Zugl.: Magdeburg, Universiẗat, Diss., 2007.

Mast cells are immune cells important in innate immunity. Besides their role in asthma and allergies, mast cells are critical effector cells against various pathogens. Mast cells are established to be protective against bacterial infections, but little is known about their functions in viral infections. Mast cells are abundant in the lungs where influenza A virus (FluA) enter the host. We measured mRNA transcription, protein translation, and synthesis of new viral particles in FluA-treated mast cells. While expression of FluA mRNA and proteins followed similar time courses in both mast cells and epithelial cells, mast cells released a near absence of FluA. We also studied mast cell cytokine release in response to FluA and other viral-associated stimuli such as TLR ligands and type I interferons. Mast cells released the cytokines IL-6 and TGF-, and chemokines CXCL8 and CCL5 in response to various viral stimuli. However, different stimuli were capable of inducing release of different mediator subsets, demonstrating the specificity of mast cell responses. Since FluA infection of mast cells produce little new FluA virions, we investigated whether FluA induces expression of antiviral proteins in mast cells. FluA treatment resulted in mast cell expression of antiviral proteins, namely myxovirus resistance protein A, protein kinase R, interferon stimulated gene 15, viral stress inducible protein 56, and endothelial nitric oxide synthase. Next, we performed co-culture experiments using FluA-infected epithelial cells with or without the addition of mast cells. Our results showed that mast cells in co-culture inhibited the expression of the viral hemagglutinin protein in FluA-infected epithelial cells. Also, preliminary results showed that addition of mast cells protected epithelial cells from FluA infection by limiting the release of FluA particles and reducing epithelial cell death. Our discovery that mast cells produce little virus and express antiviral proteins suggest that mast cells have antiviral mechanisms to restrict FluA infection. This concept was further supported by evidence that mast cells are protective against FluA infection in epithelial cells. This research provides a better understanding of mast cells in innate immunity and may reveal unique antiviral mechanisms valuable in the development of antiviral therapeutics.

Mast cells are granulocytes that require signaling from the receptor protein-tyrosine kinase Kit and its ligand stem cell factor (SCF) for their maturation and function. In addition to providing growth and survival signals, the Kit receptor is involved in crosstalk to β1 integrins leading to mast cell adhesion, spreading, and migration on fibronectin (FN). Previous studies reported the involvement of the non-receptor protein-tyrosine kinases Fps/Fes and Fer in signaling downstream of the high affinity IgE receptor in mast cells and identified cell migration defects in Fer-deficient bone marrow-derived mast cells (BMMCs). Fps/Fes also becomes phosphorylated downstream of the Kit receptor in BMMCs, and this involves the action of the Src family kinase Fyn as an upstream activator of Fps/Fes. In this study, the Fps/Fes SH2 domain was observed to bind the phosphorylated Kit receptor in vitro, suggesting that the SH2 domain plays a role in the activation mechanism of Fps/Fes. To investigate the function of Fps/Fes in Kit signaling, BMMCs were generated from wild-type and Fps/Fes-null mice. Analysis of downstream effectors revealed that Fps/Fes is required for maximal p38 MAPK signaling. Further examination of Fps/Fes-deficient BMMCs revealed increases in adhesion, spreading, and a defect in cell polarization on full-length FN (a ligand for multiple β1 integrins), compared to wild-type BMMCs. Similar phenotypes are observed using an α5β1 integrin-specific FN fragment (9-11) as the matrix. Reduced phosphorylation of the putative Fps/Fes substrate HS1 (a cortactin homologue involved in actin regulation) is observed in Fps/Fes-deficient BMMCs, compared to control cells, and this may contribute to the observed cytoskeletal defects. Restoring Fps/Fes expression in Fps/Fes-deficient BMMCs by retroviral transduction results in a rescue of cell spreading, polarization, and chemotaxis defects to levels similar to those of wild-type cells. This thesis provides novel insights into the potential mode of Fps/Fes activation downstream of the Kit receptor, and a role for Fps/Fes in regulating crosstalk between Kit and α5β1 integrins to promote cytoskeletal reorganization and motility of mast cells. / Thesis (Master, Biochemistry) -- Queen's University, 2009-01-28 15:05:04.056

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No description available.

