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1
Hoenderdos, Kim. "Modulation of neutrophil degranulation by hypoxia." Thesis, University of Cambridge, 2015. https://www.repository.cam.ac.uk/handle/1810/247459.
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Neutrophils are key effector cells of the innate immune system. They employ a number of powerful ‘weapons’ to eliminate pathogens, including an array of destructive proteins packaged into distinctive granule subsets. In addition to their microbicidal activity, these granule proteins are capable of causing substantial tissue damage if inappropriately deployed. To mitigate against this possibility, most physiological stimuli induce minimal extracellular degranulation. Sites of inflammation and infection are usually hypoxic, and it has been shown that oxygen depletion compromises neutrophil function by impairing the generation of reactive oxygen species and hence bacterial killing. The key finding reported in this thesis is that hypoxia substantially increases the release of all neutrophil granule subsets, as measured by the release of (active) hallmark proteins (elastase, myeloperoxidase, lactoferrin and matrix metalloproteinase-9). In consequence, supernatants from hypoxic neutrophils induced substantially more damage to lung epithelial cell layers than supernatants from neutrophils cultured under normoxic conditions; this damage was protein- and protease-dependent. This pattern of damage was seen consistently across lung adenocarcinoma-derived epithelial cells, primary immortalised lung epithelial cells, and primary human bronchial epithelial cells grown in physiological air-liquid interface culture. Surprisingly, the mechanism of hypoxia-augmented degranulation was found to be independent of protein synthesis and specifically, of the transcription factor HIF-1α (the ‘master-regulator’ of hypoxic responses); thus, hypoxia did not affect mRNA transcript or protein abundance of the major granule components, and hypoxia mimetics failed to recapitulate the phenotype. Inhibition of the key pathways known to be involved in neutrophil degranulation, including, phosphatidylinositol 3-kinase and phospholipase C, but not calcium flux prevented augmented granule release under hypoxia In conclusion, hypoxia induces a destructive neutrophil phenotype, with increased release of multiple histotoxic proteases. This may contribute to tissue injury and disease pathogenesis in a range of clinically important conditions.
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Admiraal, Claudia Johanna. "Eosinophil degranulation as an allergy activation marker." [S.l. : Amsterdam : s.n.] ; Universiteit van Amsterdam [Host], 2001. http://dare.uva.nl/document/59048.
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Dexter, Emily Jane. "The role of adenosine in mast cell degranulation." Thesis, University College London (University of London), 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.314292.
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Swindle, Emily Jane. "Mast cells as a source and target for nitric oxide and reactive oxygen species." Thesis, University of Liverpool, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.269602.
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Zankl, Claudia. "Stereoselektive Synthese von Sphingolipiden zur Inhibierung der Degranulation von Mastzellen." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2009. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-23408.
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Die Degranulation von Mastzellen soll durch Glycosphingolipide, welche mit der Zellmembran wechselwirken inhibiert werden. Der Sphingosingrundkörper wurde in zehn-stufigen Synthese ausgehend von N-Boc-Serin, aufgebaut. Die anschließende Glycosylierung erfolgte nach der Trichloracetimidatmethode in sehr guten Ausbeuten und stellte den Schlüsselschritt dar. Durch die Variation von unter Anderem der Amidseitenkette, der Glycosylkopfgruppe und des Sphingosingrundkörpers wurde eine Vielzahl an Derivaten für das Screening im Degranulationsassay bereitgestellt<br>The present dissertation covers the synthesis of glycosphingolipids which interact with the cell membrane in order to inhibit the degranulation of mast cells. The sphingosin body was synthesized in ten steps starting from N-Boc-Serin. The key step, the glycosylation was achieved using the trichloracetimidat method. The variation of the amid sidechain, the gylcosyl headgroup and the sphingosin body created a number of derivatives that were tested in the degranulation assay
6
Foweraker, J. E. "A study of eosinophil granule proteins and in vitro degranulation." Thesis, University of Cambridge, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.377204.
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Sinniah, Ajantha. "Annexin A1 as an endogenous regulator of mast cell degranulation." Thesis, Queen Mary, University of London, 2015. http://qmro.qmul.ac.uk/xmlui/handle/123456789/9089.
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Annexin A1 (Anx-A1) is a 37kDa protein that is secreted by some cells in response to glucocorticoids (GCs) and which mediates several of their acute anti-inflammatory effects. In addition to GCs, ‘mast cell stabilising’ cromones such as nedocromil also mobilise Anx-A1 by promoting its phosphorylation by protein kinase C (PKC) and hence its secretion, which explains their acute efficacy as anti-allergic agents. This thesis addresses a fundamental aspect of Anx-A1 in the actions of anti-allergic drugs. In this study, anti-allergic drugs such as H1 antagonists, mast cell stabilisers and ‘dual action’ drugs were first tested for their ability to enhance Anx-A1 phosphorylation in a model system using U937 cells. Biochemical and immuno-fluorescent techniques were used to study the mechanisms by which these drugs suppress mediator release from cord blood derived mast cells (CDMCs) and murine bone-marrow derived mast cells (BMDMCs) from wild type and Anx-A1 null-mice. This thesis suggest that PKC activation is crucial for Anx-A1 export in mast cells and nedocromil in the presence of dexamethasone, prolongs the duration of PKC activation and subsequently phosphorylation, externalisation and release of Anx-A1 from CDMCs. The ability of nedocromil to inhibit β-hexosaminidase, tryptase, histamine and PGD2 release are dependent on Anx-A1 in CDMCs. Interestingly, ketotifen, a ‘dual action’ drug possesses a similar pharmacological profile to nedocromil, but not promethazine, which does not act through the Anx-A1 release. Strong evidence supports the notion that the mechanisms of action of nedocromil are modulated by Anx-A1, thus the possibility that FPR2 might be involved in the acute actions of nedocromil was tested. Nedocromil inhibits the release of PGD2 through the activation of FPR2 but not the inhibition of histamine release. A possible explanation for this finding could be that Anx-A1 might be interacting with other FPR family members to exert the histamine inhibitory effects. Although only a small subset of the downstream intracellular signaling pathway of MAPK was tested, the results indicate that Anx-A1 differentially regulates the activation of p38 and JNK in CDMCs treated with nedocromil. These findings indicate a novel model system in which Anx-A1 mediates the pharmacological actions of anti-allergic drugs and thus has an important role in preventing the mast cell degranulation.
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El-Lati, Suhad George. "Morphological and biochemical aspects of degranulation of human skin mast cells." Thesis, University of Southampton, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.316384.
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Biethahn, Katharina [Verfasser]. "Regulation of Fc epsilon receptor I-mediated mast cell degranulation / Katharina Biethahn." Kiel : Universitätsbibliothek Kiel, 2011. http://d-nb.info/1043358471/34.
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HADJADJ, BADRE. "Modelisation de la degranulation des basophiles humains et etude du controle optimal." Paris 6, 1994. http://www.theses.fr/1994PA066385.
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L'objectif de notre travail consiste a proposer des modeles mathematiques associes aux phenomenes de la degranulation des basophiles humains et de la liberation de l'histamine, puis a elaborer des techniques pour un controle optimal du systeme. Cela nous permet d'etudier l'effet de hautes dilutions sur ce systeme. Dans le chapitre 1 nous avons etabli un modele de simulation representant le phenomene en fonction des dilutions homeopathiques. Dans le chapitre 2 nous avons etudie un modele compartimental lineaire tres simplifie puis nous avons introduit un controle optimal. Dans le chapitre 3 un autre modele compartimental a ete propose qui prend en compte le phenomene du retro-controle negatif de l'histamine (feed-back). Puis a nouveau nous avons etudie le controle optimal du systeme. Finalement dans le chapitre 4 nous avons etudie et applique une methode decompositionnelle, dite technique d'adomian, sur les deux systemes differentiels associes aux modeles compartimentaux etudies
11
Koranteng, Rachael Darkoa. "Effects of nitric oxide on mast cell activation." Thesis, University of Liverpool, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.343761.
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Willox, Ian. "Role of the chemokine receptor CXCR3 in human mast cell degranulation and signalling." Thesis, University of Bath, 2009. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.518109.
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The chemokine receptor CXCR3, which has three known variants (CXCR3-A, CXCR3-B and CXCR3-Alt), has been implicated in the recruitment of mast cells to tissues in many different chronic diseases with its agonists found in elevated levels in many pulmonary diseases. All three variants of CXCR3 were detected in cord blood-derived mast cells at the mRNA level. Using an antibody that is unable to distinguish individual CXCR3 isoforms, we detected a marked down-regulation of intracellular protein during maturation from progenitor cells, with no concomitant changes in the modest surface expression of CXCR3. The known CXCR3 agonists CXCL9, CXCL10 and CXCL11 as well as the reported CXCR3-B agonist CXCL4, were able to induce Akt and ERK1/2 phosphorylation, as well as partial degranulation. Responses to all agonists were inhibited by pre-treatment with selective CXCR3 antagonists and pertussis toxin. Use of novel isoform-selective inhibitors indicates that the p110 isoform of PI3K is required for degranulation and signalling responses to CXCR3 agonists. Unexpectedly, dual (but not individual) isoform inhibition of the class I and isoforms substantially inhibited signalling and degranulation responses, indicating a hitherto unrecognised synergy between these isoforms, which provide a conduit for CXCR3 signalling in mast cells.
13
Hori, Masayuki. "A CD57+ CTL Degranulation Assay Effectively Identifies Familial Hemophagocytic Lymphohistiocytosis Type 3 Patients." Kyoto University, 2017. http://hdl.handle.net/2433/225981.
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14
Brack, Eva. "Einfluss von Surfactantprotein A und D auf die ECP-Degranulation von humanen eosinophilen Granulozyten." Diss., Ludwig-Maximilians-Universität München, 2013. http://nbn-resolving.de/urn:nbn:de:bvb:19-160799.
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Die Rolle, die die hydrophoben Surfactantproteine SP-A und SP-D welche in den oberflächlichen Atemwegsflüssigkeiten zu finden sind spielen lässt sich wie folgt zusammenfassen. Zum einen, eine wichtige immunologische Funktion, indem sie zur Abwehr von Erregern beitragen. Zum anderen, bei der Regulation allergischer Reaktionen, bei welcher der eosinophile Granulozyt mit den von ihm freigesetzten Entzündungsmediatoren als wichtigste Effektorzelle zu nennen ist. Ziel dieser Arbeit war es den Einfluss der Surfactantproteine A und D auf eosinophile Granulozyten in Bezug auf ihre ECP-Degranulation zu untersuchen. In verschiedenen Versuchsreihen wurde die Freisetzung von Eosinophilem kationischem Protein (ECP) ohne (native Zellen) und nach Stimulation der Zellen mittels Fluoreszenzassay gemessen. Als Stimulanzien kamen Ca-Ionophore A23187 als eine potenter, wenn auch unphysiologischer Zellstimulus und die Immunglobuline A und G, sowie das Serum eines Allergikers als physiologische Stimuli zum Einsatz. Die Ergebnisse zeigten, dass durch die Surfactantproteine selbst keine Aktivierung der ECP-Freisetzung aus nativen eosinophilen Zellen ausgelöst werden kann. Die Degranulation stimulierter Zellen hingegen kann durch natürliches SP-A, SP-D und rekombinantes SP-D reduziert werden. Die wirksamen SP-D Konzentrationen lagen zwischen 0,05 und 5000ng/ml, wenn die Zellen mit Ca-Ionophore stimuliert wurden. Wurden IgA und IgG als Stimuli verwendet, konnte die ECP-Degranulation durch SP-A, SP-D und rekombinantes SP-D ebenfalls gehemmt werden, zeigte sich jedoch in keiner Konzentration signifikant. Bei der Stimulation mit Allergikerserum zeigten sowohl SP-A, SP-D als auch rekombinantes SP-D eine hemmende Wirkung. Zusammenfassend zeigen unsere In-vitro-Ergebnisse eine inhibitorische Wirkung von SP-A und D auf die ECP-Degranulation aus stimulierten eosinophilen Granulozyten. Dies ist mit der Hypothese einer protektiven Wirkung dieser Surfactantproteine bei Erkrankung aus dem allergischen Formenkreis vereinbar.
