 and Ki67, as well as tested its prognostic value for post-surgical survival. Forty-four canine cutaneous mast cell tumors, from 36 dogs were submitted to histopathologic and immunohistochemical analyses. Positive staining for TUNEL showed no relation with histological grade, and was not considered a good indicator for survival or mortality. Nevertheless, a positive correlation between the apoptotic indexes was found.

by Chung Lap Chan. / "March 1999." / Thesis (Ph.D.)--Chinese University of Hong kong, 1999. / Includes bibliographical references (p. 270-307). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Made available in DSpace on 2014-06-11T19:30:59Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-06-06Bitstream added on 2014-06-13T20:01:17Z : No. of bitstreams: 1 araujo_ac_dr_botib_parcial.pdf: 268121 bytes, checksum: edd3cb8f63dfdeca1bdff45c6a285127 (MD5) Bitstreams deleted on 2015-06-25T13:00:45Z: araujo_ac_dr_botib_parcial.pdf,. Added 1 bitstream(s) on 2015-06-25T13:03:12Z : No. of bitstreams: 1 000716406_20160731.pdf: 207684 bytes, checksum: 3b5f8364b84761be0ff6e1a7cadcc796 (MD5) Bitstreams deleted on 2016-08-01T11:30:02Z: 000716406_20160731.pdf,. Added 1 bitstream(s) on 2016-08-01T11:30:58Z : No. of bitstreams: 1 000716406.pdf: 1009644 bytes, checksum: 9956487a11a013c64929e4fac5497961 (MD5) / As reações alérgicas afetam grande parte da população e tem aumentado nos últimos anos. Nesse sentido, a histamina liberada pelos mastócitos é um mediador importante e a procura por compostos que inibam a liberação do referido mediador se faz necessária tendo em vista que os tratamentos disponíveis apresentam limitações. Estudos prévios demonstraram que o extrato metanólico bruto da casca do caule de Hymenaea stigonocarpa (ME) apresenta atividade inibitória sobre a liberação de histamina. Assim, o presente trabalho realizou o fracionamento biomonitorado do ME a fim de identificar a(s) frações mais ativa(s). As frações foram avaliadas em suspensão de mastócitos peritoneais de ratos Wistar machos desafiados com ionóforo A23187 e composto 48/80 (apenas as frações mais ativas). Posteriormente, a fração mais ativa foi avaliada em mastócitos sensibilizados com ovoalbumina (OVA). A dosagem de histamina foi realizada utilizando-se um sistema fluorimétrico automatizado e a análise fitoquímica por CG/EM. Os resultados mostraram que a fração acetato de etila foi a mais ativa e, na concentração de 100 g/mL inibiu em 78, 98 e 85% a liberação de histamina induzida pelo ionóforo A23187, composto 48/80 e OVA respectivamente. O fracionamento biomonitorado desta fração por cromatografia líquida sob vácuo (CLV) gerou seis sub-frações, das quais as mais ativas demonstraram ser constituídas por terpenos e ácidos graxos de cadeia longa / Allergic reactions affect most of the population and has increased in recent years. Accordingly, histamine released by mast cells is an important mediator and search for compounds that inhibit release of that mediator is necessary in order that the treatments available have limitations. Previous studies demonstrated that the crude methanol extract of the stem bark of Hymenaea stigonocarpa (ME) has an inhibitory activity on the histamine release. Thus, the present study performed the bioguided fractionation of the ME in order to identify (s) most active fractions (s). The fractions were evaluated on the histamine release from rat peritoneum mast cells challenged with ionophore A23187 and compound 48/80 (only the most active fractions). Subsequently, the most active fraction was evaluated in mast cells sensitized with ovalbumin (OVA). The dosage of histamine was performed using an automated fluorimetric system and phytochemical analysis by GC/MS. The results showed that the ethyl acetate fraction was the most active and at concentration of 100 g/mL inhibited by 78, 98 and 85% histamine release induced by ionophore A23187, compound 48/80 and OVA, respectively. The bioguided-fractionatation of this fraction by vacuum liquid chromatography (VLC) generated six sub-fractions, of which the most actives showed the presence of terpenes and long chain fatty acids

FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico / Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Angiogenesis is the development of new blood vessels from pre-existing capillaries, being an essential step in tumor growth for supplying nutrition and oxygen to cells in proliferation. A cell that may be involved in this process is the mast cell (MC), since besides the defense function, acts in the blood vessels regulation. The MC participation in the induction of angiogenesis has been suggested in various malignant tumors. The purposes of this study was to evaluate angiogenesis and mast cell density in oral epithelial dysplasia and squamous cell carcinoma (SCC). This is an observational, retrospective and quantitative study using the sample selection from the archives of the Department of Legal Medicine and Pathology and Laboratory of Oral Pathology, both from the Federal University of CearÃ. For MC evaluation , the sample was consisted of 73 paraffin blocks, distributed between SCC (n=30), epithelial dysplasia (n=23) and hyperplasias fibroepithelial (HFE) (n = 20), as control, and for angiogenesis the sample was 65 blocks, consisted of 24 SCC, 19 epithelial dysplasias and 22 HFE. Immunohistochemistry was performed using the MC-tryptase, CD31 and CD34 antibodies. For quantification, digital images were captured and then counting was performed using Image J software. The antibody staining percentage was determined using SAMM software. With regard to mast cells, there was a lower density in malignant lesions in relation to HFE and dysplasia (p = 0.0092). Evaluating angiogenesis, CD31 expression showed differences between epithelial dysplasia and SCC and between SCC and HFE, with a greater percentage of vessels in SCC (p <0.0001). However, CD34 expression did not differ between groups. The CD31 antibody was shown to be a better angiogenesis marker in oral mucosa than CD34. Increased vascularity in oral squamous cell carcinoma suggests that angiogenesis is necessary for tumor growth, increasing when the malignant transformation starts. However, no correlation was found between mast cells and angiogenesis. / AngiogÃnese à o surgimento de um novo vaso sanguÃneo a partir de capilares prÃ-existentes, sendo um passo essencial no crescimento tumoral por fornecer nutriÃÃo e oxigÃnio Ãs cÃlulas em proliferaÃÃo. Uma cÃlula que pode estar envolvida nesse processo à o mastÃcito, pois, alÃm da funÃÃo de defesa, atua na regulaÃÃo de vasos sanguÃneos. Sua participaÃÃo na induÃÃo da angiogÃnese tem sido sugerida em vÃrios tumores malignos. Os objetivos deste trabalho foram avaliar a angiogÃnese e a densidade de mastÃcitos em displasias epiteliais e no carcinoma espinocelular (CEC) de boca. Trata-se de um estudo observacional, retrospectivo e quantitativo, realizado atravÃs da seleÃÃo de amostra proveniente dos arquivos do Departamento de Patologia e Medicina Legal e do laboratÃrio de Patologia Bucal do curso de Odontologia, ambos da Universidade Federal do CearÃ. Para a avaliaÃÃo dos mastÃcitos, a amostra foi constituÃda por 73 blocos parafinados, distribuÃdos entre CEC (n=30), displasias epiteliais (n=23) e hiperplasias fibroepiteliais (HFE) (n=20), como controle, e para a angiogÃnese a amostra foi de 65 blocos, sendo 24 de CEC, 19 de displasias epiteliais e 22 de HFE. Foi realizada imunohistoquÃmica utilizando-se os anticorpos anti-triptase, para mastÃcitos e anti-CD31 e anti-CD34, para vasos sanguÃneos. Para quantificaÃÃo, foram capturadas imagens digitais e, em seguida, utilizados softwares para auxiliar na contagem dos mastÃcitos (Image J) e para determinaÃÃo do percentual de marcaÃÃo do anticorpo (SAMM). Com relaÃÃo aos mastÃcitos, houve menor densidade destes nas lesÃes malignas em relaÃÃo Ãs HFE e displasias (p=0,0092). Avaliando angiogÃnese, a expressÃo de CD31 mostrou diferenÃa entre os grupos CEC e displasia epitelial e entre CEC e HFE, havendo um maior percentual de vasos nos CEC (p<0,0001). Contudo, o CD34, nÃo mostrou diferenÃa entre os grupos. O anticorpo CD31 mostrou-se melhor marcador de angiogÃnese em mucosa oral do que CD34. O aumento da vascularizaÃÃo em CEC oral sugere que a angiogÃnese à necessÃria ao crescimento tumoral, aumentando à medida que inicia o processo de malignizaÃÃo. NÃo foi encontrada correlaÃÃo entre mastÃcitos e angiogÃnese.

