Submitted by Adriana Martinez (amartinez@unoeste.br) on 2017-02-10T16:34:06Z No. of bitstreams: 1 Fabíola Azevedo.pdf: 1121993 bytes, checksum: 7e7e5309d63e8985debf7b6b2d17469c (MD5) / Made available in DSpace on 2017-02-10T16:34:06Z (GMT). No. of bitstreams: 1 Fabíola Azevedo.pdf: 1121993 bytes, checksum: 7e7e5309d63e8985debf7b6b2d17469c (MD5) Previous issue date: 2016-12-15 / Introduction: Increased sugarcane production among the rise in use of herbicides, such as the 2,4-dichlorophenoxyacetic acid (2,4-D), might causes several harm for health. Studies that evaluate the alterations due to the use of this herbicide in the hemogram and in the cells of the nasal mucosa are very important, since the information provided helps in understanding the interactions of substances as the 2,4-D with the human organism. Objective: To analyze blood count components and inflammatory infiltrate in experimental models submitted to nebulization with 2,4-D herbicide, in different time intervals and concentrations. Method: Eighty male Swiss mice were divided into four groups (n = 20): saline, low (3,71 x 10-3 grams of active ingredient per hectare), medium (6,19 x 10-3 grams of active ingredient per hectare) and high concentrations (9,28 x 10-3 grams of active ingredient per hectare). All animals were exposed to the nebulization predefined for each group for 15 minutes during different time intervals: 24, 48, 72 and 192 hours. Blood samples were taken by intracardiac puncture and after euthanasia, the muzzle of each animal was removed for mast cell quantification through toluidine blue staining and analysis of the inflammatory infiltrate by Hematoxylin-Eosin staining (HE). Analysis of normality was conduced using the statistical package Shapiro-Wilk, and for statistical analysis of data Two-way Anova test was performed, considering statistically significant results with p <0.05. Results: All animals in the low concentration group presented microcytosis and hypochromia within 72 hours; Eosinophilia and lymphocytosis within 48 hours. Neutrophilia was observed in the high concentration group over the 192 hour interval. The mast cell count showed significantly increased values in the high concentration group, in the time interval of 48 hours, when compared to the 24 hour period. Conclusion: In conclusion, we observed that in spite of changes in haematological values, the 2,4-dichlorophenoxyacetic acid herbicide can not be considered dose-dependent and time-dependent herbicide, but it might provoke allergic reactions. / Introdução: O aumento da produção de cana-de-açúcar em conjunto com a utilização de herbicidas, como o ácido 2,4-diclorofenoxiacético (2,4-D) traz diversos agravos à saúde humana. Estudos que avaliam as alterações decorrentes do uso desse herbicida no hemograma e nas células da mucosa nasal são de grande importância, pois as informações fornecidas podem ser utilizadas com o intuito de ajudar na compreensão da interação dessa substância com o organismo humano. Objetivo: Analisar os componentes do sangue e infiltrado inflamatório em modelos experimentais submetidos à nebulização ao herbicida 2,4-D, em diferentes intervalos de tempo e concentrações. Metodologia: Foram utilizados 80 camundongos Swiss machos, divididos em quatro grupos (n=20): salina, baixa (3,71 x 10-3 gramas de ingrediente ativo por hectare), média (6,19 x 10-3 gramas de ingrediente ativo por hectare) e alta concentração (9,28 x 10-3 gramas de ingrediente ativo por hectare). Todos os animais foram expostos às nebulizações preconizadas para cada grupo por 15 minutos, em diferentes intervalos de tempo: 24, 48, 72 e 192 horas. A coleta das amostras de sangue foi através de punção intracardíaca e após a eutanásia, o focinho de cada animal foi retirado para a quantificação de mastócitos, através da coloração de azul de toluidina e análise do infiltrado inflamatório, pela coloração de Hematoxilina-Eosina (HE). Para a análise de normalidade foi utilizado o pacote estatístico Shapiro-Wilk e para a análise estatítica dos dados, utilizamos o teste Two-way Anova, considerando estatisticamente diferentes os resultados que apresentaram p< 0,05. Resultados: Todos os animais do grupo baixa concentração apresentaram microcitose e hipocromia no intervalo de 72 horas; eosinofilia e linfocitose no intervalo de 48 horas. Foi verificada neutrofilia no grupo alta concentração no intervalo de 192 horas. A contagem de mastócitos apresentou valores significativamente aumentados no grupo alta concentração, no intervalo de tempo de 48 horas, quando comparado ao de 24 horas. Conclusão: Em conclusão observamos que, apesar das alterações nos valores hematológicos, o herbicida ácido 2,4-diclorofenoxiacético não pode ser considerado dose e tempo-dependentes, mas pode provocar reações alérgicas.

