Orientadores: Gilberto de Nucci, Elen Cristina Teizem Landucci / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-11T21:27:11Z (GMT). No. of bitstreams: 1 Ferreira_Tatiane_M.pdf: 838991 bytes, checksum: fcfc9182e3cb79df03511f3867236e61 (MD5) Previous issue date: 2008 / Resumo: A habilidade do veneno bruto de Lachesis muta muta e da fosfolipase A2 básica (LmTX-I) de induzir aumento da permeabilidade microvascular na pata e pele de ratos foi investigada nesse trabalho. O veneno bruto ou LmTX-I foi injetado subplantar ou intradermicamente e o edema foi medido após intervalos de tempo específicos. O volume da pata foi medido usando um hidropletismômetro, enquanto o extravasamento na pele foi medido através do acúmulo de albumina humana marcada com 125I (injetada previamente i.v.) nos sítios de pele. A liberação de histamina dos mastócitos de ratos foi medida por espectrofluorometria. O veneno bruto (0,3-3 µg/pata) ou LmTX-I (0,1-1 µg/pata) induziu edema de pata de maneira dose-dependente. A injeção intradérmica do veneno bruto (0,003-10 µg/sítio) ou LmTX-I (0,003-0,3 µg/sítio) na pele dorsal também resultou em extravasamento de proteínas plasmáticas de maneira dose-dependente. O extravasamento plasmático induzido pelo veneno bruto foi significativamente diminuído pelo tratamento com mepiramina (antagonista de receptor H1, 6 mg/kg), ciproeptadina (antagonista H1 e 5-HT2, 2 mg/kg), LNAME (inibidor não seletivo da síntese de óxido nítrico, 100 nmol/sítio), SR140333 (antagonista de receptor NK1, 1 nmol/sítio), icatibant (antagonista de receptor B2, 0,6 mg/kg) e indometacina (inibidor não seletivo das cicloxigenases, 5 mg/kg), mas não pelo tratamento com PCA4248 (antagonista de receptor de PAF, 5 mg/kg). O extravasamento na pele induzido pela LmTX-I foi significativamente inibido pela ciproeptadina, mepiramina, indometacina e PCA4248, enquanto que L-NAME, SR140333 e icatibant não tiveram efeito. Tanto o veneno bruto quanto a LmTX-I induziram a liberação de histamina de mastócitos peritoneais de ratos de maneira concentração-dependente. Em conclusão, o veneno de L. m. muta e a LmTX-I aumentam a permeabilidade microvascular por mecanismos envolvendo a ativação de mastócitos e a formação de metabólitos do ácido araquidônico. A resposta induzida pelo veneno bruto também envolve liberação de substância P, bradicinina e óxido nítrico enquanto a resposta induzida pela LmTX-I conta com a participação do PAF. / Abstract: The ability of crude venom and a basic phospholipase A2 (LmTX-I) from Lachesis muta muta venom to increase the microvascular permeability in the rat paw and skin has been investigated. Crude venom or LmTX-I were injected subplantarly or intradermically, after which oedema was measured at selected times thereafter. Paw volume was measured using a hydropletismometer, whereas skin extravasation at the skin sites was measured as accumulation of i.v. injected 125I-human serum albumin. Histamine liberation from rat mast cell was measured spectrofluorometrically. Crude venom (0.3-3 µg/paw) or LmTX-I (0.1-1 µg/paw) induced a dose-dependent rat paw oedema. Intradermal injection of crude venom (0.03-10 µg/site) or LmTX-I (0.003-0.3 µg/site) in the dorsal skin also resulted in dosedependent plasma extravasation. Crude venom-induced plasma extravasation was significantly inhibited by the histamine H1 receptor antagonist mepyramine (6 mg/kg), the histamine/5-hydroxytriptamine antagonist cyproheptadine (2 mg/kg), the nitric oxide synthesis inhibitor N?-L-nitro-arginine methyl ester (L-NAME; 100 nmol/site), the tachykinin NK1 receptor antagonist SR140333 (1 nmol/site), the bradykinin B2 receptor antagonist Icatibant (0.6 mg/kg) and the cyclooxygenase inhibitor indomethacin (5 mg/kg). The platelet-activating factor receptor antagonist PCA4248 (5 mg/kg) had no significant effect. LmTX-I-induced skin extravasation was significantly inhibited by cyproheptadine, mepyramine, indomethacin and PCA4248, while L-NAME, SR140333 and Icatibant had no effect. Additionally, both Lachesis muta muta venom and LmTX-I concentration-dependently induced histamine release from rat peritoneal mast cells. In conclusion, Lachesis muta muta venom and LmTX-I increase microvascular permeability by mechanisms involving mast cell activation and both had arachidonic acid metabolites participation. Crude venom-induced responses also involves substance P, bradykinin and nitric oxide release, whether LmTX-I-induced response involves PAF. / Mestrado / Mestre em Farmacologia