15
Ip, Chun-him, and 葉俊謙. "Neutrophil elastase: switch from stored form to unopposed form following neutrophil degranulation in inflamedenvironments." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2011. http://hub.hku.hk/bib/B47164955.
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Herzog, Gloria [Verfasser]. "Degranulation und Freisetzung von radikalen Sauerstoffspezies durch Eosinophile Granulozyten im nekrotischen Milieu / Gloria Herzog." Ulm : Universität Ulm. Medizinische Fakultät, 2013. http://d-nb.info/1036925072/34.
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Lee, Jai-Wei. "The effect of recombinant human interleukin-1ß and interleukin-8 on bovine neutrophil migration and degranulation." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp03/MQ50813.pdf.
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Lee, Jai-Wei 1970. "The effect of recombinant human interleukin-1b and interleukin-8 on bovine neutrophil migration and degranulation /." Thesis, McGill University, 1999. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=21587.
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The objective of this study was to investigate the effect of recombinant human interleukin-1beta (rHIL-1beta) and interleukin-8 (rHIL-8) on bovine neutrophil migration and degranulation. An in vitro co-culture system was used to study bovine neutrophil migration. This simulative system allowed studying neutrophil migration across endothelium (bovine aorta endothelial cells), extracellular matrix (ECM), and epithelium (MAC-T) in the correct sequences and directions. Quantification of neutrophil migration was carried out by assaying the activity of myeloperoxidase, a major enzyme of neutrophils. Degranulation of azurophilic, specific, and tertiary granules was studied by measuring releases of myeloperoxidase, lactoferrin, and gelatinase, respectively. The results showed that bovine neutrophils were able to migrate across the simulative co-culture system in response to zymosan activated serum. Recombinant HIL-8 was demonstrated to have a dose-dependent effect on bovine neutrophil migration. Furthermore, rHIL-8 had a dose-dependent effect directly on degranulation of azurophilic and specific granules, but not on tertiary granules. On the other hand, rHIL-1beta only had a significant effect on degranulation of azurophilic granules when the concentration of 100 ng/ml was used. The dose effect of rHIL-1beta on specific degranulation was much stronger. Moreover, the effect of 100 ng/ml rHIL-1beta was augmented when the rHIL-1beta containing solution was preincubated with MAC-T monolayers for four hours. This indicated that MAC-T cells might generate other degranulating factors in response to the stimulation of rHIL-1beta. These MAC-T-derived degranulating factors did not have effect on the release of tertiary granule contents.
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Walsh, Sarah K. "Studies on the role of mast cell degranulation in mediating the cardioprotective effects of endothelin-1." Thesis, Robert Gordon University, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.436353.
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Zarnegar, Behdad. "Mechanisms triggering the recruitment of mast cell progenitors to the lung and regulation of mast cell degranulation." Doctoral thesis, Uppsala universitet, Institutionen för medicinsk biokemi och mikrobiologi, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-306115.
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Mast cells stem from the bone marrow and migrate via the blood as mast cell progenitors. Upon arrival in peripheral tissues, they develop into mast cells. These rare immune cells have numerous granules that contain large amounts of pro-inflammatory mediators. Mast cells accumulate at certain sites in the asthmatic lung, and once activated they release mediators that are thought to induce symptoms. In mouse models of allergic airway inflammation, the increase in lung mast cells in asthma can be mimicked and is mainly caused by the recruitment of mast cell progenitors to the lung. However, whether other types of lung inflammation stimulate the recruitment of mast cell progenitors to the lung was unknown until now. Here, using a murine model of influenza A virus infection, this type of virus was demonstrated to trigger an extensive recruitment of mast cell progenitors to the lung, most likely through the induction of VCAM-1 expression in the lung endothelium. Thereafter, some influenza-induced mast cell progenitors developed into an intermediate mast cell stage before they matured into mast cells. However, upon the resolution of inflammation, the mast cells that accumulated in the lung upon influenza infection were gradually lost. Because the recruitment of mast cell progenitors started early after influenza infection, the role of innate immune signals in inducing the recruitment of mast cell progenitors was addressed. The intranasal administration of either Poly I:C or IL-33 was sufficient to induce an increase in lung mast cell progenitors in a TLR3- or ST2-dependent fashion. However, the influenza-induced recruitment of mast cell progenitors to the lung occurred independently of TLR3 and ST2. VAAT/SLC10A4 is a member of the solute carrier family of proteins that is expressed in nerve cells and mast cells. In this study, murine VAAT was localized to mast cell granules and regulated the IgE/antigen-mediated release of granule-associated mediators and ATP. However, the absence of VAAT did not affect IgE/antigen-mediated de novo synthesis of cytokines and lipid mediators. Additionally, mice lacking VAAT had attenuated passive cutaneous anaphylaxis reactions and scratched less frequently in response to compound 48/80 injections, suggesting that VAAT regulates reactions for which mast cells are implicated in vivo.
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CHEN, XIAOJING. "Control of Secondary Granule Release in Neutrophils by Ral GTPase." Digital Archive @ GSU, 2011. http://digitalarchive.gsu.edu/biology_diss/96.
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Neutrophil (PMN) inflammatory functions, including cell adhesion, diapedesis, and phagocyto-sis, are dependent on the mobilization and release of various intracellular granules/vesicles. In this study, I found that treating PMN with damnacanthal, a Ras family GTPase inhibitor, resulted in a specific release of secondary granules, but not primary or tertiary granules, and caused dy-sregulation of PMN chemotactic transmigration and cell surface protein interactions. Analysis of the activities of Ras members identified Ral GTPase as a key regulator during PMN activation and degranulation. In particular, Ral was active in freshly isolated PMN, while chemoattractant stimulation induced a quick deactivation of Ral that correlated with PMN degranulation. Over-expression of a constitutively active Ral (Ral23V) in PMN inhibited chemoattractant-induced secondary granule release. By subcellular fractionation, I found that Ral, which was associatedwith the plasma membrane under the resting condition, was redistributed to secondary granules after chemoattractant stimulation. Blockage of cell endocytosis appeared to inhibit Ral transloca-tion intracellularly. In conclusion, these results demonstrate that Ral is a critical regulator in PMN that specifically controls secondary granule release during PMN response to chemoattrac-tant stimulation.
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Haider, Waqar Yunus. "The role of the dileucine motif in Helix VIII of the BLT1 receptor and RhoA in neutrophil degranulation." Mémoire, Université de Sherbrooke, 2010. http://savoirs.usherbrooke.ca/handle/11143/4024.
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Neutrophil degranulation involves a number of well-orchestrated structural and biochemical events. We have investigated the mechanism of intracellular signalling involved in neutrophil degranulation that was mediated by the high affinity leukotriene (LT)B[subscript 4] receptor, BLT1. The model systems used were consisted of Peripheral blood neutrophils as well as promyeloid PLB-985 cells, stably transfected with human BLT1 cDNA (PLB-BLT) or a substitution mutant (2L(304-305)/A) of the distal dileucine motif in helix VIII of BLT1, and differentiated into a neutrophil-like phenotype. The degranulation of these cells was measured in the presence and absence of factors that would affect the signaling pathway. The results show that Degranulation responses to LTB[subscript 4] were similar for differentiated PLB-BLT1 and neutrophils. However, the degranulation response of cells bearing the dileucine mutation in helix VIII of BLT1 was significantly reduced in response to LTB[subscript 4]. Pretreatment of differentiated PLB-BLT1 cells and neutrophils with Y-27632, a pharmacological inhibitor of p160-ROCK, the down-stream effector of the small GTPase RhoA, abrogated their degranulation in response to LTB[subscript 4]. The degranulation defect observed with the dileucine mutation was corrected by transient transfection of the cells bearing the mutation with a constitutively active form of RhoA. Taken together, our results suggest an essential role for the distal dileucine motif in helix VIII of BLT1 involving RhoA which allows normal neutrophil degranulation in response to LTB[subscript 4].
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Brack, Eva [Verfasser], and Matthias [Akademischer Betreuer] Griese. "Einfluss von Surfactantprotein A und D auf die ECP-Degranulation von humanen eosinophilen Granulozyten / Eva Brack. Betreuer: Matthias Griese." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2013. http://d-nb.info/104214737X/34.
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Tahmasebi, Ava, and Jennifer Skytén. "An in vitro Study of Drug-induced Degranulation of Human HMC-1 Mast Cells and Rat RBL-2H3 Cells." Thesis, Umeå universitet, Tandläkarutbildning, 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-97850.
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Mast cells and basophils participate in biological responses such as inflammation and allergic reactions and contain potent mediators such as histamine and cytokines. Some drugs, for example opioids, are known to trigger mast cell degranulation with the release of immunogenic mediators that may cause severe side effects. The aim of this study was to investigate if the human mast cell line HMC-1 is a useful model to study codeine-induced degranulation in vitro. The rat basophilic leukemia RBL-2H3 cell line was used as a reference. Time- and concentration-dependent effects of codeine, compound 48/80, and A23187 on the degranulation of HMC-1 cells and RBL-2H3 cells were investigated by measuring extracellular levels of β-hexosaminidase and [3H]serotonin. Neither codeine nor compound 48/80 produced any significant basophil or mast cell degranulation, whereas the calcium ionophore A23187 triggered a degranulation of both cell lines. In conclusion, none of the cell lines are useful as relevant, robust and reproducible in vitro models to study drug-induced mast cell degranulation.
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Spindler, Markus [Verfasser], and Markus [Gutachter] Bender. "The role of the adhesion and degranulation promoting adapter protein (ADAP) in platelet production / Markus Spindler ; Gutachter: Markus Bender." Würzburg : Universität Würzburg, 2020. http://d-nb.info/1221963236/34.
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Geng, Dianliang. "Preparation of Divalent and Trivalent Antigens for Immunogical Studies on Degranulation of Mast Cells and Preparation of Ceragenins for Antiviral Studies Against Vaccinia Virus." BYU ScholarsArchive, 2007. https://scholarsarchive.byu.edu/etd/1589.
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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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Karhu, T. (Toni). "Isolation of novel ligands for MAS-related G protein-coupled receptors X1 and X2, and their effect on mast cell degranulation." Doctoral thesis, Oulun yliopisto, 2017. http://urn.fi/urn:isbn:9789526216331.