Mast cells are key players of the innate immune system due to their location at the interface of host and pathogen encounters, such as on mucosal surfaces or the skin. Secreting a variety of compounds, they communicate with other immune cells in a highly specific manner. Subsequently, reinforcements against foreign invaders are recruited while also defending the host, using bacteriolytic effector molecules. One type of pathogens which are competent challengers of the host’s immune system are Streptococci, causing a burden for humans and animals. Streptococcus equi subspecies equi is one example, a highly contagious horse pathogen with a silent carrier subset, causing “strangles”, a disease resulting in equine morbidity and mortality all over the world. The present study aimed to explore the virulence factors in S. equi responsible for immune system activation, represented by mast cells. Knockout mutants of the genes aroB, hasA, pyrC, recA, sagA and a combination of those, including a deletion strain of all superantigens (seeHILM), were co-cultivated with murine bone-marrow-derived mast cells (BMMCs). Mast cells alone and S. equi strain 4047 (wild-type) were used as controls. It was shown that 4 h after encounter of the bacteria, BMMCs responded with IL-6, TNF-α and MCP-1 secretion, indicating an inflammatory response to all strains except against the sagA mutant (ΔsagA) or the multi-deletion strain, the latter lacking several virulence factors including sagA. These results were confirmed at the mRNA-level where IL-6, TNF-α and Nr4a3 gene expression was significantly upregulated in BMMCs after 4 h incubation with wild-type S. equi. In contrast, when BMMCs were co-cultivated with sagA-deficient S. equi, no detectable upregulation was seen. These results were further confirmed in peritoneal-derived mast cells. After 24 h no secretion of cytokines was detected in response to ΔsagA mutants, in contrast to the strong cytokine output in response to wild-type S. equi. To elucidate the role of SagA, the precursor of streptolysin S (SLS), lysis was determined, and it was observed that ΔsagA does not lyse mast cells in contrast to wild-type with intact SLS. Transwell-based experiments indicated a partially contact-dependent response of mast cells to bacteria. Taken together, this study shows for the first time that SLS is the major mast cell activator produced by S. equi. I suggest the possible mechanism of cell death by lysis and reprogrammed signaling pathways of the host by sublytic concentrations of SLS, resulting in damage associated pattern-mediated signaling as well as auto- and paracrine amplification by inflammatory cytokines and other messenger molecules.

Human mast cells are often found perivascularly and at mucosal sites and may play crucial roles in the inflammatory response. Recent studies have suggested a prominent role for mast cells in host defense. In this study, we analyzed the effects of a common airway pathogen, Moraxella catarrhalis and a commensal bacterium, Neiserria cinerea, on activation of human mast cells. Human mast cell leukemia cells (HMC-1) were activated with either phorbol myristate acetate (PMA) and calcium ionophore or with varying concentrations of heat-killed suspensions of bacteria. Supernatants were assayed for the cytokines interleukin-4 (IL-4), granulocyte macrophage colony stimulating factor (GM-CSF), IL-6, IL-8, IL-13 and monocyte chemotactic protein-1 (MCP-1). Nuclear proteins were isolated and assayed by electrophoretic mobility shift assay (EMSA) for nuclear factor kappaB (NF-κB) nuclear binding activity. In some experiments, NF-κB inhibitor, Bay-11 was added to determine functional significance. Both M. catarrhalis and N. cinerea induced mast cell activation and selective secretion of two key inflammatory cytokines, IL-6 and MCP-1. This was accompanied by NF-κB activation. Neither spun bacterial supernatants nor bacterial lipopolysaccharide induced cytokine secretion, suggesting need for direct bacterial contact with mast cells. Scanning electron microscopy revealed active aggregation of bacteria over mast cell surfaces. The NF-κB inhibitor, Bay-11, inhibited expression of MCP-1. These findings suggest the possibility of direct interactions between human mast cells and common bacteria and provide evidence for a novel role for human mast cells in innate immunity.

Chronic pressure overload (PO) is associated with cardiac hypertrophy and altered autonomic control of cardiac function, in which the latter may involve adaptations in central and/or peripheral cardiac neural control mechanisms. To evaluate the specific remodeling of the intrinsic cardiac nervous system following pressure overload, the descending thoracic aorta artery of the guinea pig was constricted ∼20%, and the animals recovered for 9 wk. Thereafter, atrial neurons of the intrinsic cardiac plexus were isolated for electrophysiological and immunohistochemical analyses. Intracellular voltage recordings from intrinsic cardiac neurons demonstrated no significant changes in passive membrane properties or action potential depolarization compared with age-matched controls and sham-operated animals, but afterhyperpolarization duration was increased in PO animals. Neuronal excitability, as determined by the number of action potentials produced with depolarizing stimuli, was differentially increased in phasic neurons derived from PO animals in response to exogenously applied histamine compared with sham and age-matched controls. Conversely, pituitary adenylate cyclase-activating polypeptide-induced increases in intrinsic cardiac neuron evoked AP frequency were similar between control and PO animals. Immunohistochemical analysis demonstrated a two-fold increase in the percentage of neurons immunoreactive for neuronal nitric oxide synthase in PO animals compared with control. The density of mast cells within the intrinsic cardiac plexus from PO animals was also increased twofold compared with preparations from control animals. These results indicate that congestive heart failure associated with chronic pressure overload induces a differential remodeling of intrinsic cardiac neurons and upregulation of neuronal responsiveness to specific neuromodulators.