As doenças periodontais afetam os tecidos de suporte dos dentes e são desencadeadas por microrganismos que possuem a capacidade de invadir os tecidos periodontais. A evolução desta doença é influenciada pela resposta inflamatória e imunológica do hospedeiro e envolve a participação de diversos tipos celulares. Atualmente, existem evidências de que os mastócitos, além de outras funções, possuem a capacidade de eliminar bactérias, através da fagocitose. Assim sendo, este estudo teve por objetivo avaliar a capacidade fagocítica dos mastócitos frente ao periodontopatógeno A. actinomycetemcomitans, além de comparar sua capacidade fagocítica com a dos macrófagos, considerados fagócitos profissionais. Para este fim, foram realizados ensaios fagocíticos in vitro utilizando mastócitos e macrófagos murinos, desafiados ora com A. actinomycetemcomitans ora com Escherichia coli, opsonizados ou não, sob diferentes proporções célula: bactérias. Após 1 hora de desafio, as células foram coradas com laranja de acridina e avaliadas qualitativamente utilizando-se microscópio de varredura confocal a laser e quantitativamente através do microscópio de fluorescência convencional. Nossos resultados demonstraram que os mastócitos murinos se mostraram eficientes quanto a sua capacidade fagocítica frente a A. actinomycetemcomitans. Os valores percentuais de mastócitos com A. actinomycetemcomitans internalizados, na ausência de opsonização com complemento, foram maiores que aqueles na presença da opsonização, sugerindo a participação de receptores opsoninas-independentes no reconhecimento deste patógeno pelos mastócitos, além do receptor de complemento tipo 3 (CR3). Comparando os dois tipos celulares, verificou-se que ambas as células apresentaram importante atividade fagocítica contra A. actinomycetemcomitans, porém os valores percentuais de mastócitos com bactérias internalizadas sem complemento foram maiores que aqueles de macrófagos com bactérias internalizadas com complemento, em uma das proporções (1:10). Os resultados deste trabalho sugerem o papel dos mastócitos como fagócitos profissionais na patogênese da doença periodontal induzida por placa dentobacteriana. / Periodontal diseases affect the supporting tissues of the teeth and are triggered by microorganisms which are capable of invading periodontal tissues. The evolution of this disease is influenced by inflammatory and immune response of the host and involves the participation of different cell types. Currently, there is evidence that mast cells, among other functions, have the ability to eliminate bacteria by phagocytosis. Thus, this study aimed to evaluate the phagocytic ability of mast cells against the periodontopathogens A. actinomycetemcomitans, and compare with the phagocytic capacity of macrophages, which are considered professional phagocytes. Therefore, in vitro phagocytic assays were conducted using murine mast cells and macrophages, challenged with A. actinomycetemcomitans or Escherichia coli, at the same time, opsonized or not, under different proportions cell: bacteria. After 1 hour of challenge, cells were stained with acridine orange and qualitatively assessed by using a confocal laser scanning electron microscope and quantitatively by the conventional scanning fluorescence microscope. The results demonstrated that phagocytic ability of murine mast cells was effective against A. actinomycetemcomitans. The percentages of mast cells with A. actinomycetemcomitans internalized in the absence of opsonization with complement, were higher than those in the presence of opsonization, suggesting the involvement of opsonin-independent receptors in recognition of this pathogen by mast cells, as well as complement receptor type 3 (CR3). Comparing the two cell types, it was observed that both cells showed significant phagocytic activity against A. actinomycetemcomitans, however, the percentages of mast cells with internalized bacteria without complement were higher than those of macrophages with internalized bacteria with complement, in one of the proportions (1:10). The results suggest the role of mast cells as professional phagocytes in the pathogenesis of periodontal disease induced by dental plaque.