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Abstract The mast cells are an integral part of the human immune system. They are important modulators of inflammatory and physiological processes. Mast cells exert their functions through degranulation and release of inflammatory mediators, such as histamine, proteases and cytokines. There are two main pathways leading to the mast cell activation, the immunoglobulin-dependent and the immunoglobulin-independent pathway. The latter pathway can be triggered by several non-immunological stimuli, and two novel receptors responsible for the activation have been identified, the MAS-related G protein-coupled receptor X1 (MRGPRX1) and X2. The MRGPRX1 and MRGPRX2 have two established functions: i) they trigger the degranulation of mast cells and ii) they are involved in pain perception and itch on a specific subset of sensory neurons. These receptors are not expressed in all of the populations of mast cells, only in the tryptase and chymase containing mast cells, contributing to the mast cell heterogeneity. Unlike most G protein-coupled receptors, the MRGPRX1 and MRGPRX2 are quite non-selective, binding an ever growing list of different ligands. Their ligands include endogenous neuropeptides, host defense peptides and protein fragments, as well as synthetic compounds such as different antibiotics. Their endogenous ligands could be a triggering signal in some mast cell-related diseases by degranulating mast cells and thereby inducing inflammation. Due to the non-selectivity of MRGPRX1 and MRGPRX2, they probably still have many hitherto unknown ligands. The aim of this study was to isolate novel endogenous ligands for the MRGPRX1 and MRGPRX2 from human tissues with the “reverse pharmacology approach” and to determine their potential to degranulate mast cells. The starting materials for the isolation, human platelets and plasma, contained MRGPRX1 and MRGPRX2 activating compounds. From the human plasma, three fragments of albumin able to activate the MRGPRX2 were isolated and sequenced. These fragments were dose-dependently activating the MRGPRX2 and degranulating mast cells. Two MRGPRX1 activating hemoglobin β-chain fragments were isolated from human platelets. These fragments were dose-dependently activating the MRGPRX1, but had no effect on mast cell degranulation<br>Tiivistelmä Syöttösolut on tärkeä osa ihmisen immuunijärjestelmää. Ne ovat tärkeitä tulehdus- ja fysiologistenprosessien säätelijöitä. Syöttösolujen vaikutus välittyy degranulaation ja siinä vapautuvien tulehdusvälittäjäaineiden kautta. Vapautuviin aineisiin lukeutuu esim. histamiini ja lukuisia sytokiinejä, sekä proteaaseja. Syöttösolujen aktivaatio voi tapahtua immunoglobuliineista riippuvaa tai immunoglobuliineista riippumatonta reittiä pitkin. Monet ei-immunologiset tekijät voivat laukaista jälkimmäisen reitin ja kaksi uutta tähän vaikuttavaa G-proteiinikytkentäistä reseptoria on löydetty, MAS-related G protein-coupled receptor X1 (MRGPRX1) ja X2. MRGPRX1:llä ja MRGPRX2:lla on kaksi tunnettua tehtävää: i) ne laukaisevat syöttösolujen degranulaation ja ii) ne osallistuvat kivun ja kutinan aistimiseen tietyissä tuntohermoissa. Näitä reseptoreita ei ilmennetä kaikissa syöttösoluissa, vaan ainoastaa tryptaasia ja kymaasia sisältävissä syöttösoluissa, ja täten osaltaan selittävät syöttösolujen monimuotoisuutta. Useimmista G-proteiinikytkentäisistä reseptoreista poiketen MRGPRX1 ja MRGPRX2 ovat laajakirjoisia, sitoen monia erilaisia ligandeja. Ligandeihin kuuluu endogeenisia neuropeptidejä, antimikrobiaalisia peptidejä ja proteiinin fragmentteja, sekä synteettisiä yhdisteitä kuten erilaisia antibiootteja. Reseptoreiden endogeeniset ligandit voivat toimia laukaisijana jossain syöttösoluihin liittyvissä sairauksissa, degranuloidessaan syöttösoluja ja aiheuttaen paikallisen tulehdustilan. Reseptoreiden laajakirjoisuudesta johtuen niillä on oletettavasti monia vielä tuntemattomia ligandeja. Tämän tutkimuksen tarkoitus oli eristää uusia endogeenisiä ligandeja MRGPRX1:lle ja MRGPRX2:lle ihmisen kudoksista ”kääteisfarmakologista lähestymistapaa” hyödyntäen ja selvittää ligandien kyky syöttösolujen degranulaatioon. Lähtömateriaalina käytetyt ihmisen verihiutaleet ja plasma sisälsivät MRGPRX1:ta ja MRGPRX2:ta aktivoivia yhdisteitä. Plasmasta eristettiin ja sekvensoitiin kolme albumiinin fragmenttia, jotka aktivoivat MRGPRX2:ta. Nämä fragmentit aktivoivat MRGPRX2:ta ja degranuloivat syöttösoluja annosriippuvaisesti. Kaksi MRGPRX1:tä aktivoivaa hemoglobiinin β-ketjun fragmenttia eristettiin ihmisen verihiutaleista. Nämä fragmentit tunnistettiin hemorfiineiksi ja ne aktivoivat MRGPRX1:tä annosriippuvaisesti, mutta eivät vaikuttaneet syöttösolujen degranulaatioon
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Santos, Marcela de Souza. "Estudo da sinalização de mastócitos mediada por IgE: desenvolvimento de inibidores e efeito de níveis reduzidos de fosfatidilinositol 4,5-bifosfato." Universidade de São Paulo, 2012. http://www.teses.usp.br/teses/disponiveis/60/60138/tde-04072012-162611/.
Full textAbstract:
As doenças alérgicas alcançaram proporções mundialmente epidêmicas. A ativação de receptores para IgE, Fc RI, em mastócitos, é o mecanismo chave para a iniciação e propagação das respostas patofisiológicas dos processos alérgicos. Após a interação destas células com um alérgeno, há a ativação de uma cascata de eventos de sinalização, a qual resulta na secreção de mediadores alérgicos pré-formados, através de um processo regulado de exocitose, além da síntese e secreção de mediadores lipídicos e citocinas. Desta forma, a inibição da responsividade dos mastócitos, quando ativados por um alérgeno, representa uma via importante para o desenvolvimento de novos candidatos a fármacos com indicação antialérgica. Neste sentido, este trabalho buscou, num primeiro momento, contribuir com a compreensão dos papéis desempenhados por um glicerofosfolipídeo de membrana, fosfatidilinositol 4,5-bifosfato (PtdIns(4,5)P2), em eventos de sinalização em mastócitos, mediados por IgE. Este trabalho permitiu destacar a importância de PtdIns(4,5)P2 como um regulador chave das respostas de Ca2+ e das alterações de morfologia de mastócitos estimulados por um alérgeno. Foi observado ainda que níveis reduzidos de PtdIns(4,5)P2 determinaram a inibição do processo de endocitose de receptores Fc RI ativados, um evento crucial para a redução da transdução de sinais. Estes resultados não somente trazem um ganho de conhecimento acerca dos detalhes que orquestram os eventos de sinalização em mastócitos estimulados por um alérgeno, como também apontam que a regulação dos níveis de PtdIns(4,5)P2 pode certamente ser apontada como alvo para o desenvolvimento de novas moléculas inibidoras da ativação mastocitária. A segunda etapa deste trabalho teve como objetivo avaliar o potencial inibitório de alguns compostos de origem natural e sintética sobre a degranulação de mastócitos, evento em que mediadores alérgicos, como a histamina, são secretados, em resposta ao estímulo celular. Inicialmente, avaliou-se o efeito inibitório de um conjunto de arilcumarinas sintéticas, estruturalmente relacionadas, sobre a degranulação. Um número significativo de moléculas foram ativas e, dentre elas, algumas substituições junto à estrutura do anel 3-fenilcumarínico, como as hidroxilações das posições 6, 2\'e 5\', puderam ser identificadas como importantes para o potencial bioativo. Finalmente, o trabalho apresentou uma molécula de origem natural, como um potente inibidor da degranulação de mastócitos e da secreção de citocinas. Trata-se da piridovericina, um metabólito secundário, isolado do fungo entomopatogênico Beauveria bassiana. Dessa forma, tanto as cumarinas, como a piridovericina podem ser apontadas como compostos de partida de grande potencial para o desenvolvimento de novos fármacos anti-alérgicos.<br>Allergic diseases have approached epidemic proportions worldwide. The activation of IgE receptors, Fc RI, from mast cells, is the key event for the initiation and propagation of pathophysiological responses involved in the allergic processes. The interaction between mast cells and allergens triggers a signaling cascade, which results in secretion of pre-formed allergic mediators, through regulated exocytosis, in addition to the synthesis and secretion of lipid mediators and cytokines. In this way, the inhibition of mast cell responsiveness, upon allergen stimulation, represents an important pathway for the development of new antiallergic drug candidates. Thus, the present work tried, firstly, to gain better insight of the roles played by phosphatidylinositol 4,5-biphosphate (PtdIns(4,5)P2) during IgE-mediated mast cell signaling. This work highlighted the importance of PtdIns(4,5)P2 as a key regulator of Ca2+ responses and mast cell morphological alteration, when activated by an allergen. It was observed that reduced levels of PtdIns(4,5)P2 determined the inhibition of activated Fc RI endocytosis, a crucial event for signal transduction termination. Those results not only improve the actual knowledge in mast cell signaling but also point out the regulation of PtsIns(4,5)P2 levels as a target to be pursued during the development of new inhibitors of mast cell activation. The second part of this work aimed to evaluate the capacity of both synthetic and natural compounds to inhibit mast cell degranulation, characterized by the release of granule-contained allergic mediators, such as histamine, upon cell stimulation. Initially, the inhibitory effect of a set of structurally related synthetic arylcoumarins was evaluated. A significant number of molecules were active, and a few substitutions within such molecules could be pointed as important for the biological activity, such as the hydroxylation of carbons 6, 2\' e 5\' of the 3-phenylcoumarin ring. Lastly, this work presents a natural compound as a potent inhibitor of mast cell degranulation and cytokine secretion. The refered compound is pyridovericin, a secondary metabolite isolated from the entomophatogenic fungus Beauveria bassiana. Therefore, both the coumarin derivatives and pyridovericin can be regarded as lead compounds for the development of new anti-allergic drugs.
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Oliveira, Mariana Bellini. "Avaliação do potencial antialérgico de flavonóides: estudo sinergístico e influência de sistemas lipossomais." Universidade de São Paulo, 2013. http://www.teses.usp.br/teses/disponiveis/60/60138/tde-06092013-094238/.
