Background: Mast cells are known to play a role in the pathophysiology of asthma. Determining their contribution to the development of asthma symptoms has been difficult as they remain tissue-resident and do not usually migrate into the airway lumen for detection using expectorated sputum. Objectives: We investigated the presence and activity of mast cells in the blood and sputum of healthy controls and patients with moderate to severe asthma and the relationship with clinical characteristics of asthma and their associated microenvironment. Methods: Cell-free sputum supernatant was used to detect levels of soluble tryptase and T2 and non-T2 cytokines by ELISA. RNA/cDNA isolated from sputum cells measured expression levels of eosinophil and mast cell-specific genes by digital PCR. Relevant clinical characteristics and measurements of lung function, airway hyperresponsiveness, FeNO, blood eosinophils, IgE and tryptase were collected. Results: Tryptase was detectable in the fluid phase portions of sputum, irrespective of the inflammation based on the differential cell count, and was significantly different than healthy controls. Eosinophil and mast cell-specific genes were detected in sputum cells at levels significantly different than healthy controls. Sputum tryptase was not associated with any phenotype or severity of asthma but identified some associations with clinical characteristics. It is associated with a unique cytokine signature. Conclusion: Differences were seen between eosinophilic and non-eosinophilic phenotypes in sputum supernatant and sputum cells. Eosinophil and mast cell-specific genes were detected in sputum cells but were not associated with asthma severity. Greater levels of cytokines IL-4 and IL-13 in sputum, suggest presence of mast cells in the airway epithelium that contribute to mucus secretion observed in patients with uncontrolled symptoms of asthma. Further investigations hope to identify the relationship of mast cells with the quantification of mucus in these patients to understand and confirm those with a predominant mast cell component. / Thesis / Master of Science (MSc) / In patients with asthma, mast cells are one of the inflammatory cells normally associated with allergies however, they also function by non-allergic mechanisms (bacteria, viruses, fungus, pollutants). Inflammatory cells cause swelling of the bronchial tubes, tightening/spasm of the bronchial muscles, and mucus production. The level of inflammation is measured in lung fluids such as sputum, but the mast cell remains in the lung tissue and therefore difficult to detect. When mast cells are activated they release mediators such as tryptase, that travel through the lung tissue. Tryptase was detected in the sputum of patients with moderate to severe asthma, with levels significantly greater than in healthy people. The level of sputum tryptase was independent of other inflammatory cells such as eosinophils, and was associated with biomarkers related to excess secretion of mucus. Further research hopes to understand if the mucus is related to the activity of the mast cell.