Alopecia areata (AA) is a chronic inflammatory disease of hair follicles manifesting as patchy areas of hair loss on the scalp and body. Development of AA is associated with pen- and intra-follicular inflammation of anagen stage hair follicles, primarily by CD4+ and CD8+ cells. We hypothesized that if cell-mediated cytotoxicy against hair follicles is to be a component of the hair loss disease mechanism, increased expression of genes and products typical of cytotoxic cells, as well as increased apoptosis activity within affected hair follicles, would be expected to occur in the lesional skin compared to the normal skin. Furthermore, we studied gene expression levels of multiple cytokines and characteristic chemokines, using the C3FI/HeJ mouse model of AA. mRNA expression levels of granzyme A, granzyme B, perform Fas, Fas ligand, TNF-cL, TNF-aRl and R2, TRAIL, TRAILR, TRAMP, Thi-, Th2-, and Th17-associated cytokines, as well as multiple chemokines were compared between the skin, draining lymph nodes, thymus and spleens of normal and AA-affected mice using quantitative reverse transcriptase PCR. FasL, granzyme A, granzyme B, pro- and anti-inflammatory cytokines were all highly up-regulated in the skin of AA-affected mice. Immunohistochemical studies of the skin revealed that, although greater numbers of granzyme B and FasL expressing cells were present in AA affected skin, the cells were morphologically diffusely distributed and not exclusively located within the focal pen- and intrafollicular infiltrate. The majority of these cells were further characterized as mast cells, which were also found in substantially greater numbers in the skin of mice with AA compared to their normal haired controls. Almost no perform expressing cells were identified in AA affected mouse skin and TUNEL staining suggested relatively limited apoptosis activity in hair follicle keratinocytes. In conclusion, while granzymes and FasL may play important roles in disease development, the profiles and patterns of expression are not consistent with direct cell-mediated cytotoxic action against the follicular epithelium in chronic mouse AA. Potentially, hair growth inhibiting cytokines may play a more dominant role in AA development than previously thought. Furthermore, mast cells, with their increased presence around hair follicles in the AA affected mouse skin and their ability to express granzyme B and FasL, are suggested as potential key players in the pathogenesis of AA. / Medicine, Faculty of / Medicine, Department of / Experimental Medicine, Division of / Graduate