Full textAbstract:
Os problemas decorrentes de doenças alérgicas é tema em destaque atualmente devido ao aumento de pessoas que vêm apresentando sintomas e sofrendo as consequências de mudanças aceleradas nos costumes da nossa sociedade. Apesar das diversas terapias oferecidas, pacientes alérgicos ainda sofrem com efeitos colaterais decorrentes do uso de medicamentos anti-alérgicos. Assim, a busca por novas alternativas que possam amenizar os sintomas alérgicos torna-se relevante. As substâncias naturais tem sido alvo de intensa investigação devido às propriedades farmacológicas no tratamento de diversas doenças incluindo as alérgicas. Neste contexto, este trabalho foi focado no extrato Vimang e seu componente majoritário, a mangiferina (Mgf), que tem apresentado atividades biológicas diversas. Sabendo-se que a atividade biológica de um extrato pode ser potencializada pelo sinergismo entre seus componentes, foi avaliado o efeito sinérgico de substâncias isoladas do Vimang, tais como mangiferina, quercetina, catequina, ácido gálico e benzóico. Esses componentes foram combinados e as misturas avaliadas quanto à capacidade em inibir a desgranulação mastocitária. O Vimang, a quercetina e mangiferina inibem a desgranulação mastócitária enquanto catequina, ácido gálico e benzóico não são ativos; e porisso, não foram incluídos na determinação do isobolograma de aditividade, adotado no estudo de sinergismo. O isobolograma de aditividade mostra sinergismo para as concentrações de mangiferina iguais a 5, 10, 20 e 40 ?M e quercetina iguais a 0,2, 1 e 1,6 ?M; e mostra antagonismo para mangiferina 80 ?M e quercetina 0,4 e 0,8 ?M. Tais resultados sugerem que altas concentrações de mangiferina atrapalham a interação sinérgica entre estas substâncias e altas concentrações de quercetina exercem um efeito contrário, ou seja, favorecem o sinergismo entre quercetina e mangiferina. A baixa solubilidade de flavonóides em meio fisiológico levou ao estudo da interação da Mgf com lipossomos de Dimiristoil-L-?-Fosfatidilcolina (DMPC) para aplicação nos ensaios biológicos. A Interação da Mgf com lipossomos de DMPC, bem como a estabilidade lipossomal, foram avaliadas quanto aos efeitos de pH, força iônica e presença de colesterol. O pH influencia a eficiência de incorporação (EI) da Mgf que é maior em pHs ácidos. Em soluções de pH ácido ou fisiológico há aumento de tamanho do lipossomo na presença de Mgf, o que reforça a hipótese de interação desta com a bicamada lipídica. O aumento da força iônica, na ausência de Mgf, diminui a estabilidade do lipossomo. Na ausência de sal, a Mgf favorece a formação de agregados e diminui a estabilidade dos lipossomos e na presença de sal, a Mgf previne a agregação e estabiliza os agregados lipossomais. A EI da Mgf nos lipossomos de DMPC preparados pelo método da injeção etanólica, em pH fisiológico, foram ao redor de 13%. A tentativa de otimizar a EI foi realizada pela adição de colesterol em diferentes proporções lipídio:colesterol. Um aumento de 13% para 17% foi observado para a proporção DMPC:colesterol (9:1). A Mgf livre inibe a desgranulação dos mastócitos de forma concentração-dependente; mas a presença de lipossomos, preparados pelo método da injeção etanólica não altera significativamente esse perfil. A EI da mangiferina em lipossomos de DMPC:colesterol (9:1), preparados pelo método do filme lipídico, foi igual a 45%. Os resultados de potencial antialérgico, para este caso, mostraram que os lipossomos de DMPC favorecem o potencial antialérgico da Mgf em concentrações acima de 25 ?M. Esses resultados abrem perspectivas na busca de sistemas lipossomais mais estáveis e que possam ser aplicados em ensaios biológicos in vitro e in vivo.<br>The problems due to allergic diseases are currently a highlighted topic due to the increase of people who are showing symptoms and suffering the consequences of rapid changes in the habits of our society. Despite various therapies offered, allergic patients still suffer side effects from the use of anti-allergic drugs. Thus, the search for new alternatives that can alleviate the allergic symptoms becomes relevant. Natural substances have been the subject of intense research due to the pharmacological properties in the treatment of several diseases, including allergic ones. In this context, this work was focused on Vimang extract and its major component, the mangiferin (Mgf), which has shown several biological activities. Given that the biological activity of an extract can be enhanced by the synergism between its components, the synergistic effect of compounds isolated from Vimang such as mangiferin, quercetin, catechin, gallic acid and benzoic acid was evaluated. These components were combined and the mixtures tested for their ability to inhibit mast cell degranulation. The Vimang, quercetin and mangiferin inhibit mast cell degranulation while catechin, gallic acid and benzoic acid are not active and for that reason they were not included in the determination of the isobologram adopted in the synergism study. The isobologram of additivity shows synergism for the concentration of mangiferin equal to 5, 10, 20 and 40 ?M and quercetin equal 0.2, 1 and 1.6 ?M, and shows antagonism for mangiferin 80 ?M and quercetin 0.4 and 0.8 ?M. The low solubility of flavonoids in physiological medium led to the study of the interaction of Mgf with liposomes of L-?-dimyristoyl-phosphatidylcholine (DMPC) for application in biological assays. The interaction of Mgf with DMPC liposomes as well as the liposomal stability was evaluated considering the effects of pH, ionic strength and cholesterol presence. The pH influences the incorporation efficiency (IE) of mangiferin, which is higher in acidic pHs. In solutions of acidic or physiological pH, the liposome size increases in the presence of Mgf, which reinforces the hypothesis of interaction of Mgf with the lipid bilayer. The increased ionic strength in the absence of Mgf decreases the stability of the liposome. In the absence of salt, Mgf favors the formation of aggregates and decreases the stability of the liposomes. When in presence of salt, Mgf prevents the aggregation and stabilizes the liposomal aggregates. The IE of mangiferin in DMPC prepared by the ethanol injection method, at physiological pH, were around 13%. The attempt to optimize the IE was performed by the addition of cholesterol in different proportions lipid:cholesterol. An increase from 13% to 17% was observed for the proportion of DMPC:cholesterol (9:1). Free mangiferin inhibits mast cells degranulation in a dose-dependent manner, but the presence of liposomes prepared by the ethanol injection method does not significantly change this profile. The IE of mangiferin in liposomes of DMPC:cholesterol (9:1), prepared by the method of lipidic film was equal to 45%.The results of antiallergic potential in this case show that DMPC liposomes favor the mangiferin antiallergic potential at concentrations above 25 mM. These results open perspectives in the search for more stable liposomal systems and can be applied to biological assays in vitro and in vivo.
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Callejon, Daniel Roberto. "Estudo da interação de galectina-1 e mastócitos." Universidade de São Paulo, 2008. http://www.teses.usp.br/teses/disponiveis/60/60135/tde-30072008-104422/.
Full textAbstract:
A galectina-1 (Gal-1) pertence à uma família de proteínas ligantes de ?-galactosídeos e participa de vários processos biológicos, tais como a modulação da resposta inflamatória. Os mastócitos desempenham um importante papel em eventos inflamatórios e alérgicos. Entretanto, o impacto da Gal-1 na biologia dos mastócitos é pouco conhecido. Neste trabalho, foram analisados os aspectos morfológicos e funcionais de células RBL-2H3 tratadas com Gal-1. Além disso, foi investigado o efeito da ausência de Gal-1 endógena sobre a desgranulação in vivo. A avaliação da interação de Gal-1Texas-Red com células RBL-2H3, por citometria de fluxo, indicou que esta lectina ligou-se às superfícies dessas células de modo dose-dependente. A Gal-1 foi capaz de promover a exposição de fosfatidilserina (FS, um marcador de apoptose) nas superfícies dessas células por meio de reconhecimento de carboidratos. Entretanto, as células RBL-2H3 tratadas com Gal-1 não apresentaram apoptose e/ou necrose, como indicado pelos resultados obtidos dos ensaios de TUNEL, DNA laddering, hipodiploidia, citotoxicidade e microscopia eletrônica de transmissão. As análises de microscopia eletrônica de varredura e Differential Interference Contrast (DIC) de células tratadas com Gal-1 (10 µM-1 hora) indicaram que essa lectina induziu ondulações apenas nas porções apicais das membranas plasmáticas de células RBL-2H3. Além disso, a Gal-1 modulou negativamente a formação de ondulações nas superfícies de células RBL-2H3 estimuladas via Fc?RI. A distribuição de componentes de Lipid Rafts relacionados ao processo de ativação celular via Fc?RI (Lyn, LAT e GD1b) foi modificada pelo tratamento dessas células com Gal-1 (10 µM), por 1 hora. As células RBL-2H3 tratadas com Gal-1(10µM-45 minutos) apresentaram níveis de liberação da enzima ?-hexosaminidase (?-HEX) semelhantes ao do controle negativo, sugerindo que essa lectina não promove a desgranulação de células RBL-2H3. Por outro lado, a Gal-1 inibiu a desgranulação de células RBL-2H3 ativadas via Fc?RI. O valor máximo de inibição (80%) da liberação de ?-HEX foi atingido quando as células foram tratadas com 10 µM de Gal-1 por 24 horas. Este efeito inibitório não foi detectado quando as células foram tratadas com Gal-1 na presença de ?-D-Tiogalactopiranosídeo (TDG), quando estimuladas com ionóforo de cálcio ou tratadas com a forma monomérica da Gal-1. Os dados de microscopia confocal mostraram que os grânulos secretórios de células submetidas ao procedimento de estimulação via Fc?RI, foram fracamente marcados com o anticorpo AD1 e apresentaram uma distribuição citoplasmática difusa. Entretanto, células tratadas com Gal-1 (10 µM - 24 horas) e estimuladas via Fc?RI foram intensamente marcadas com anticorpo AD1 e mostraram um padrão perinuclear, como detectado em células não estimuladas. De modo interessante, camundongos deficientes para o gene de Gal-1 quando submetidos ao ensaio de anafilaxia passiva cutânea (PCA) apresentaram uma reação significativamente maior que os animais selvagens. Com base no conjunto de resultados obtidos sugere-se que a Gal-1 pode participar da homeostase de mastóctios sem provocar apoptose e/ou necrose dessas células. Além disso, a Gal-1 pode modular o processo de exocitose de mastócitos por meio das propriedades lectínica e de dimerização dessa proteína e esse efeito modulatório parece estar associado a eventos de sinalização celular anteriores ao influxo de cálcio.<br>Galectin-1 (Gal-1) belongs to a family of ?-galactoside-binding proteins and is involved in several biological processes, including modulation of the inflammatory response. Mast cells play a critical role in allergic and inflammatory events, however, little is known about the impact of Gal-1 on mast cell biology. In this study, we examined the role of Gal-1 in mast cell function using RBL-2H3 (Rat Basophilic Leukemia) and galectin-1 deficient mice. We report that Gal-1 recognized glycoconjugates on mast cells and this interaction promotes phosphatidylserine (PS) exposure in the absence of cell death or apoptosis. Morphological analysis of Gal-1-treated RBL-2H3 cells, by scanning electron microscopy and Differential Interference Contrast (DIC), indicated that Gal-1 induces modifications on cell membranes and modulation of ruffles formation on cell surface of stimulated RBL-2H3 cells. Interesting, Gal-1 treatment of RBL-2H3 cells, with or without stimulation, promotes alterations in the distribution of the components (Lyn, LAT and GD1b) of Lipid Rafts. Gal-1 did not promote degranulation on RBL-2H3 with or without prior sensitization with IgE. However, Gal-1 treatment inhibits the cell degranulation mediated by via Fc?RI and this effect was time and dose-dependent. Also, this inhibition was related to carbohydrate recognition domain and required Gal-1 dimerization. Importantly, confocal microscopy analysis showed that Gal-1 was distributed in cytoplasm close to secretory granules stained AD-1 antibody on RBL-2H3 with or without prior stimulation with Fc?RI. In addition, we found significantly increased passive cutaneous anaphylaxis reaction in Gal-1 deficient mice. The results demonstrated that Gal-1 may participate of homeostasis and exocytose in mast cells suggesting that Gal-1 can have important role in allergic and inflammatory process.
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Geng, Dianliang. "Preparation of divalent and trivalent antigens for immunological studies on degranulation of mast cells and preparation of ceragenins for antiviral activity studies against vaccinia virus /." Diss., CLICK HERE for online access, 2008. http://contentdm.lib.byu.edu/ETD/image/etd2257.pdf.
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Côte, Marjorie. "Caractérisation moléculaire et fonctionnelle de la syntaxine 11 et de son partenaire MUNC18-2, impliqués dans la lymphohistiocytose familiale." Paris 5, 2011. http://www.theses.fr/2011PA05T059.