Cyathostomins are potentially life threatening parasitic nematodes of adult horses and are highly prevalent worldwide. Infected animals may be asymptomatic or show clinical signs of weight loss, diarrhoea and colic. Third and fourth stage larvae spend a large proportion of their lifecycle encysted in the large intestinal wall where they cannot currently be detected ante mortem. Mast cells are commonly found at interfaces to the external environment, such as the rectum, and these cells and the proteinases they produce have been implicated in protective host immune responses against nematode infection in animals. Previous studies have demonstrated an increase in caecal mast cell proteinase expression during cyathostomin infection. Prior to this study, there were two known equine mast cell proteinases, which had been purified and characterised from a mastocytoma (equine tryptase [eqTRYP] and equine mast cell proteinase-1 [eqMCP-1]). However, as many mammalian species express multiple closely-related chymases it was hypothesised that other equine mast cell proteinases exist that have not yet been characterised and which may be more closely associated with the level of worm burden. The primary objective of this study was to investigate the recruitment of mast cells to the large intestine in cyathostomin infected horses and the expression of mast cell proteinases in response to infection. A further aim was to evaluate the potential of associated mast cell proteinase assays or rectal biopsy mast cell enumeration for utility in diagnostic tests to estimate cyathostomin mucosal burden. A secondary objective was to explore the existence of further mast cell proteinases and the relationship of these enzymes to cyathostomin mucosal burden. Optimised sampling protocols, parasitological, histological and immunohistochemistry techniques were performed to enumerate cyathostomin mucosal burden and to characterise the mast cell populations in the caecum, right ventral colon (RVC) and rectum of naturally infected horses (n=28). Mast cell populations correlated throughout the intestine, providing further evidence of the common mucosal system. EqMCP-1 and eqTRYP labelled mast cells were identified throughout the large intestine. Significant positive linear relationship existed between rectal proteinase-labelled mucosal mast cell populations and both the combined total cyathostomin mucosal burden (CTMB; eqMCP-1, p=0.018; eqTRYP, p=0.048) and the combined total luminal burden (CTLB; eqMCP-1, p=0.009; eqTRYP, p=0.007). Concentrations of eqMCP-1 and eqTRYP in (i) serum, (ii) local serum from venous blood draining the large intestine, and (iii) large intestinal tissue homogenates were assessed using ELISA. There was no significant correlation identified between local and peripheral serum proteinase concentrations suggesting that peripheral serum proteinase levels are not representative of the local proteinase response. There was however a significant negative relationship between peripheral serum eqMCP-1 concentrations and the CTMB, which could relate to the activation and sequestering of proteinases within the gut lumen. Concentrations of eqMCP-1 and eqTRYP measured in local serum did not significantly positively correlate with cyathostomin mucosal burden. There was a significant association observed between intestinal tissue levels of eqMCP-1 and eqTRYP and the CTMB in the RVC (p < 0.023), providing support for their role in the immune response. Four proteinase sequences, equine tryptase (TLP1), Granzyme B-like (GZMBL), putative equine Mast Cell Proteinase-1 (CLP1) and Granzyme(BGH)-like (GZM(BGH)L), were sequenced and the local transcription levels of each of these enzymes assessed using quantitative reverse-transcription PCR. The expression of TLP1 was closely correlated with GZMBL expression, and there was a significant positive relationship observed between TLP1 and GZMBL transcript levels and combined total mucosal burden in the RVC. Both GZM(BGH)L and CLP1 transcript levels were also positively correlated with each other, but the levels of these transcripts were not statistically correlated to any of the cyathostomin parasitological measures assessed here. This work has provided the basis for further rectal biopsy studies to examine the important dynamics of the mast cell response to cyathostomin infection. The results from this thesis, with the demonstration of novel proteinases, are encouraging for further investigation into equine mast cell proteinases and their role in cyathostomin infections.

N/A, thesis is written in chapters. / Thesis / Master of Science (MSc)

No description available.

The mechanisms that mediate acute exacerbations in chronic inflammatory diseases such as periodontitis are not understood. IL-8 is a potent chemoattractant for neutrophils in acute inflammatory lesions. We investigated the role of fibroblast-mast cell interactions on short-term IL-8 release. Human gingival fibroblasts were co-cultured with human mast cells (HMC-1). After co-culture, the concentration of IL-8 was measured by ELISA. HMC co-cultured with fibroblasts increased IL-8 secretion by >6-fold, which required intercellular contact and was blocked by the gap junction inhibitor BGA. Thapsigargin-induced elevations of intracellular calcium increased IL-8 levels by 15-fold. Chemotaxis of human neutrophils was significantly enhanced in response to conditioned medium from co-cultures. Calcein-dye transfer showed intercellular, gap junction communication between HMC and fibroblasts that was dependent in part on β1 integrins. We conclude that mast cells adhere to fibroblasts and promote IL-8 secretion, thereby enhancing neutrophil chemotaxis and possibly the perpetuation of the inflammatory response.

The mechanisms that mediate acute exacerbations in chronic inflammatory diseases such as periodontitis are not understood. IL-8 is a potent chemoattractant for neutrophils in acute inflammatory lesions. We investigated the role of fibroblast-mast cell interactions on short-term IL-8 release. Human gingival fibroblasts were co-cultured with human mast cells (HMC-1). After co-culture, the concentration of IL-8 was measured by ELISA. HMC co-cultured with fibroblasts increased IL-8 secretion by >6-fold, which required intercellular contact and was blocked by the gap junction inhibitor BGA. Thapsigargin-induced elevations of intracellular calcium increased IL-8 levels by 15-fold. Chemotaxis of human neutrophils was significantly enhanced in response to conditioned medium from co-cultures. Calcein-dye transfer showed intercellular, gap junction communication between HMC and fibroblasts that was dependent in part on β1 integrins. We conclude that mast cells adhere to fibroblasts and promote IL-8 secretion, thereby enhancing neutrophil chemotaxis and possibly the perpetuation of the inflammatory response.

