Made available in DSpace on 2014-06-11T19:33:27Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-03-05Bitstream added on 2014-06-13T21:06:09Z : No. of bitstreams: 1 calderon_c_dr_botfmvz.pdf: 668199 bytes, checksum: 39ff86991440908435624d410c3ad500 (MD5) / Universidade Estadual Paulista (UNESP) / 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. / 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.

Orientador: Renée Laufer Amorim / Coorientador: Carlos Roberto Daleck / Banca: Mirela Tinucci Costa / Banca: Sabryna Gouveia Calazans / Banca: Luiz Henrique de Araújo Machado / Banca: Renata Alonso Sobral / Resumo: O mastocitoma (MCT) cutâneo é uma das neoplasias malignas mais comuns em cães, representa 11% dos tumores de pele nesta espécie e apresenta comportamento biológico variável. Este trabalho dá continuidade a pesquisas com biomarcadores prognósticos em MCTs cutâneos em cães. Foi realizado um estudo em busca de critérios complementares ao exame clínico (ultrassonografia pelo método Doppler), que auxiliem na determinação do potencial de recidiva e metástase do tumor, correlacionando-os com a expressão da proteína KIT, densidade microvascular (DMI), tamanho, número de tumores, tempo de evolução, ulceração, tempo de sobrevida e graduação histopatológica. Além disso, foi realizada uma análise dos parâmetros clínicos, incluindo dados epidemiológicos, achados histológicos e moleculares e a correlação destes com o comportamento biológico do MCT. Foram avaliados 20 cães, que totalizaram 28 tumores. A ultrassonografia (US) pelo método Doppler permitiu a identificação de vasos em 54% dos tumores. Não houve correlação entre a presença de vasos e a DMI, a localização da proteína KIT, os graus histológicos, o tempo de evolução, o tamanho, a ocorrência de recidivas e metástases, e o tempo de sobrevida, mas sim com a presença de ulceração tumoral. Observou-se correlação estatística entre o grau histológico, a DMI, a presença de ulceração e o número de tumores e também da expressão de Ki-67 com os padrões de marcação da proteína KIT. O grau histológico no MCT cutâneo canino não deve ser avaliado isoladamente, mas sim em conjunto com a expressão da proteína KIT, DMI, proliferação celular, presença de ulceração, número de tumores e ocorrência de recidivas e metástases. Indicamos que estudos adicionais... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Cutaneous mast cell tumor (MCT) is one of the most common malignant neoplasms in dogs, representing 11% of skin tumors in this species and shows a variable biological behavior. This article maintains the search on prognostic biomarkers in cutaneous MCTs in dogs. A study was conducted to find additional criteria to the clinical examination (Doppler ultrasound) to add in determining the potential for recurrence and metastasis of tumors, correlating them with KIT protein expression, intratumoral microvessel density (IMD), size, number of tumors, duration time, ulceration, survival rates and histological classification. In addition, we performed an analysis of clinical parameters, including epidemiological data, histological and molecular ones and correlate them with the biological behavior of MCT. We evaluated 20 dogs, in a total of 28 tumors. The Doppler ultrasound (US) allowed the identification of vessels in 54% of tumors. There was no correlation between the presence of vessels and IMD, KIT protein location, histological grades, duration time, tumor size, recurrence and metastasis, and survival rates, but with ulceration. We have observed statistical correlation between histological grade, IDM, presence of ulceration and number of tumors and also the expression of Ki-67 with patterns of KIT protein. The histological grade in canine cutaneous MCT should not be assessed in isolation but in conjunction with expression of KIT protein, IDM, cell proliferation, presence of ulceration, number of tumors and recurrences and metastases rates. We suggest that additional studies should be done to further evaluate the use of Doppler US as a noninvasive method to chacarterize the vascularization and blood flow in... (Complete abstract click electronic access below) / Doutor