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La Lymphohistiocytose Familiale (LHF) est une pathologie immunitaire de transmission autosomique récessive. Elle est caractérisée par une activation lymphocytaire et macrophagique exagérée, ces cellules infiltrant les différents organes du patient et les détruisant en produisant de grandes quantités de cytokines inflammatoires. Chez les patients atteints de LHF, des défauts en perforine (LHF2), Munc13-4 (LHF3) et syntaxine 11 (LHF4) ont été précédemment décrits. Ces gènes codent pour des protéines impliquées dans lʼactivité cytotoxique des lymphocytes T et des cellules natural killer (NK). Mon travail de thèse permet de montrer que le gène codant pour la protéine Munc18-2 (ou syntaxin-binding protein 2, STXBP2) est muté chez un certain nombre de patients présentant une LHF, permettant la caractérisation dʼune nouvelle forme génétique dénommée LHF de type 5 (FHL5). Les lymphoblastes de ces patients ont une expression fortement diminuée ou indétectable de la protéine Munc18-2. Un défaut dʼexocytose des granules cytotoxiques est mis en évidence dans les cellules NK de patients, après que les granules ont polarisé au niveau de la synapse immunologique. Ce défaut est complémenté par lʼexpression ectopique de Munc18-2. Nous montrons également que la syntaxine 11 est le principal partenaire de Munc18-2 dans les lymphocytes, son expression stable nécessitant la présence de Munc18-2. Certains patients présentant une LHF5 et associant à leur désordre immunitaire une pathologie intestinale, nous ont fait rechercher un rôle de Munc18-2 au niveau des les cellules épithéliales de lʼintestin. Munc18-2 est localisé à la membrane apicale des cellules épithéliales intestinales et il pourrait ainsi réguler la fusion de compartiments intracellulaires avec la membrane plasmique. Pour approcher le rôle de Munc18-2 dans la polarisation et le trafic intracellulaire des cellules épithéliales de lʼintestin, nous avons utilisé la lignée Caco2 dans laquelle nous avons éteint lʼexpression de Munc18-2. Cependant, nos résultats ne permettent pas à lʼheure actuelle de définir un rôle précis de Munc18-2 au niveau de lʼintestin à lʼaide de cette lignée de cellules épithéliales, probablement du fait dʼune expression résiduelle de la protéine. Notre travail permet donc dʼidentifier un défaut en Munc18-2 à lʼorigine de la LHF5 et un rôle essentiel de cette protéine à une étape tardive de la voie de sécrétion des granules cytotoxiques en sʼassociant à la syntaxine 11, elle-même responsable dʼune forme génétique de LHF. Outre lʼintérêt fondamental que représente la compréhension des mécanismes dʼexocytose, notre travail participe à une meilleure compréhension de la physiopathologie de la lymphohistiocytose familiale et de sa composante intestinale lorsquʼelle résulte dʼun défaut en Munc18-2<br>Familial hemophagocytic lymphohistiocytosis (FHL) is a genetically heterogeneous autosomal recessive immune disorder characterized by the occurrence of uncontrolled activation of lymphocytes and macrophages infiltrating multiple organs. Disease-causing mutations in the perforine (FHL2), Munc13-4 (FHL3) and syntaxin 11 (FHL4) genes have been identified in individuals with FHL. This study shows that the gene encoding for the syntaxin-binding proteins 2 (STXBP2, also known as Munc18-2) is mutated in a subset of patients with FHL, designated as FHL5. Lymphobasts isolated from these patients have a severe decreased in Munc18-2 expression. NK cells exhibited impaired cytotoxic granules exocytosis although perforin-containing granules were able to polarize toward the immunological synapse. This defect could be overcome by ectopic expression of wild type Munc18-2. Furthermore, this work provide evidence that syntaxin 11 is the main partner of Munc18-2 in lymphocytes, as its expression required the presence of Munc18-2. These data indicate that Munc18-2 is required at a late step of the secretory pathway for the release of cytotoxic granules by binding to syntaxin 11, which deficiency also causes FHL. Moreover, some FHL5 patients associate to their immune disturbance an intestinal disorder. This additional phenotype strongly suggests that Munc18-2 has a non-redundant function in gut epithelial cells. Munc18-2 is localized to the apical membrane of intestinal epithelial cells and seems to regulate the fusion of intracellular compartments to the plasma membrane. The function of Munc18-2 in the polarization and intracellular trafficking of epithelial cells was thus investigated using Caco2 cell line in which Munc18-2 gene was silenced. So far, no precise role of Munc18-2 in intestinal cell can be evidenced. Beside the fundamental interest of this study in understanding the molecular mechanisms regulating the exocytic machinery from different cells, our work contributes to a better understanding of the pathophysiology of the LHF and its intestinal expression when resulting from Munc18-2 deficiency
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Krajewski, Anna Christina. "Die Regulation der Synthese und Freisetzung von FGF-2 aus humanen dermalen Mastzellen." Doctoral thesis, Humboldt-Universität zu Berlin, Medizinische Fakultät - Universitätsklinikum Charité, 2006. http://dx.doi.org/10.18452/15404.
Full textAbstract:
Die Synthese und -Freisetzung von FGF-2 aus humanen dermalen Mastzellen Fibroblast growth factor-2 (FGF-2) ist ein Mitglied einer großen Familie von Wachstumsfaktoren. FGF-2 fördert das Wachstum und die Entwicklung von Blutgefäßen (Angiogenese) und nimmt somit Einfluss auf Wundheilungsprozesse, Gewebeentwicklung, als auch verschiedene pathologische Vorgänge im Organismus. Mastzellen wurden lange Zeit allein als Effektorzellen der Typ I Allergiereaktion betrachtet. Mittlerweile betrachten man sie auch als wichtige Zellen für die Gewebe Homöostase und Wundheilung. In der vorliegenden Arbeit wurden MC aus humenen dermalen Gewebe isoliert, um deren FGF-2 Synthese und –Freisetzung zu untersuchen. Die Zellen wurden mit verschiedenen proinflammatorischen Mediatoren stimuliert. FGF-2 wurde mit ELISA und PCR Methoden bestimmt. Durch Stimulationen mit a–IgE, SP, IL–4, IL–6 und IL–8 wurde eine gesteigerte FGF–2–Synthese induziert. Weiterhin zeigten die vorliegenden Ergebnisse, dass bei der Degranulation der MC FGF-2 freigesetzt wird, auch wenn der zugrunde liegende Mechanismus für die Freisetzung weiterhin unklar bleibt. UVA1–und PUVA1–Bestrahlung hatten einen inhibieren Effekt auf die Sekretion des Proteins.<br>Synthesis and release of FGF-2 from human dermal mast cells Fibroblast growth factor-2 (FGF-2) is a member of a large family of proteins. FGF-2 stimulates the growth and development of new blood vessels (angiogenesis) that contribute to the pathogenesis of several diseases (i.e. atherosclerosis), normal wound healing and tissue development. Mast cells are traditionally viewed as effector cells of immediate type hypersensitivity reactions. There is, however, a growing body of evidence that the cells might play an important role in the maintenance of tissue homeostasis and repair. In this present investigation we isolated MC from human tissue to investigate their FGF-2 synthesis and release after stimulation with different proinflammatory mediators. To detect FGF-2 we used ELISA and PCR technique. We could show the up-regulation of FGF-2 synthesis after stimulation with a-IgE, SP, IL-4, IL-6 and IL-8. Within the degranulation of MC there was a release of FGF-2 even though the mode of release still remains unclear. Through UV-light radiation we could show a downregulation of FGF-2 release.
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Fuzissaki, Carolina Nakau. "Estudo da modulação de funções efetoras de neutrófilos humanos por derivados cumarínicos: avaliação do efeito biológico sobre a produção de espécies reativas de oxigênio e a desgranulação." Universidade de São Paulo, 2009. http://www.teses.usp.br/teses/disponiveis/60/60138/tde-04012010-213823/.
Full textAbstract:
Os neutrófilos são células fagocíticas do sistema imune inato com mecanismos especializados de digestão de patógenos, complexos imune e detritos celulares, que são mediados principalmente por espécies reativas de oxigênio (EROs) e enzimas proteolíticas. Entretanto, a ativação maciça de neutrófilos leva a uma liberação exacerbada de enzimas e EROs para o meio extracelular, o que pode ultrapassar a capacidade de defesa tecidual, composta por antioxidantes e antiproteinases, e lesar o tecido, bem como amplificar o processo inflamatório observado em algumas doenças inflamatórias, autoimunes e infecciosas. O envolvimento de neutrófilos na fisiopatologia de tais doenças tem atraído o interesse na pesquisa de novas substâncias com propriedades antioxidantes e imunomodulatórias. Neste trabalho, foi avaliado o efeito modulatório de onze derivados de cumarinas hidroxiladas e acetoxiladas sobre duas funções efetoras de neutrófilos humanos (produção de EROs e desgranulação), bem como a citotoxicidade dessas substâncias. Além disso, a relação estrutura-atividade foi analisada. Para tais investigações, o sangue venoso foi coletado de voluntários saudáveis e os neutrófilos foram isolados pelo método da gelatina. O metabolismo oxidativo dos neutrófilos foi desencadeado por zimosan opsonizado com soro humano normal (ZIops) ou forbol-12-miristato-13-acetato (PMA), e a resposta celular foi avaliada pelos ensaios de quimioluminescência dependente de lucigenina (QLluc) ou de luminol (QLlum). Para a realização desses ensaios, foram padronizadas as seguintes condições experimentais: concentração das sondas luminol e lucigenina; concentração do solvente dimetilsulfóxido; tempo de leitura da QLuc e QLlum. Posteriormente, a atividade antioxidante das cumarinas frente ao radical 2,2-difenil-1-picrilhidrazil (DPPH) foi avaliada espectrofotometricamente em 510 nm. Além disso, foi avaliado o efeito das cumarinas na desgranulação dos neutrófilos induzida por n-formil-metionil-leucil-fenilalanina (fMLP), utilizando-se a enzima elastase como marcador, bem como o efeito dessas substâncias na atividade da elastase liberada pelos neutrófilos. Ambos os ensaios foram realizados através da quantificação de p-nitroanilina liberada após a quebra de um substrato específico para essa enzima, em 405 nm. A toxicidade das cumarinas sobre os neutrófilos foi avaliada pela exclusão do azul de tripan e pela medida da liberação de lactato desidrogenase. Observou-se que, tanto para as células estimuladas por ZIops quanto por PMA: (i) a maioria das cumarinas inibiu a QLluc e a QLlum de maneira dependente da concentração, sendo que as mais ativas (C, D) possuíam grupos orto-diidroxi; (ii) quatro cumarinas (A, B, F, G) inibiram a QLluc, mas aumentaram a QLlum; (iii) a cumarina não-substituída (K) não teve efeito modulatório significativo sobre a QLlum ou QLluc; (iv) ordem de efeito inibitório das demais substâncias (E, H, I, J) foi dependente do número e posição dos grupos substituintes, bem como do tipo de sonda quimioluminescente (luminol ou lucigenina) e do estímulo utilizado. Verificou-se também que três cumarinas (C, D, H) tiveram atividade antioxidante significante frente ao radical livre DPPH. Além disso, quatro das substâncias avaliadas (A, B, E, G) inibiram a desgranulação dos neutrófilos, mas nenhuma delas (A - K) interferiu na atividade da elastase. A análise do conjunto de resultados obtidos sugere que o número e posição dos grupos hidroxil e acetil no esqueleto cumarínico foram importantes para a modulação do metabolismo oxidativo de neutrófilos humanos, sendo que, dependendo do tipo de EROs medida, tais características estruturais podem levar a um efeito anti- ou pró-oxidante. Além disso, o efeito modulatório das cumarinas sobre as funções efetoras dos neutrófilos foi dependente o tipo de estímulo utilizado, e não foi mediado pela toxicidade dessas substâncias, nas condições ensaiadas. Sendo assim, os resultados obtidos podem auxiliar no entendimento dos requisitos estruturais das cumarinas necessários para uma modulação eficiente das funções efetoras dos neutrófilos envolvidas em doenças inflamatórias.<br>Neutrophils are phagocytic cells from the innate immune system with highly developed mechanisms for intracellular digestion of pathogens, immune complexes and cell debris, which are mainly mediated by reactive oxygen species (EROs) and proteolytic enzymes. However, massive neutrophil activation lead to release of large amounts of enzymes and EROs to the extracellular milieu, that may overpower the tissue defense systems, composed of antioxidants and antiproteinases, and damage the tissue, as well as contribute to the amplification of the inflammatory process found in some inflammatory, autoimmune and infectious diseases. The involvement of neutrophils in the physiopathology of such diseases has attracted the interest in the search of new compounds with antioxidant and immunomodulatory properties. In this work, we evaluated the modulatory effect of eleven hydroxylated and acetoxylated coumarin derivatives in two effector functions of human neutrophils (EROs production and degranulation) as well as the cytotoxic effects of these compounds. In addition, the structure-activity relationship was analyzed. Venous blood was collected from healthy volunteers and neutrophils were isolated by the gelatin method to perform our experiments. The neutrophil oxidative metabolism was triggered by normal human serum-opsonized zymosan (ZIops) or phorbol-12-myristate-13-acetate (PMA), and the cellular response was evaluated by the lucigenin (QLluc)- or luminol (QLlum)-amplified chemiluminescence assays. In order to conduct this study, the following experimental conditions had to be established: concentration of the chemiluminescence probes luminol and lucigenin; concentration of the solvent dimethylsulfoxide; the QLluc and QLlum reaction time. Afterwards, the antioxidant activity against 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) was evaluated spectrophotometrically at 510 nm. Moreover, the effect of coumarins in the n-formyl-metionyl-leucyl-phenylalanine (fMLP)-induced neutrophil degranulation was evaluated by using elastase as marker, and the effect of these compounds in the elastase activity were also evaluated. Both assays were performed by measuring the elastase-mediated p-nitroaniline release from a specific substrate (405 nm). Toxicity of coumarins to the neutrophils was evaluated by trypan blue exclusion and measurement of lactate dehydrogenase release. Considering both, ZIops and PMA-stimulated neutrophils, we observed that: (i) most of coumarins inhibited the QLluc and the QLlum in a concentration-dependent manner, being the orto-dihydroxylated (C, D) the most active ones; (ii) four coumarins (A, B, F, G) inhibited the QLluc but increased the QLlum; (iii) the unsubstituted coumarin (K) had no significant modulatory effect on QLlum or QLluc; (iv) the rank order of inhibitory effect among the other compounds (E, H, I, J) was dependent on the number and position of substituents, as well as the type of chemiluminescent probe (luminol or lucigenin) and stimulus used. In addition, we observed that three coumarins (C, D, H) had a significant antioxidant activity against DPPH, and four compounds (A, B, E, G) inhibited the neutrophil degranulation, but none of the tested coumarins (A - K) interfered in the elastase activity. Taken together, our results suggest that the number and position of the hydroxyl and acetyl groups in the coumarin moiety were important to modulate the human neutrophil oxidative metabolism. Depending on the type of EROs measured, such structural features can lead to an anti- or pro-oxidant effect. Furthermore, the modulatory effect of coumarins on the neutrophil effector functions was dependent on the type of stimulus used, but it was not mediated by toxicity of these compounds to the neutrophils, under the assessed conditions. Therefore, the results described herein may be helpful to understand the structural requirements of coumarins to reach an efficient modulation of the neutrophil effector functions involved in inflammatory diseases.