No description available.

Spiral artery (SA) remodeling is a fundamental process during early pregnancy that involves the action of vascular smooth muscle cells (VSMCs), maternal immune cells, but also fetal extravillous trophoblast cells (EVTs) and the extracellular matrix (ECM). Mast cells (MCs) and their mediator chymase, have been identified as prominent players for a sufficient SA remodeling process at the fetal-maternal interface in vivo. In contrast, endocrine disrupting chemicals (EDC), especially Bisphenol A (BPA) and 17-α-ethinylestradiol (EE2), have been shown to have a negative impact on SA remodeling in animal models in vivo. However, neither the mechanisms underlying the positive effects of MCs and chymases for the remodeling process, nor the interference of EDCs on SA remodeling, have been elucidated. The purpose of the present work was to evaluate the effect of MCs and recombinant human chymase CMA1 (rhuCMA1) on the phenotype and/or behavior of VSMCs, EVTs and ECM in vitro. Moreover, the influence of BPA and EE2 on the functionality of EVTs in vitro was observed. Using an immortalized human trophoblast cell line (HTR8/SV-neo), a mouse trophoblast cell line (SM9-2), human primary uterine vascular smooth muscle cells (HUtSMCs) and mouse primary uterine smooth muscle cells (SMCs), we assessed the effects of the human MC cell line HMC-1, the mouse mast cell line MC/9 and rhuCMA1 on VSMCs, EVTs and ECM. Additionally, the HTR- 8/SV-neo cells functionality was evaluated after treatment with BPA or EE2. We found that mouse MC/9 cells induce fibronectin expression and migration in SMCs. Furthermore MC/9 cells increase the proliferation and migration of SM9-2 cells. Both human HMC-1 cells and rhuCMA1 stimulate the migration, proliferation, and change of synthetic/contractile marker expression in HUtSMCs. In addition, HMC-1 cells increase the proliferation and migration of HTR8/SVneo trophoblast cells while having an impact on the expression of tissue remodeling genes. HTR8/SVneo cells presented increased migration rates along with decreased expression of the matrix-metalloproteinase regulator genes (TIMPs) upon treatment with rhuCMA1. Moreover, BPA interfered with HTR-8/SV-neo cell proliferation and reduced MMP2 expression in HTR-8/SV-neo. Interestingly, EE2 had no impact on proliferation or migration but suppressed the MMP2 expression in HTR-8/SV-neo cells. The obtained results indicate that MCs, and partly their mediator chymase CMA1, shape the phenotype and modulate the functionality of VSMCs and EVTs. Collectively, possible mechanisms by which MCs and specifically rhuCMA1 promote SA remodeling were identified. The findings are relevant for the understanding of this crucial step in pregnancy and thus, for the comprehension of dysregulated pathways that can lead to pregnancy complications like fetal growth restriction and preeclampsia. Moreover, this work contributes to the knowledge about how EDCs impact on early pregnancy and highlights the high risk of EDCs exposure disturbing the fundamental reproductive process of SA remodeling.

Mast cells are tissue resident members of the immune system. They have a wide range of functions and receptors including the FcεRI receptor, which gets activated by binding to IgE bound to an antigen. When the cells are activated in this manner, a process termed the LysRS- Ap4A-MITF signalling pathway occurs, resulting in the translocation of the Lys tRNA synthetase into the nucleus and an activation of its moonlighting activity - the production of diadenosine tetraphosphate (Ap4A). Ap4A is a dinucleoside polyphosphate, a type of ubiquitous molecule present in all domains of life. They are made up of two nucleosides joined together by a 5' to 5' phosphodiester bridge of variable lengths. Recently, these molecules have been shown to serve as non-canonical initiating nucleotides during bacterial transcription, where they function as 5' RNA caps, similar to the well-known 7- methylguanosine eukaryotic mRNA cap. In this thesis, I present proof of existence of Ap 4A capped RNA in mast cells, a previously unknown 5' RNA structure in eukaryotic cells, and I attempt to pinpoint its role in the activation of these cells and in the wider context of mast cell mediated immune response. Keywords: mast cells, RNA caps, Dinucleoside polyphosphates, Ap 4A, RNA modification, IgE, FcεRI receptor, Lysine tRNA synthetase