Orientador: Ana Lia Anbinder / Coorientadora: Noala Vicensoto Moreira Milhan / Banca: Fábio Luiz Coracin / Banca: Monica Ghislaine Oliveira Alves / Resumo: Granuloma piogênico (GP) é uma lesão de origem inflamatória que ocorre frequentemente em pele e cavidade oral. Existem dois subtipos histológicos: o tipo não lobular (GPNL), que é caracterizado por proliferação vascular semelhante a tecido de granulação, sem padrão de organização; e o tipo lobular (GPL) que se caracteriza pela organização dos vasos em agregados lobulares, separados por feixes de tecido conjuntivo. O objetivo deste estudo foi revisar todos os casos de granulomas piogênicos do nosso serviço de patologia bucal, a partir do ano 2000, reclassificar e correlacionar as características clínicas, microscópicas e imuno-histoquímicas com os subtipos da lesão. No levantamento foram encontrados no arquivo 197 casos diagnosticados como granuloma piogênico e hemangioma lobular capilar. Após revisão das lâminas, 9 casos foram reclassificados, e 19 foram excluídos, restando 169 casos, sendo 62 de GPL e 107 de GPNL. Foram coletados ainda dados como sexo, idade, local da lesão, tipo de nódulo, ocorrência de trauma prévio e hipótese diagnóstica clínica. Reações imuno-histoquímicas (GLUT-1, CD34, D2-40, AML e Mast cell) de 22 casos, sendo 11 lobulados e 11 não lobulados. A média de idade de acometimento foi de 38,59±16,96 anos, com 55,62% dos casos ocorrendo em pacientes do sexo feminino (10,12% durante gravidez), com maior acometimento em gengiva (39,64%), 44,97% dos nódulos eram do tipo pediculado e 13,02% relataram trauma mecânico prévio. O GPNL ocorre mais em gengiva, enquant... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Pyogenic granuloma (GP) is an inflammatory lesion that occurs frequently in the skin and oral cavity. There are two histological subtypes: the non-lobular type (NLCH), which is characterized by vascular proliferation similar to granulation tissue, without organization pattern; and lobular capillary hemangioma (LCH) characterized by the organization of vessels in lobular aggregates, separated by bundles of connective tissue. The purpose of this study was to review all cases of pyogenic granulomas of our oral pathology service, from the year 2000, to reclassify and correlate the clinical, microscopic and immunohistochemical characteristics with the subtypes of the lesion. In the survey, 197 cases diagnosed as pyogenic granuloma and lobular capillary hemangioma were found in our files. After review, 9 cases were reclassified, and 19 were excluded, remaining 169 cases, being 62 LCH and 107 NLCH. Data such as sex, age, site of lesion, type of nodule, previous trauma and clinical diagnostic hypothesis were also collected. Immunohistochemical reactions (GLUT-1, CD34, D2-40, SMA and Mast cell) of 24 cases, 11 LCH and 11 NLCH. Mean age was 38.59±16.96 years, with 55.62% of cases occurring in female patients (10.12% during pregnancy), with a greater involvement in gingiva (39.64%), 44.97% of the nodules were pedunculated and 13.02% reported previous mechanical trauma. The NLCH occurs more in gingiva, while LCH affects more lips (p<0,05). The number of microvessels (CD34 positive), SMA ... (Complete abstract click electronic access below) / Mestre

Mast cells are known to play an important role in allergic events and in other inflammatory reactions through varied intracellular signaling transduction proteins. RasGRP4 is a mast cell-restricted guanine nucleotide exchange factor (GEF) and diacylglycerol (DAG)/phorbol ester receptor. Interleukin (IL) -13, a critical cytokine for allergic inflammation, exerts its effects through a complex receptor system including IL-4Rα, IL-13Rα1 and IL-13Rα2. IL-13Rα2 has been reported to be a decoy receptor for IL-13. My experiments indicate that the mast cell specific RasGRP4 protein regulates the level of IL 13Rα2 and controls IL-13/ IL 13Rα1-mediated intracellar signaling events in mast cells. Phosphorylation of STAT6 plays an important