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Joulia, Régis. "Etude de la dynamique de dégranulation des mastocytes et analyse de l'effet des éicosanoïdes dans la coopération entre mastocytes et lymphocytes T Helper." Thesis, Toulouse 3, 2016. http://www.theses.fr/2016TOU30191/document.
Full textAbstract:
Les mastocytes sont des cellules immunitaires présentes dans tous les tissus de l'organisme. Ils ont été depuis longtemps associés aux réponses allergiques, mais ces cellules sont aussi des acteurs majeurs de la réponse inflammatoire. La dégranulation des mastocytes, ou exocytose des granules sécrétoires, est un mécanisme d'action important de ces cellules. Au laboratoire, nous avons mis au point une méthode innovante qui nous permet de suivre en temps réel la dynamique de cette dégranulation. Cette méthode repose sur l'utilisation de l'avidine couplé à un fluorochrome qui se lie spécifiquement à la matrice des granules. Nous avons recherché les modalités de la dégranulation lorsque les mastocytes sont activés par un ligand cellulaire comme des cellules cibles recouvertes d'anticorps. Nous avons pu mettre en évidence, de façon surprenante, que les mastocytes dégranulent de manière polarisée contre une cellule cible opsonisée avec des anticorps de type IgE ou IgG en mettant en place un nouveau mécanisme que nous avons nommé ADDS (Antibody Dependent Degranulatory Synapse). L'ADDS est caractérisée par une signalisation du récepteur RFc et une dépolymérisation du cytosquelette cortical d'actine locales. De plus, cette synapse peut aussi avoir lieu lorsque le mastocyte est au contact d'un parasite opsonisé comme Toxoplasma Gondii, et induit la mort du parasite et la libération de cytokines et chimiokines pro-inflammatoires. Nous avons ensuite analysé la dynamique de la dégranulation à l'échelle " single cell " et sa régulation par des facteurs inflammatoires. Grâce à une approche de " cell barcoding " et de cytométrie en flux en temps réel, nous avons pu mettre en évidence que la dégranulation induite par l'agrégation des RFc?I était contrôlée par deux mécanismes : un premier qui règle le nombre de mastocytes qui dégranulent et un second qui régule l'intensité de la dégranulation. L'interleukine 33 peut finement potentialiser ces deux mécanismes en augmentant le pourcentage de mastocytes qui dégranulent et l'intensité de la dégranulation. L'IL-33 induit ainsi l'émergence de cellules hautement inflammatoires. Dans un second axe de recherche, nous avons étudié quel pouvait être l'impact des prostaglandines dans le dialogue entre mastocytes et lymphocytes T CD4+ Helper (TH). Nos résultats indiquent un rôle insoupçonné de la prostaglandine D2 et la prostaglandine E2 comme des acteurs importants pour la production d'interleukine 17 par les LTH. En conclusion, mon travail de thèse nous a permis de révéler l'existence de la synapse dégranulatoire des mastocytes, de nouveaux mécanismes contrôlant la dégranulation et d'identifier les mastocytes comme une source importante de prostaglandines impliquées dans la polarisation des LTH<br>Mast cells are tissue-resident immune cells particularly enriched in regions exposed to the external environment. They have been associated, for a long time, with allergic disorders but these cells are also major effectors of inflammatory response. The degranulation process, or granule exocytosis, is one of the main effector functions of mast cells and it has been implicated in various biological processes. In our laboratory, we have developed a new method to monitor live mast cell degranulatory response. This approach is based on fluorescent avidin that binds selectively mast cell granule matrix. Thanks to this method, we have investigated the degranulatory response following mast cell activation by cell bound antigens. We have shown that mast cells can undergo polarized degranulation toward IgE- or IgG-opsonized cells in a new mechanism we named ADDS (Antibody Dependent Degranulatory Synapse). The ADDS is characterized by a local signaling of FcR receptors and cortical actin depolymerisation. Moreover, this synapse takes place when mast cells are triggered by opsonized parasite Toxoplasma Gondii. It leads to the death of the parasite and the release of pro-inflammatory cytokines and chemokines. We next analyzed the mast cell degranulatory response dynamics at the single cell level and its regulation by alarmin IL-33. Using cutting-edge "cell barcoding" approach and live flow cytometry, we showed that Fc?RI mediated degranulatory response is controlled by two mechanisms: a first one that sets the frequency of degranulated mast cells and a second one that regulates the magnitude of the degranulation. The IL-33 fine tunes these two mechanisms by augmenting both the frequency of degranulated mast cells and the extent of individual mast cell degranulation. Our results indicate that interleukin 33 induces the emergence of high inflammatory mast cells. In a second axis of research, we analysed the influence of prostaglandins during the cooperation between mast cells and CD4+ T helper cells. Our results indicate that prostaglandin D2 and E2 produced by mast cells are important inducers of interleukin 17 by CD4+ T helper cells. Taken together, my thesis work revealed that mast cells can form degranulatory synapses, new mechanisms that control the degranulatory response and identified mast cells as a source of pro-IL-17 prostaglandins during their cooperation with CD4+ T helper cells
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Kämpe, Mary. "Eosinophil Inflammation in Allergic Disease : Clinical and experimental studies in allergic asthma and allergic rhinitis." Doctoral thesis, Uppsala universitet, Lungmedicin och allergologi, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-130949.
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Allergic diseases are chronic inflammatory conditions, characterised by eosinophil inflammation systemically and in target organs, where cytotoxic granule proteins are responsible for tissue injury. Allergic rhinitis is known to be a risk factor for the development of asthma, yet not all with rhinitis develop asthma. The overall aim was to investigate the involvement of eosinophils in allergic rhinitis and allergic asthma in vivo and in experimental settings, with a focus on differences between rhinitis and asthma. Birch pollen allergy was used as a model and patients were studied during pollen season and after nasal and bronchial allergen challenge. During pollen season and at baseline, allergic rhinitis and allergic asthma had the same degree of systemic eosinophil inflammation. Despite this, impairment in lung function during season and increased bronchial responsiveness at baseline were more common in the asthmatics. Systemic inflammation was more pronounced after seasonal exposure than after experimental challenge. Allergic rhinitis and allergic asthma had the same degree of eosinophil airway inflammation after bronchial challenge, but only the asthmatics had increased bronchial responsiveness measured as PD20 for birch allergen. Allergen primed eosinophils were investigated in vitro for C3b-induced degranulation after seasonal and experimental challenge. The released amount of eosinophil granule proteins was within the same range for all three allergen challenge models with just minor differences in propensity for degranulation between rhinitics and asthmatics. Signalling through PI3K for degranulation was studied with the specific inhibitor Wortmannin. PI3K signalling for eosinophil degranulation was clearly involved in allergic rhinitis and allergic asthma irrespective of the model for allergen exposure. Asthmatics demonstrated less inhibition of degranulation through PI3K during pollen season, indicating that other pathways contribute to eosinophil degranulation in allergic asthmatics. Conclusion: Allergic rhinitis and allergic asthma present with the same degree of systemic and local eosinophil inflammation. The eosinophils are primed for degranulation equally and follow the same pathway through PI3K for degranulation. Our data indicates that eosinophil inflammation per se is not sufficient for the development of asthma.
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Karawajczyk, Malgorzata. "Mechanisms of granule protein mobiliation in blood eosinophils." Doctoral thesis, Uppsala University, Department of Medical Sciences, 2000. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-502.
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<p>Serum levels of eosinophil granule proteins namely ECP, EPO and EPX, which are stored in the matrix of specific granules, were shown to correlate with the course of disease in disorders involving eosinophils. The concentration of eosinophil proteins in serum is the result of their release <i>in vivo</i> and <i>ex vivo</i> during the sampling procedure. Generally, eosinophils release the content of their specific granules in three ways: exocytosis, piecemeal degranulation (PM) or cytolysis. Which of them is operating in circulating eosinophils has not yet been defined. The aim of this thesis was to study the mechanisms of granule protein release from blood eosinophils in respect of protein subcellular localization and cell ultrastructure.</p><p>In patients with bacterial infections, serum levels of ECP but not EPO increased, while in patients with viral infections both proteins remained within the range of healthy controls. G-CSF is a cytokine involved in the response mechanism to bacterial but not viral infections. Administration of G-CSF to healthy subjects induced an elevation of eosinophil numbers and a preferential increase of serum EPX and ECP in comparison to EPO.</p><p>The model of PM consists of the stepwise transportation of specific granule contents from the granules towards the plasma membrane. We observed that administration of G-CSF to healthy subjects and the allergen exposure of allergic subjects during the pollen season, caused changes in the ultrastructure of eosinophil specific granules such as loosening of the matrix, granule matrix lucency and ragged losses of their core. Similar alterations of morphology had been previously described for eosinophils undergoing PM.</p><p>ECP, EPX and EPO were localized not only in the specific granules but also in extra-granular compartments as shown both by immuno electron microscopy and subcelular fractionations, An extra-granular EPX compartment was present in healthy as well as in allergic and in hypereosinophilic subjects, and there were no significant differences in its size between the groups. The size of the extra-granular compartments of ECP and EPO was increased in allergics during the season, and these compartments were clearly separate from that of EPX. Results of this show the differential mobilization ofgranule proteins in blood stream eosinophils serum and indicates PM as its mechanism.</p>
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Ahrens, Frank. "Wirkung, Permeation und Katabolismus von Histamin an isolierten Dickdarmepithelien des Schweins." Doctoral thesis, Universitätsbibliothek Leipzig, 2004. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-37580.