Objective. The aim of this study was to determine the effects of interleukin-10 on mast cell development from bone marrow progenitors.Materials and Methods. Unseparated mouse bone marrow cells were cultured in IL-3+SCF, giving rise to mast cells and monocytes/macrophages. The addition of IL-10, and the use of Signal Transducer and Activator of Transcription (STAT)3-deficient bone marrow cells were employed to measure the effects of IL-10 and STAT3 expression on cell viability, proliferation, and differentiation. Bax-deficient and Bcl-2 transgenic bone marrow cells were used to determine the importance of the mitochondria in IL-10-mediated effects.Overview. Mast cells arise from hematopoietic stem cells and continue development in either connective tissue or mucosa. Th2 cytokines have been implicated in the regulation of mast cell development and subsequent function. Mast cells have also been shown to be essential players in many Th2 immune responses. In the following study we investigate the effects of the Th2 cytokine IL-10 on mast cell development from isolated bone marrow progenitors. The addition of IL-10 to whole murine bone marrow greatly reduced cell numbers and altered the phenotype of the developing progenitor cells. The reduction in cell numbers was due to apoptosis, as judged by DNA fragmentation and caspase activation. The apoptosis observed included alteration in mitochondrial membrane potential. Furthermore, apoptosis could be reduced by the overexpression of Bcl-2 or by ablating p53 expression. Utilizing a flox/cre system we found that IL-10 mediated apoptosis required expression of Stat-3, since Stat-3 deficient bone marrow cells did not undergo apoptosis in response to IL-10. In this study we also observed significant alterations in the mast cell growth factor receptors IL-3R and c-kit. The loss of these growth factor receptors may explain the apoptosis induced by IL-10. These data demonstrate the potent regulatory capabilities of Th2 cytokines on mast cells, a central effector in the Th2 response.

Inflammatory response in the airway may lead to asthma. Asthma may develop during the childhood in some asthmatic patients. Both environmental and genetic factors may influence the onset and progress of asthma. It is well-known that there may be complex neural innervation and reflex mechanisms between trachea and esophagus. Intraesophgeal infusion of 1N HCl could lead to tracheal inflammation by activating neural reflex pathway and cause tachykinin-like substance to release. In this study, we first sensitized rats with 1ml of OVA-Al［OH］3 mixture containing 200£gg OVA via intraperitoneal injection on days 1, 2, 3 and 11, then perform intraesophageal infusion of ovalbumin to see whether stimulation of esophagus in sensitized rat model could involve inflammatory response in the lower airways. Animals were perfused with saline and fixative at various time points and the esophagus and airway tissues were processed for the subsequent analysis. We observed the extent of plasma leakage and migration of leukocytes in the lower airway. India ink was used to label the leaky blood vessels.The magnitude of plasma leakage was expressed by the area density of India ink-labeled blood vessels. The results showed that the intraesophageal infusion of ovalbumin 75 mg/kg caused an increase in plasma leakage in the lower airways. The plasma leakage peaked at 30 min, the area density of plasma leakage in trachea was 22.43 ¡Ó 3.34¢H; and 20.57 ¡Ó 4.91¢H in right bronchus; 18.47 ¡Ó 5.03¢H in left bronchus and 27.85 ¡Ó 2.71¢H in epiglottis. The extent of leakage gradually diminished 3 hours after ovalbumin infusion. However, a second increased plasma leakage peaked at about 4 hours of ovalbumin infusion. Tissue sections clearly showed degranulation of mast cells in OVA infusion group. Experimental data showed that pretreatment with either bilateral vagotomy, or mepyramine, the histamine H1 receptor antagonist, significantly inhibited the inflammatory response in the lower airways induced by intraesophageal infusion of OVA. In conclusion, there were clearly two phases, early and late phase responses, in inflammatory response in OVA-sensitized rats receiving intra-esophageal OVA challenge. The underlying mechanisms may involve vagal C-fibers and histamine H1 receptors.

No description available.

Made available in DSpace on 2014-06-11T19:31:11Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-11-11Bitstream added on 2014-06-13T19:41:10Z : No. of bitstreams: 1 costa_ss_dr_botfmvz.pdf: 1745465 bytes, checksum: 6163d7d6432dccda5910a889a705ab7b (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / 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... / 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)