role in airway hyperresponsiveness and asthma. The development of therapeutics that can regulate RasGRP4 could be used to modulate the IL-13-induced phosphorylation of STAT-6 that may be used as therapy in patients with asthma. SLE is a complex, heterogeneous systemic autoimmune disease characterized by the presence of high levels of autoantibodies. Dysregulation of RasGRP1, a Ras active gene, in mice resulted in a SLE-like disorder. Yasuda and coworkers demonstrated that a defective isoform of RasGRP1 (Δ11) was present in a subset of patients with SLE. My experiments indicate that RasGRP1 upregulates the expression of IL2RG in T cells. In contrast the Δ11 RasGRP1 isoform expressed in a subset of SLE patients leads to defective expression of IL2RG. The IL2RG chain is a common chain which forms part of a number of different receptors eg. IL-2, 4, 7, 9, 15, 21. IL-2 as well as IL-21, which shares sequence homology with IL-2, has been reported to be involved in the generation of regulatory T cells (Tregs). In SLE patients, CD4 + CD25+ Tregs, which play an essential role in controlling immunologic tolerance to self-antigens and preventing autoimmunity, are significantly decreased when compared with healthy controls. The accumulative evidence suggests that the defective isoform of RasGRP1 (Δ11) downregulates expression of IL2RG in SLE patients?? T cells and this could effect the generation of CD4 + CD25+ Tregs. This may be another immunological mechanism in loss of tolerance observed in patient with SLE.

Mast cells (MCs) play a central role in inflammation by releasing mediators following activation. S100A8 and S100A9 are abundantly expressed in inflammatory sites such as asthmatic lung, sunburnt skin and atherosclerosis where MCs are involved in pathogenesis; roles of S100A8 in MC function are undetermined. The aims of this thesis were to determine effects of S100A8 on MC activation, particularly provoked by IgE and UVB. Initially, effects of UVB on MC activation were investigated as detailed functions were unclear. Cord blood-derived human mast cells (CBMCs) were treated in vitro with varying doses of UVB and production of multiple cytokines and viability investigated. UVB exposure selectively increased levels of IL-8 (CXCL8), and to a less extent IL-1β, but not eight other cytokines tested. New protein synthesis partially contributed and IL-8 production was p38 MAPK-dependent. UVB dose-dependently induced MC apoptosis indicating a potential regulatory mechanism of MC function. The ability of recombinant S100A8, S100A9 or S100A8/9 heterodimer to modulate IgE/antigen (DNP/anti-DNP)-mediated activation of a murine MC line, and of bone marrow-derived (mBM) MC activation was determined. The S100s did not directly induce degranulation or induce IL-6. S100A8 significantly inhibited DNP/anti-DNP-provoked degranulation, and IL-6 and TNF mRNA and protein induction. S100A8 did not alter FcεRIα expression. S100A9 was less effective; and the S100A8/9 complex was also suppressive. S100A8 only weakly suppressed non-specific MC degranulation. Mutation of Cys41 in S100A8 negated its suppressive activity. Because S100A8 scavenges oxidants via this reactive Cys residue, we propose that this may mediate its ability to downmodulate IgE-dependent MC responses. Similar to the thiol scavenger N-acetyl-L-cysteine, S100A8 but not the Ala41 mutant, attenuated DNP/anti-DNP-provoked LAT phosphorylation. However, the disulfide-bonded S100A8 dimer and S100A8 containing a sulfinamide bond between Cys41 and Lys34/35 also reduced MC activation, indicating an additional pathway(s). S100A8 did not suppress antigen/IgE-induced responses of CBMC possibly because these may not truly reflect fullymature human tissue MCs. S100A8 did not alter UVB-induced IL-8 release by CBMCs, or affect apoptosis. Murine S100A8 may have anti-inflammatory properties by regulating MC activation in an activator-specific manner, at least partially by scavenging ROS to suppress intracellular signalling.