Mast cells (MC) are present in nearly all tissues in the body and participate in many physiological processes including allergy, tissue remodelling, fibrosis, angiogenesis, and autoimmunity. They can be activated by many stimuli, including allergic and innate immune stimulation. When activated, MC release mediators through which they can regulate inflammatory processes. Recently, we have discovered that rat and human MC express the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), the gene responsible for Cystic Fibrosis (CF). We showed that CFTR had functional activity in MC and its expression was differentially regulated by IFNg. In this thesis, we compared CFTR expression between MC and epithelial cells (EC) by Western blot analysis and found that CFTR expression in MC is similar to that in EC, but there are some differences which suggest either glycosylation or post-transcriptional/translational differences between MC and EC. We also explored the role of CFTR in human MC secretion from various cellular compartments, in response to various stimuli. When we blocked CFTR using pharmacological inhibitors, there was an inhibition of cAMP-dependent Cl- flux. Our data also shows that CFTR pharmacological inhibition had no effect on IgE/anti-IgE-mediated b-hexosaminidase or eicosanoid release from MC. When we stimulated MC with either IgE/anti-IgE or the adenosine receptor agonist NECA (3 uM) for 24h in the presence of CFTR inhibitors, secretion of several mediators appeared to be dysregulated including IL-8, MIF, IL-13, IL-16, PAI-1 and CCL1. To add to these findings, we also used short hairpin RNA (shRNA) to reduce CFTR expression in MC. CFTR deficient MC were unresponsive to NECA and showed reduced constitutive IL-6 secretion. Finally, we cultured MC from CF and non-CF donor peripheral blood progenitors and compared several phenotypic and functional aspects of the cells. We saw no difference in growth, protease content and surface marker expression between CF and non-CF MC, but stimulation of the cells with IgE/anti-IgE or Pseudomonas aeruginosa appeared to differentially induce cytokine synthesis and secretion from CF and non-CF MC. These findings suggest that MC function is dysregulated in CF and that CF MC may be involved in the pathophysiology of CF. / Experimental Medicine

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.

Thesis (Ph.D.)--Boston University / PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you. / The action of progesterone and estrogen on uterine mast cells of the ovariectomized rat was studied. A single injection of estradiol-17B (0.25 μg) produced a highly significant (P < O.OO1) reduction in both mesometrial and antimesometrial mast cell populations. The mean ± S.E. number of mesometrial and antimesometrial mast cells in the control animals was 19.2 ± 1.3 and 6.3 ± 0.7 respectively, while in the estrogen treated animals the respective values at 15 hours post treatment were 4.1 ± 0.5 and 2.9 ± 0.4. Estrogen treatment also resulted in considerable degranulation of the mast cells. In comparison, progesterone, when administered as a single injection (5 mg) did not produce a drastic reduction of the mast cells. Fifteen hours after its administration, progesterone had produced only a moderate reduction of the mast cell population; however, the decline was significantly smaller than those values obtained at the same time interval following estrogen treatment. Since it is known that progesterone treatment for at least 48 hours, followed by estrogen constitute the basic hormonal sequence for decidualizationin the rat, experiments were designed to study possible relationship between mast cell population and deciduoma development. Results obtained from these experiments demonstrate quite clearly that maximal decidual response was possible only among animals treated over a 48 hour period with progesterone (5 mg/ 24 hours) followed by a small (0.25 μg) injection of estradiol-17B and then traumatized 15 hours later (at a time when mast cell population was reduced to the lowest level). Thus, the hormonal treatment which resulted in the lowest level of mast cell numbers also permitted the largest deciduoma development. Shelesnyak (1957) proposed that histamine play a vital role in decidualization in the rat. On the other hand, it has been shown that mast cell degranulation with the accompanying loss of metachromasia is related to histamine release (Thon, 1967). In order to evaluate the role of estrogen as opposed to that of histamine in decidualization, animals were treated with estrogen and progesterone in addition to an estrogen antagonist -CN-55,945-27. The data from these experiments indicated only a moderate decidual response among the treated animals. In addition, the mast cell population in the uterine wall of these animals, at the time of traumatization, was considerably reduced and degranulated. Thus, uninterrupted estrogen action seems to be necessary for the establishment of sensitivity for maximal deciduoma development. In another set of investigations, uterine mast cells were depleted by administration of compound 48/80. Animals depleted of their mast cells and then treated with progesterone, estrogen, followed by trauma developed only small to moderate deciduomata. However, when rats were allowed to recover for seven days (at which time over 50% of the mast cells had reappeared) and then given the treatment as the preceding group, massive full length deciduoma were produced. The evidence suggests that maximal uterine sensitivity to decidualization is possible only after adequate hormonal treatment, and only in uteri not depleted of mast cells. / 2031-01-01

Chronic Obstructive Pulmonary Disease (COPD) is a common lung disease characterized by progressive and irreversible airway obstruction, and mainly caused by a chronic exposure to lung irritants. As of 2010, 384 million people suffered from COPD worldwide. It is widely accepted that a chronic inflammatory response is integral to COPD pathogenesis and linked to disease progression. The cellular mediators and molecular mechanisms of COPD-associated inflammation are not completely understood and are difficult to emulate in animal models, which hinders the development of better treatments. In this study, experimental COPD and its associated inflammation were induced in mice using a 4-week protocol involving intranasal administration of LPS and elastase. Model validation on wild-type mice yielded COPD-like disease judging from flow cytometric analyses with and pulmonary function testing. After 4 weeks of exposure to LPS and elastase, mice developed classic aspects of COPD such an increase in lung-infiltrating cells, (e.g. neutrophils, CD4+and CD8+ T-cells). Acute inflammation in the form of substantial neutrophilia was due to the last LPS administration, whereas the observed eosinophilia and elevated counts of mast cell populations, CD4+ and CD8+ T-cells were due to the cumulative effects of LPS and elastase. The nature of COPD-associated inflammation in mast cell deficient mice was investigated in two experiments. Our first experiment suggested a mild protective role of mast cells, a finding not reproduced in the second experiment possibly due to expired elastase. Our study suggests that mast cells are not required for COPD-associated inflammation.

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