Abstract Both immunosuppressive and nifedipine medication have been associated with drug-induced gingival overgrowth. There are several hypothetical mechanisms for drug-induced gingival overgrowth, such as the influence of genetic predisposition, alterations in gingival tissue homeostasis, especially in the function of fibroblasts, and drug-induced action on growth factors. Clinical studies have also shown that, those with poor oral hygiene status drug-induced gingival overgrowth is more prevalent and severe than those with good oral hygiene status. The working hypothesis was that immunosuppressive medication and/or nifedipine medication affects inflammatory cell profile and mitotic activity of keratinocytes in human overgrown gingiva. We studied gingival samples, collected from nifedipine-medicated cardiac outpatients and immunosuppression-medicated organ-transplant recipients. Patients were placed into four groups: 1) the immunosuppression group, patients receiving cyclosporin-A (CsA), azathioprine (AZA) and prednisolone (Pred) 2) the immunosuppression plus nifedipine group, patients receiving CsA, AZA, Pred. and nifedipine 3) the nifedipine group patients receiving only nifedipine and 4) the non-medicated control group. All of the samples related to moderate to severe degrees of gingival overgrowth, covering half to two thirds of the clinical crown. The aim of the study was to investigate the occurrence of Langerhans cells, macrophages, mast cells and mitotic activity of keratinocytes in human drug-induced overgrown gingiva, and consequently to assess their possible role in the pathogenesis of drug-induced gingival overgrowth. We found that immunosuppressive medication increased the numbers of reparative macrophages (RM3/1) and decreased the numbers of tryptase- and chymase-positive mast cells (MCTC) cells. We have also shown that immunosuppressive and nifedipine medication decreased the numbers of Langerhans cells (CD1a) and increased the numbers of 27E10-macrophages parallelly. Additionally we found increase in the mitotic activity of gingival keratinocytes and even two-fold thickening of gingival epithelium in immunosuppressive and nifedipine medication-induced gingival overgrowth as compared with healthy gingiva. Immunosuppressive medication activated gingival epithelium (27E10 expression in gingival keratinocytes) more than nifedipine medication. In conclusion, our results suggest that gingival overgrowth among immunosuppressive- and nifedipine-medicated patients is related to alteration of tissue homeostasis. First, this suggestion is supported by changes found in the numbers of cells that directly affect connective tissue turnover, e.g. reparative macrophages (RM3/1) and mast cells. Changes in the numbers of these cells could alter the cytokine- and growth factor-profile, which affects fibroblast function. Secondly, we found changes in the numbers of cells involved in regulation of inflammation, e.g. Langerhans cells and monocytes as compared with healthy controls. Immunosuppressive medication could directly activate gingival keratinocytes. We suggest that our findings mainly reflect the effects of immunosuppressive medication, but the role of inflammation cannot be excluded. The changes observed above represent differences of the pathogenesis of drug-induced gingival overgrowth between immunosuppressive and nifedipine medication. It must be however remembered that drug-induced gingival overgrowth is a result of multicausal intrinsic and extrinsic factors. Age, gender, concomitant medication with multiple drugs, plaque accumulation, and genetic disposition are additional risk factors. The abnormal distribution of specific immune system cell subpopulations does not alone prove a functional relationship to gingival overgrowth.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22686号 / 医博第4630号 / 新制||医||1045(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 生田 宏一, 教授 竹内 理, 教授 杉田 昌彦 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

4 Abstract Mast cells are involved in variety of immunological processes, but they are mostly known for their role in allergy and asthma. As asthma and allergy are serious diseases with spreading tendency during last decades, mast cells are subject of intensive research. It is expected that studies of mast cell signalling pathways will contribute to our understanding of the nature of these diseases and help to design efficient treatment strategies. In an attempt to identify genes responsible for asthma disease, genome-wide screening methods have been currently applied. Using these methods, mutations in ORMDL3 (Orosomucoid1-like) protein were found out as a high risk asthma factor. ORMDL3 is a member of evolutionary conserved ORMDL family, comprising in mammals also of ORMDL1 and ORMDL2. Physiological function of these proteins is poorly understood and it has not been studied in mast cells. We decided to study the role of ORMDL proteins in mast cells. Lentiviral delivery system was optimised for generation of stable knock-downs (KD) of all three members of the ORMDL family in primary mast cells. The ORMDL gene expression was measured by improved qPCR (quantitative PCR) reaction buffers. We found that all ORMDL genes are expressed in mast cells in order ORMDL3 > ORMDL2 > ORMDL1. Next, we investigated the...

The activation of bone marrow-derived mast cells (BMMCs) induces a number of cell processes such as degranulation, proliferation and cytoskeleton rearrangements. Although microtubules are important in these processes, molecular mechanisms that control changes in microtubule organisation during cell activation are unknown. Activation of BMMCs can be achieved in several ways. Under physiological conditions, the aggregation of IgE receptors (FcRI) on the surface of BMMCs leads to the initiation of specific signaling pathways. Cells can be also activated nonspecifically by a tyrosine phosphatase inhibitor pervanadate, or by thapsigargin that inhibits Ca2+ ATPase pumps located on the endoplasmic reticulum. In this diploma thesis it was found out that rapid morphological changes can be monitored when BMMC are immobilised on the fibronectin before their activation. It was proved that specific and nonspecific activation events lead to microtubule reorganization, as well as to generation of a large number of microtubule-dependent protrusions. In the course of FcRI aggregation, generation of microtubule protrusions depends on the activity of Src family protein tyrosine kinases and on the intracellular Ca2+ concentration. STIM1, an endoplasmic reticulum Ca2+ sensor, which participates in the activation of...