Following the discovery of mast cells (or mastzellen) by the prolific physician researcher, Paul Ehrlich, many advances have improved our understanding of these cells and their fascinating biology. The discovery of immunoglobulin E and receptors for IgE and IgG on mast cells heralded further in vivo and in vitro studies, using molecular technologies and gene knockout models. Mast cells express an array of inflammatory mediators including tryptase, histamine, cytokines, chemokines, and growth factors. They play a role in many varying disease states, from atopic diseases, parasitic infections, hematological malignancies, and arthritis to osteoporosis. This review will attempt to summarize salient evolving areas in mast cell research over the last few centuries that have led to our current understanding of this pivotal multifunctional cell.

Chronic urticaria (CU) is a mast cell-driven chronic inflammatory disease with a female predominance. Since CU affects mostly females in reproductive age, pregnancy is an important aspect to consider in the context of this disease. Sex hormones affect mast cell (MC) biology, and the hormonal changes that come with pregnancy can modulate the course of chronic inflammatory conditions, and they often do. Also, pregnancy-associated changes in the immune system, including local adaptation of innate and adaptive immune responses and skewing of adaptive immunity toward a Th2/Treg profile have been linked to changes in the course of inflammatory diseases. As of now, little is known about the effects of pregnancy on CU and the outcomes of pregnancy in CU patients. Also, there are no real-life studies to show the safety of urticaria medications during pregnancy. The recent PREG-CU study provided the first insights on this and showed that CU improves during pregnancy in half of the patients, whereas it worsens in one-third; and two of five CU patients experience flare-ups of their CU during pregnancy. The international EAACI/GA²LEN/EuroGuiDerm/APAAACI guideline for urticaria recommends adopting the samemanagement strategy in pregnant and lactating CU patients; starting treatment with standard doses of second-generation (non-sedative) H1 antihistamines, to increase the dose up to 4-folds in case of no response, and to add omalizumab in antihistamine-refractory patients; but also emphasizes the lack of evidence-based information on the safety and efficacy of urticaria treatments during pregnancy. The PREG-CU study assessed treatments and their outcomes during pregnancy. Here, we review the reported effects of sex hormones and pregnancy-specific immunological changes on urticaria, we discuss the impact of pregnancy on urticaria, and we provide information and guidance on the management of urticaria during pregnancy and lactation.

Head and neck cancer is a heterogeneous group of tumours, of which certain subgroups such as cancer of the mobile tongue frequently are associated with a relatively poor prognosis due to the high risk of regional failure and mortality rates that haven’t improved in a significant way over the last 3 decades, despite advancements in both diagnostics and treatment. Today we lack means to assess the biological aggressiveness of each individual tumour, which varies largely. Treatment comprises of surgery with additional radiotherapy and medical therapies in more advanced tumours. The focus in this thesis is on molecular biomarker expression in head and neck cancer and especially in association with radiotherapy. Increased knowledge paves the way to a more individualized cancer treatment aiming for better outcome and less overtreatment and sequelae. The aims of this thesis was: To map the effects of radiotherapy in both tumour and adjacent tissue for the possible markers hyaluronan, EGFR and mast cells. To investigate whether the expression of hyaluronan in the epithelium and connective tissue stroma and EGFR in the tumour correlates with the risk for developing cervical metastasis in N0 patients, and to find out whether the 3-year tumour-specific survival rates correlates with the expression of HA in the epithelium and EGFR in the tumour. To establish an animal model for radiation-induced mucositis and to use that model to examine the pattern of invading inflammatory cells. To investigate whether the expression of podoplanin in tongue cancer correlates with the risk for cervical metastasis and to determine whether the total amount of lymph vessels in the diagnostic biopsy has any impact on the clinical outcome. To investigate the differences in the metabolome of tongue cancer cell lines with different radiosensitivity. The most important findings of this thesis were: The expression of EGFR and hyaluronan hade the same pattern of expression in both tumour and adjacent tissues before radiotherapy. The expression of EGFR was increased in the epithelium of the adjacent tissue close to the tumour after radiotherapy. The intensity of the staining of hyaluronan was correlated to the 3-year survival rates in patients with tongue cancer. An experimental model for radiation-induced oral mucositis in rat was established and in this model a temporal pattern of macrophage invasion with two different subtypes of macrophages was found. There were no correlation between the expression of podoplanin in the tumour tissue and the cervical metastasis rate in patients with tongue cancer, but the younger patients were more likely to have a higher expression of podoplanin in their tumour than elder patients. Tongue cancer cell lines with different radiosensitivity respond to irradiation with different patterns of metabolic expressions.