Full textAbstract:
Bei Schweinen lassen sich im Anfangsteil des Dickdarms hohe Konzentrationen an Histamin nachweisen. Zum einen wird viel exogenes Histamin in der Ingesta durch Bakterien gebildet. Zum anderen befindet sich im proximalen Kolon viel endogenes Histamin, welches in verschiedenen Populationen von Mastzellen gespeichert ist. Beide Histaminquellen stellen eine potenzielle Gefahr für die Gesundheit des Tieres dar. Sollte Histamin in den vorhandenen Mengen in die Blutzirkulation übertreten, müsste mit dem Tod des Tieres gerechnet werden. Da unter normalen Bedingungen bei Schweinen keine pathophysiologischen Reaktionen auf die hohen Histaminkonzentrationen im Darm beobachtet werden können, muss auf eine effektive Darmbarriere geschlossen werden. Weil weder diese Barriere bisher untersucht wurde, noch bekannt war, welche Wirkung Histamin in diesem Darmteil des Schweins besitzt, sollte in dieser Arbeit Wirkung, Permeation und Katabolismus von Histamin an isolierten Epithelien des proximalen Kolons mit Hilfe der Ussing-Kammer-Technik untersucht werden. Die Zugabe von Histamin zur serosalen Seite der Epithelien führte zu einem schnellen Anstieg des Kurzschlussstroms. Im Gegensatz zu zahlreichen anderen Untersuchungen an Darmepithelien, in denen eine H1-vermittelte Wirkung von Histamin gefunden wurde, wurde die Wirkung am proximalen Kolon des Schweins über H2-Rezeptoren vermittelt. Die Änderung des Kurzschlussstroms nach Histaminzugabe resultierte aus einer Chloridsekretion. Eine Chloridsekretion scheint somit eine generelle Wirkung von Histamin auf Darmepithelien zu sein, unabhängig von der Art des Wirkungs-vermittelnden Rezeptortyps. Histamin wurde aus den Epithelpräparationen spontan und nach Stimulation von Mastzellen freigesetzt. Obwohl nach Mastzellstimulation eine hohe Histaminfreisetzung beobachtet werden konnte, war dieses Histamin nicht an der sich aus der Stimulation ergebenen elektrophysiologischen Reaktion des Epithels beteiligt. In Fluxstudien mit radiaktiv markiertem Histamin wurde eine konzentrationsabhängige Histaminpermeation über das Epithel festgestellt. Diese Permeation ist von mukosal nach serosal scheinbar parazellulär lokalisiert. Dagegen scheint bei der Permeation von serosal nach mukosal ein transzellulärer Anteil vorhanden zu sein, da eine aktive Sekretion von Histamin in das Darmlumen festgestellt werden konnte. Während der Permeation von Histamin über das Epithel wurde in Abhängigkeit von der vorgegebenen Konzentration zwischen 80% und 100% des permeirenden Histamins verstoffwechselt. Somit besteht eine effektive Darmbarriere gegenüber exogenem Histamin, die sich aus einer geringen Permeation und einer hohen intraepithelialen Verstoffwechselung von Histamin zusammensetzt. Beide für den Abbau von Histamin in Frage kommenden Enzyme, Diaminoxidase (DAO) und Histamin-N-Methyltransferase (HNMT), sind am Katabolismus von Histamin beteiligt. Während in der Literatur die DAO als das bedeutendste Enzym des Histaminkatabolismus am Darm angegeben wird, ist am proximalen Kolon des Schweins die HNMT wichtiger für den Histaminabbau. Beide Enzyme bewerkstelligen sowohl den Abbau von endogen freigesetztem Histamin als auch von transepithelial permeierendem Histamin. Somit hätte eine Hemmung dieser Enzyme, die durch eine Vielzahl von Stoffen, darunter gebräuchliche Arzneimittel, hervorgerufen werden kann, dramatische Konsequenzen. In diesem Fall würde der Körper in hohen Maßen sowohl von endogenem als auch von exogenem Histamin aus dem Darm belastet werden<br>In the oral part of pig large intestine, high amounts of luminal histamine can be found due to bacterial production. Further more, histamine is abundantly present in the intestinal wall, where it is stored in different populations of mast cells. Both sources of histamine, exogenous and endogenous, are very dangerous for the body, because histamine is able to elicit systemic effects when it is spilt over in the systemic circulation. Under normal conditions no pathophysiological reactions can be observed in pigs due to the high amounts of histamine in the gut. Therefore, it must be concluded that there is a very effective barrier against luminal histamine. However, neither the barrier function has been characterized yet, nor is there any data available on the action of histamine in this part of the porcine gut. Therefore, the aim of this study was to investigate the effect, permeation and catabolism of histamine in isolated epithelia of the proximal colon by using the Ussing chamber technique. Addition of histamine to the serosal side induced a rapid rise in short-circuit current. In contrast to many studies investigating the action of histamine in other gut epithelia, in the pig proximal colon histamine acts via H2 receptors. Histamine induced a chloride secretion, which seems to be a common mechanism of gut epithelia, independent from histamine receptor type involved. Endogenous histamine was liberated spontaneously from the epithelia in small amounts. High amounts of histamine were found after a mast cell stimulation. However, this histamine did not participate in a concurrent electrophysiological reaction of the epithelia. In flux studies with radioactively labeled histamine, a transepithelial permeation of histamine was observed in a dose dependent manner. This permeation was located on the paracellular pathway in the mucosal-to-serosal direction. In the serosal-to-mucosal direction a, at least in part, transcellular pathway must be concluded from the observed histamine secretion into the gut lumen. Among 80% and 100% of histamine was catabolised dose-dependently during permeation. Therefore, the very effective gut barrier against histamine is based on a low paracellular permeation and a high intraepithelial catabolism of histamine. The histamine-degrading enzymes, diamine oxidase (DAO) and histamine N-methyltransferase (HNMT), took both part in the catabolism of histamine. While in literature DAO is called the bottleneck of histamine degradation in the gut, HNMT seems to be more significant in pig proximal colon. DAO and HNMT are important for the catabolism of exogenous and endogenous histamine. Therefore, inhibition of these enzymes, which is possible by numerous drugs, would have dramatic consequences. In that case, high amounts of histamine would be able to reach the systemic circulation
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Chorvátová, Michaela. "Vliv elektrických pulzů na lidské krevní fagocyty." Master's thesis, Vysoké učení technické v Brně. Fakulta chemická, 2019. http://www.nusl.cz/ntk/nusl-401914.
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The phagocytic cells circulating in the bloodstream play a key role in both the defense of the body and the pathology of inflammatory diseases. Thus, targeting their functions has potential to modulate an immune response, especially during the inflammatory phase. This master's thesis was focused on the influence of electric pulses on the most abundant phagocyte population in human peripheral blood, namely neutrophils. The theoretical part describes the role of neutrophils in the development of the immune response and the effects of the electric field on various cells. Consequent part of the thesis was the optimization of the electrical stimulation of neutrophils using a unique platform with a network of gold electrodes. In stimulated cells by electrical pulses, activation of selected signaling pathways, degranulation, ROS production, citrullination of histone H3 and expression of surface markers were monitored. Overall, electrical stimulation was observed to induce neutrophil activation but only electrical pulses of size 1 V were found to be statistically significant in the case of ROS production and 10 mV and 100 mV electrical pulses in the case of metalloproteinase MMP8 degranulation. The absence of significant effects in the most observed parameters was probably due to unwanted activation of neutrophils in control samples.
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Ferreira, Isabel Cristina Costa Vigato. "Fc?R e CR3 no lúpus eritematoso sistêmico: variantes polimórficas e sua influência na fagocitose e desgranulação dos neutrófilos." Universidade de São Paulo, 2012. http://www.teses.usp.br/teses/disponiveis/60/60135/tde-05102012-085559/.
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O lúpus eritematoso sistêmico (LES) é uma doença autoimune e a suscetibilidade às infecções está associada as suas anormalidades imunológicas e a sua terapia imunossupressora e citotóxica. Dentre as alterações que predispõem às infecções no LES estão anormalidades moleculares e funcionais dos neutrófilos: clearance e fagocitose ineficientes de imunocomplexos (IC) e bactérias, neutropenia, defeitos na quimiotaxia, redução do burst oxidativo e redução da expressão de receptores para IgG (Fc?R) e para complemento (CR). Além disso, os polimorfismos genéticos dos Fc?R têm sido associados com as disfunções imunes do LES. Os Fc?R são importantes mediadores das funções efetoras do neutrófilo e atuam em sinergismo com os CR (CR1 e CR3). O polimorfismo dos genes Fc?RIIA e Fc?RIIIB determina a expressão de variantes alélicas com diferenças funcionais, as quais podem influenciar as respostas biológicas e a suscetibilidade e o prognóstico das doenças infecciosas. Em particular, o alótipo Fc?RIIa-R131, tem menor afinidade para a IgG2, o que resulta em prejuízo na fagocitose mediada por esta imunoglobulina. A IgG2 é essencial contra bactérias encapsuladas e os pneumococos são responsáveis por 6-18% das infecções bacterianas no LES. O objetivo deste estudo foi investigar a influência dos polimorfismos genéticos dos Fc?R na fagocitose e desgranulação dos neutrófilos, associados ao polimorfismo do CR3, e à ocorrência de infecções bacterianas no LES. Os genótipos foram determinados por reações da polimerase em cadeia para os polimorfismos das variantes alélicas; a fagocitose e desgranulação foram estimuladas por IC contendo IgG (IC-IgG) e por IC-IgG e complemento (IC-IgG/SHN); a fagocitose de IC por neutrófilos e a expressão dos Fc?R e CR3 foram avaliadas por citometria de fluxo; e a desgranulação dos neutrófilos estimulada por IC foi medida pela liberação de elastase e lisozima. Os resultados mostraram: maior frequência para o genótipo R-131 no LES; associação do genótipo HNA-4a negativo para o CR3 com a suscetibilidade para o LES e associação do HNA-4a positivo com proteção; fagocitose menor em neutrófilos de pacientes com LES com genótipo HR-131 comparados aos neutrófilos do grupo controle com genótipo R-131 (IC-IgG e IC-IgG/SHN); no polimorfismo do Fc?RIIIb, a fagocitose e a lisozima foram menores em neutrófilos de pacientes com LES com genótipo HNA-1b e maior para HNA-1a/1b comparados aos controles com os respectivos genótipos (IC-IgG/SHN); a ocorrência de infecções foi mais frequentemente associada à presença do alelo R-131 e HNA-1b; nenhuma diferença foi observada para o polimorfismo HNA-4a do CR3, bem como para e elastase. Este estudo contribui para o entendimento das anormalidades nas funções dos neutrófilos no LES e para a identificação de indivíduos, cujo polimorfismo dos Fc?R e CR3 possa conferir suscetibilidade ou proteção às infecções.<br>Systemic lupus erythematosus (SLE) is an autoimmune disease in which disease-related and genetic factors and immunosuppressive and cytotoxic therapies all contribute to an increased susceptibility to infections. Factors predisposing to infection include defects in chemotaxis, abnormalities in neutrophil phagocytic activity, decreased immune complex (IC) and bacteria clearance, neutropenia, reduced oxidative burst, abnormalities in the expression of Fc?R (Fc?R) and complement (CR) receptors. Recent data have provided evidence that genetic polymorphism of Fc?R is associated with immune abnormalities and risk to development of SLE. Fc?R can mediate neutrophil effector functions and play a synergistic action with CR. Fc?RIIa and Fc?RIIIb display functionally relevant genetic polymorphisms, which allelic variants can influence the biological responses and the susceptibility to and course of infectious diseases. In particular, the presence of the Fc?RIIa-R131 allotype results in lower affinity binding to IgG2, a subclass of IgG specific for encapsulated bacteria. Since pneumococci accounts for 6-18% of all bacterial infections in SLE, the R131 allele can be relevant as a risk factor for infections. The aim of this study was to investigate the influence of the Fc?R polymorphisms on the phagocytosis and degranulation of neutrophils, associated with CR3 polymorphism, and bacterial infections in SLE. Genotypes were determined by polymerase chain reactions for the polymorphisms of the allelic variants; the phagocytosis and the degranulation were stimulated with IC-containing IgG (IC-IgG) and IC-IgG and complement (IC-IgG/NHS); the phagocytosis of IC by neutrophils and expression of Fc?R and CR3 were evaluated by flow cytometry, and degranulation of neutrophils stimulated with IC was measured by the release of lysozyme and elastase. The results showed a higher frequency for the genotype R-131 in SLE and association of genotype HNA-4a negative for the CR3 with susceptibility to SLE and association of HNA-4a with protection; the phagocytosis was lower in neutrophils from patients with SLE with HR-131 genotype than those in neutrophils from control group with genotype R-131 (IC-IgG and IC-IgG/NHS); in the Fc?RIIIb polymorphism, the phagocytosis and lysozyme were lower in neutrophils from patients with SLE with genotype HNA-1b and higher for HNA-1a/1b than those controls with the respective genotypes (IC-IgG/NHS); the occurrence of infections was more frequently associated with the presence of the allele R-131 and HNA-1b; no difference was observed for polymorphism HNA-4a of the CR3, neither for elastase. This study can contribute for the understanding of neutrophil abnormalities in SLE and identifying genetic markers that would predict patients Who are at high risk for infections.