Microtubules (MTs) are highly dynamic structures essential for the spatio-temporal intracellular organization and transport, signal propagation, cell differentiation, motility and division. To perform these roles, MTs create arrangements capable of fast and precise adaptation to various signals. MTs are under the control of many factors regulating MT nucleation, stability and dynamics. Bone marrow-derived mast cells (BMMCs) are important immune system cells, which can cause serious diseases if their functions are deregulated. Although MT reorganization during BMMC activation is well established, the molecular mechanisms that control their remodelling are largely unknown. In the presented thesis we functionally characterised GIT1/βPIX signalling proteins, PAK1 kinase, and Ca2+ signalling in the regulation of MT nucleation in BMMCs and other cell types. We also elucidated the function of miltefosine (hexadecylphosphocholine), a promising candidate for the treatment of mast cell-driven diseases. We found that GIT1/βPIX signalling proteins are γ-tubulin-interacting proteins associating with centrosomes in BMMCs. MT nucleation is positively regulated by GIT1 and Ca2+ , whereas βPIX is a negative regulator of MT nucleation in BMMCs. Cytosolic Ca2+ affects γ-tubulin properties and stimulates the...

Chronic myocardial infarction (CMI) is associated with remodeling of the ventricle and evokes adaption in the cardiac neurohumoral control systems. To evaluate the remodeling of the intrinsic cardiac nervous system following myocardial infarction, the dorsal descending coronary artery was ligated in the guinea pig heart and the animals were allowed to recover for 7-9 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 configuration compared with age-matched controls and sham-operated animals. The intrinsic cardiac neurons from chronic infarcted hearts did demonstrate an increase in evoked action potential (AP) frequency (as determined by the number of APs produced with depolarizing stimuli) and an increase in responses to exogenously applied histamine compared with sham and age-matched controls. Conversely, pituitary adenylate cyclase-activating polypeptide (PACAP)-induced increases in intrinsic cardiac neuron-evoked AP frequency were similar between control and CMI animals. Immunohistochemical analysis demonstrated a threefold increase in percentage of neurons immunoreactive for neuronal nitric oxide synthase (NOS) in CMI animals compared with control and the additional expression of inducible NOS by some neurons, which was not evident in control animals. Finally, the density of mast cells within the intrinsic cardiac plexus was increased threefold in preparations from CMI animals. These results indicate that CMI induces a differential remodeling of intrinsic cardiac neurons and functional upregulation of neuronal responsiveness to specific neuromodulators.

CHAPTER 1 Aggregation of receptors for IgE (Fc RI) causes mast cells and basophils to release preformed contents of granules, including histamine and a variety of enzymes. This process, called degranulation plays a central role in allergic reactions. Methods to study this process are to create multivalent ligands which can interact with the receptors and, in turn, lead to aggregation of the receptors. We prepared a series of fluorophore-labeled divalent and trivalent antigens to study the degranulation of mast cells. Trivalent antigens proved to be much better stimulators for degranulation of mast cells than divalent antigens. These results indicate that aggregates formed by trivalent antigens are more complicated than those of divalent antigens. CHAPTER 2 Membrane-active antibiotics include antimicrobial peptides (AMPs) and a class of amphiphilic steroids termed ceragenins. Recent studies of membrane-active antibiotics show that cationic, facially amphiphilic molecules could disrupt bacterial membranes. It was found recently that some antibiotics, including AMPs and ceragenins, may share both antibacterial and antiviral activity. We prepared a series of ceragenins to optimize the antiviral activity of ceragenins against vaccinia virus (VV). The results show that ceragenins exhibit potent activity against VV, protect keratinocytes against VV-mediated cell death, and preferentially target the virus. It also shows that antibacterial and antiviral activities do not correlate with each other. Although ceragenins show good antiviral activity against VV, the mechanism for this activity still remains unclear.

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