Mast cells (MCs) are long-lived immune cells, which were reported to play an important role in initiating innate and adaptive immune responses against various infections. MCs accumulate in high numbers in the stroma and at the invasion front of various human cancers, suggesting a possible contribution by MCs to tumour growth. Experimental studies using crosses of MC-deficient Kit-mutant mouse strains with mouse models of epithelial cancers have provided evidence for important MC tumour-promoting functions. However, the complex alterations of the immune system that characterize Kit-mutant mice in addition to their MC deficiency, limit the interpretation of these findings. Numerous key observations made in Kit mutant mice were not reproduced in novel, Kit-independent mouse models of MC deficiency. Thus, the impact of MCs on tumour biology remains unclear. The aim of this study is to clarify the contribution of MCs to the biology of Human papilloma virus (HPV)-induced skin cancer in a Kit-independent mouse model of MC deficiency. In K14-HPV16 transgenic mice, HPV oncogenes are constitutively expressed in the epidermis resulting in epidermal hyperplasia with 100% penetrance and squamous cell carcinoma in about 50% of the animals. A cross to a Kit-mutant line suggested that MCs are important tumour promoters in this model. We crossed K14-HPV16 mice to M5Cre R-DTA line, in which MCs are constitutively depleted with high efficiency and selectivity. Unexpectedly, the loss of MCs neither affected keratinocyte proliferation indices nor altered keratinocyte apoptosis at any stage of HPV-induced neoplasia. Furthermore, the loss of MCs did not result in any detectable changes in composition and gene expression of the inflammatory hematopoietic cell infiltrate in the tumour stroma. This shows that, contrary to current belief, MCs have no important function in orchestrating the tumour micro milieu. In keeping with this finding, MC deficiency resulted in no detectable difference in the incidence growth or grading of SSC in K14-HPV16 transgenic mice. Collectively, these results show that, despite their high density in HPV-induced neoplasia, MC have no role in cancerogenesis or neoplastic progression in the K14-HPV16 mouse model. Our findings also emphasize the importance of novel Kitindependent mouse models in the investigation of MC in vivo functions.

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.

Mouse mast cell chymases are granule-associated serine proteinases with chymotrypsin-like substrate specificities. cDNAs for two new chymases were isolated from a cDNA library constructec using mRNA from ABFTL-6 mouse mast cells by screening with a rat mast cell proteinase cDNA. The deduced amino acid sequence of mouse cymase 1 consists fo a 226 amino acid catalytic portion and a 21 amino acid preprosequence. Chymase 1 is unusual in that an Asn occurs in the substrate binding pocket, a feature that has not been observed in any other serine proteinase. Also, chymase 1 is expected to have a large positive charge (+13) at physiological pH. A partial cDNA for chymase 2 encodes 177 residues of the carboxy terminal portion of a second proteinase distinct from chymase 1. Chymase 2 cDNA contains highly conserved intron/exon junction, a high positive charge (+17) and a novel, second potential N-glycosylation site. Transcripts for both chymases are found in ABFTL-6 mast cells, but only chymase 2 mRNA is in mouse connective tissue mast cells. These data suggest that these chymases have distinct enzymatic properties and tissue-specific patterns of gene expression.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20221号 / 医博第4180号 / 新制||医||1019(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 椛島 健治, 教授 中川 一路, 教授 生田 宏一 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

<p>Mast cells are key effector cells in allergic and inflammatory diseases. However, their primary role is most likely in host defence against parasitic and bacterial infections. Mast cells are a particularly rich source of serine proteases. These proteases belong to the chymase or the tryptase family, which are encoded from the mast cell chymase and the multigene tryptase loci, respectively. To better understand the biological functions and the molecular evolution of these enzymes we have studied the organisation of these two loci in species ranging from fish to human. We show that the mast cell chymase locus has evolved from a single founder gene to a complex locus during the past 200 Myr of mammalian evolution. Forty-five fish candidate genes for hematopoietic serine proteases were also identified. However, in phylogenetic analyses none of them grouped with individual branches holding mammalian mast cell chymase locus genes, indicating an independent parallel evolution in fish. </p><p>Studies of the evolution of the multigene tryptase locus showed that this locus has been highly conserved between marsupials and eutherians. However, no genes belonging to the individual subfamilies identified in eutherians could be identified in fish, amphibians or in birds, which also here indicates parallel evolution.</p><p>To study the evolution of specific cleavage specificities associated with these proteases, the extended cleavage specificity of opossum α-chymase was determined and found to be nearly identical to human mast cell chymase and the major mouse mast cell chymase mMCP-4. This indicates a strong pressure to maintain this specificity during mammalian evolution.</p><p>Basophils are rare blood cells with functions similar to mast cells that when mature almost completely lack mRNA. To study the proteome and to primarily characterize the granule protein content of basophils, an <i>in vitro</i> purification protocol was developed to obtain transcriptionally active umbilical cord blood-derived basophil precursors.</p>

Mast cells are key effector cells in allergic and inflammatory diseases. However, their primary role is most likely in host defence against parasitic and bacterial infections. Mast cells are a particularly rich source of serine proteases. These proteases belong to the chymase or the tryptase family, which are encoded from the mast cell chymase and the multigene tryptase loci, respectively. To better understand the biological functions and the molecular evolution of these enzymes we have studied the organisation of these two loci in species ranging from fish to human. We show that the mast cell chymase locus has evolved from a single founder gene to a complex locus during the past 200 Myr of mammalian evolution. Forty-five fish candidate genes for hematopoietic serine proteases were also identified. However, in phylogenetic analyses none of them grouped with individual branches holding mammalian mast cell chymase locus genes, indicating an independent parallel evolution in fish. Studies of the evolution of the multigene tryptase locus showed that this locus has been highly conserved between marsupials and eutherians. However, no genes belonging to the individual subfamilies identified in eutherians could be identified in fish, amphibians or in birds, which also here indicates parallel evolution. To study the evolution of specific cleavage specificities associated with these proteases, the extended cleavage specificity of opossum α-chymase was determined and found to be nearly identical to human mast cell chymase and the major mouse mast cell chymase mMCP-4. This indicates a strong pressure to maintain this specificity during mammalian evolution. Basophils are rare blood cells with functions similar to mast cells that when mature almost completely lack mRNA. To study the proteome and to primarily characterize the granule protein content of basophils, an in vitro purification protocol was developed to obtain transcriptionally active umbilical cord blood-derived basophil precursors.

<p>Mast cells (MCs) are increasingly recognized as powerful sentinel cells responsible for modulating the early immune responses to a wide range of infectious agents. This protective role is attributable in part to their preponderance at the host-environment interface and their innate capacity to rapidly release modulators of immune cell trafficking which promotes the early recruitment of pathogen-clearing immune cells from the blood. However, host-adapted pathogens had been a critical threat to human for a long time because they have evolved mechanisms directed at overcoming protective immunity. </p><p>In this work, we outline <italic>Salmonella enterica</italic> serovar Typhimurium has evolved a novel mechanism to inactivate peripheral MCs resulting in limited neutrophil responses at infection sites in early stage of infection. Because of the delay in bacterial clearance at the point of entry, <italic>Salmonella</italic> are able to multiply and rapidly disseminate to distal sites. Suppression of local MCs' degranulation restricted outflow of vascular contents into infection sites, thus facilitating bacterial spread. </p><p>We discover MC suppression is mediated by the Salmonella Protein Tyrosine Phosphatase (SptP), which shares structural homology with <italic>Yersinia</italic> YopH. Interestingly, SptP, not only shares homology with phosphatases found in MCs, they are also homologous to YopH an effector protein expressed by plague causing <italic>Yersinia pestis</italic>. We show that YopH had MC suppressing abilities as SptP suggesting that this activity is shared among some of the more virulent bacterial pathogens. The functionally relevant domain in SptP is its enzymatic site and that it works by dephosphorylating the vesicle fusion protein N-ethylmalemide-sensitive factor (NSF) and by blocking phosphorylation of Syk, which is located in downstream and upstream of tyrosine phosphorylation signaling pathway in MCs. </p><p>Without SptP, orally challenged <italic>S.</italic> Typhimurium failed to suppress MC degranulation and exhibited limited colonization of the mesenteric lymph nodes. Administration of SptP to sites of Escherichia coli infection markedly enhanced its virulence. Thus, SptP-mediated inactivation of local MCs is a powerful mechanism utilized by <italic>S.</italic> Typhimurium to impede early innate immunity. This finding provides a logical explanation for why previous attempts by others to demonstrate a protective role for MCs against <italic>Salmonella</italic> infections have resulted in equivocal results. </p><p>Taken together, this work highlights an overlooked virulence mechanism possessed by certain host adapted pathogens to avoid the host's innate immune system. Additionally, this innate immune-quelling property of SptP may hold future promise in tempering harmful inflammatory disorders in the body of an immune competent host.</p> / Dissertation