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Ahrens, Frank. "Wirkung, Permeation und Katabolismus von Histamin an isolierten Dickdarmepithelien des Schweins." Doctoral thesis, [S.l.] : [s.n.], 2003. http://dol.uni-leipzig.de/pub/2003-37.
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Sampaio, Marlos Cortez. "Efeitos da MT-I, uma fosfolipase A2, isolada do veneno de Bothrops asper em mastócitos: ativação e sinalização intracelular envolvida na desgranulação." Universidade de São Paulo, 2015. http://www.teses.usp.br/teses/disponiveis/42/42133/tde-05102015-203252/.
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Os efeitos da MT-I, uma fosfolipase A2 isolada do veneno de Bothrops asper (VBa), foram avaliados em mastócitos (MC) em cultura, quanto à: i) desgranulação e liberação de prostaglandina E2 (PGE2); ii) papel da atividade catalítica na desgranulação; iii) papel da PLD, PLC, cPLA2, iPLA2, PI3K, MAPK, PKC, PTK, ERK1/2, Junk, Gαi, Gαq e do cálcio na desgranulação; iv) expressão gênica de citocinas Th1 e Th2, e v) alterações ultraestruturais em MC. Os resultados mostraram que a MT-I, em concentrações não citotóxicas, causou a desgranulação de MC. Este efeito foi parcialmente dependente da atividade catalítica e dependente da cPLA2, PLC, PLD e PI3K, mas não da iPLA2, ERK1/2, p38MAPK, PKC, MEK, Junk, Gαi e Gαq. O cálcio intra e extracelular (CRAC e LTCC) estão envolvidos neste efeito da MT-I. Ainda, a MT-I induziu a síntese e liberação da PGE2, expressão de genes de citocinas Th1 e Th2, aumento do número de vesículas citoplasmáticas e de endocitose dependente de clatrina. O VBa também causou a desgranulação de MC sugerindo que a MT-I é relevante para este efeito.<br>The effects of Myotoxin-I (MT-I), a phospholipase A2 (PLA2) from Bothrops asper venom (BaV) in cultured mast cells (MC) were evaluated focusing: i) degranulation and prostaglandin E2 (PGE2) release; ii) role of PLA2 catalytic activity in degranulation; iii) role of PLD and PLC, cPLA2 and iPLA2, PI3K, MAPK, PKC, PTK, ERK1/2, Junk, Gαi and Gαq protein and calcium in degranulation; iv) gene expression of Th1 and Th2 cytokines, and v) MC ultrastructural alterations. Results showed that MT-I, at non-cytotoxic concentrations, caused MC degranulation. This effect was partially dependent on its catalytic activity and dependent on cPLA2, PLC, PLD and PI3K, but not iPLA2, ERK1/2, p38MAPK, PKC, MEK, Junk, Gαi nor Gαq. Both intra and extracellular calcium (CRAC and LTCC) are involved in MT-I-induced degranulation. Furthermore, MT-I induced synthesis and release of PGE2, gene expression of Th1 and Th2 cytokines, increased numbers of cytoplasmic vesicles and clathrin-dependent endocytosis. BaV also caused MC degranulation suggesting that MT-I is relevant for this effect.
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Awad, Ali. "Les cellules Natural Killer (NK) dans l’allergie : effet de la chimiokine CCL18 sur les cellules NK humaines et rôle des cellules NK sur les éosinophiles." Thesis, Lille 2, 2014. http://www.theses.fr/2014LIL2S002/document.
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Les maladies allergiques sont en constante augmentation tant en prévalence qu’en gravité. Les éosinophiles sont fortement impliqués dans le dommage et le dysfonctionnement tissulaire et participent à l’entretien de l’inflammation allergique. Différentes cellules de l’immunité innée sont impliquées dans le contrôle de la réaction allergique. Parmi elles, les cellules NK, connues essentiellement pour leurs fonctions anti-tumorales et anti-microbiennes, pourraient réguler différents aspects de la réaction. Dans le sang périphérique de patients asthmatiques, les cellules NK présentent des capacités cytotoxiques accrues, ainsi qu’une prédominance de cellules NK2 comparativement à la prédominance de cellules NK1 chez les sujets non allergiques. Chez des patients atteints de dermatite atopique, le nombre et la cytotoxicité des cellules NK périphériques sont diminués, ainsi que leur capacité à produire de l’IFN-g. De plus, le dialogue entre les cellules NK et les cellules dendritiques est moins efficace chez le sujet asthmatique, menant ainsi à une capacité réduite de production d’IFN-g par les cellules NK. Dans des modèles murins d’inflammation pulmonaire, la déplétion en cellules NK par l’anti-NK1.1 ou l’anti-ASGM1 avant l’immunisation inhibe l’éosinophilie pulmonaire, l’infiltrat des LT CD3+ et l’augmentation des taux d’IL-4, IL-5 et IL-12 dans le LBA. Néanmoins, la déplétion avec l’anti-ASGM1 après l’établissement de l’inflammation éosinophilique retarde sa résolution, suggérant un rôle double des cellules NK dans l’inflammation allergique. Le recrutement et la fonction des cellules NK humaines dans l’allergie par le biais de l’analyse in vitro du rôle de CCL18 sur les cellules NK a été analysé. Cette chimiokine est préférentiellement produite au niveau du poumon et possède une double fonction dans la pathologie allergique puisqu’elle recrute les LTh2, mais également les LT reg et génère des DCs tolérogènes capables d’induire des LT reg, uniquement chez des donneurs non allergiques. Nous avons évalué la réponse des cellules NK de sujets allergiques vis-à-vis de CCL18 et l’avons comparée à celle de cellules NK provenant de sujets non allergiques. Nos travaux ont montré que CCL18 attire in vitro les cellules NK de sujets non allergiques et induit leur cytotoxicité, de façon dépendante des protéines G. Par contre, les cellules NK de sujets allergiques ne répondent pas au CCL18. La deuxième partie du travail s’est basée sur l’hypothèse d’un dialogue entre les cellules NK et les éosinophiles qui modifierait leurs fonctions respectives. Des cellules NK et des éosinophiles autologues ont été cocultivés pendant 3 et 12h, à différents ratios. Nous avons montré que les cellules NK activent directement les éosinophiles comme en témoignent l’augmentation de la libération de l’ECP, l’EDN, et de l’expression du CD63, du CD69 et la diminution de l’expression du CD62L sur les éosinophiles. De plus, les cellules NK induisent l’apoptose et la mortalité des éosinophiles dès la première heure de coculture. Cependant l’apoptose et la mortalité des cellules NK ne sont pas modifiées. La fixation des cellules NK empêche presque totalement l’activation et l’apoptose des éosinophiles, suggérant l’implication de molécules de surface et peut être de facteurs solubles. Les interactions entre molécules de surface restent à déterminer, et l’IFN-g et le TGF-β ne sont pas impliqués. Cependant, les voies de signalisation p38MAPkinase, ERK, JNK et PI3kinase interviennent dans l’activation des éosinophiles. La voie mitochondriale et ROS sont impliquées dans l’apoptose des éosinophiles induite par les cellules NK.En résumé, ces travaux ont permis de montrer que les cellules NK de sujets allergiques présentent un dysfonctionnement dans la réponse vis-à-vis de CCL18 comparativement aux sujets non-allergiques. De plus, nos résultats suggèrent que les cellules NK pourraient réguler l’inflammation à éosinophiles en induisant leur activation et/ou leur apoptose<br>Allergic diseases are steadily increasing both in prevalence and severity. Known physiopathological mechanisms involve the induction of a Th2 response by dendritic cells, leading to IgE production and inflammation, in particular linked to the recruitment of eosinophils. Eosinophils are heavily involved in injury and tissue dysfunction and contribute to the maintenance of inflammation. Different cells of innate immunity were shown to be involved in the control of allergic reaction. Among them, (NK) cells, primarily known for their anti-tumor and anti-microbial functions, may regulate different aspects of allergic reaction as suggested by studies in humans or mice. In the peripheral blood of patients with asthma, NK cells exhibit increased cytotoxic capacity, and a predominance of NK2 cells compared to the prevalence of NK1 cells in non-allergic subjects. In patients with atopic dermatitis, the number and cytotoxicity of peripheral NK cells are reduced, as well as their ability to produce IFN-g. Moreover, the dialogue between NK cells and dendritic cells is less effective in asthmatic patients, leading to a reduced capacity of IFN-g production by NK cells. In murine models of pulmonary inflammation, depletion of NK cells by anti-NK1.1 or anti-ASGM1 before immunization inhibits pulmonary eosinophilia, the infiltration of CD3+ T cells and increased levels of IL-4, IL-5 and IL-12 in the bronchoalveolar lavage. However, depletion with anti-ASGM1 after the establishment of eosinophilic inflammation delays its resolution, suggesting a dual role of NK cells in allergic inflammation.We studied the recruitment and function of human NK cells in allergy through in vitro analysis of the role of CCL18 on NK cells. This chemokine is preferentially produced in the lungs and has a dual role in allergic diseases since it recruits Th2 cells but also regulatory T cells and generates tolerogenic DCs capable of inducing regulatory T cells only from non-allergic donors. We evaluated the response of NK cells in allergic subjects towards CCL18 and compared it to that of NK cells from non-allergic donors. We showed that CCL18 attracts NK cells from non-allergic subjects and induces their cytotoxicity in a G protein dependent pathway. However, NK cells from allergic subjects did not respond to CCL18. This chemokine has no effect on the proliferation of NK cells, but may negatively regulate IFN-g production.The second part of the thesis is based on the hypothesis of a dialogue between NK cells and eosinophils which would modify their respective functions. NK cells and autologous eosinophils were cocultured during 3 and 12 hours, at different ratios. We showed that NK cells directly activate eosinophils as evidenced by the increased release of ECP, eosinophil derived neurotoxin EDN, and the expression of CD63, CD69, and reduced expression of CD62L on living eosinophils. In addition, coculture with NK cells induced apoptosis and mortality of eosinophils in the first hours of coculture. However, apoptosis and death of NK cells were not changed. Fixation of NK cells prevented almost completely the activation and apoptosis of eosinophils, suggesting the involvement of surface molecules, however soluble factors cannot be excluded. These interactions require cell contact, but the molecules involved remain to be determined. Concerning soluble factors, IFN-g and TGF-β are not involved in these mechanisms. However, the signaling pathways p38MAPkinase, ERK, JNK and PI3-kinase are involved in eosinophils activation. Concerning eosinophil apoptosis induced by NK cells, the mitochondrial pathway is more involved than the caspase pathway.In summary, our studies show that NK cells from allergic patients exhibit a defect in their response towards CCL18 compared to non-allergic subjects. In addition, these results suggest that NK cells may regulate eosinophilic inflammation by inducing their activation and / or apoptosis
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Lahtinen, Mika. "NO Effect on Inflammatory Reaction in Extracorporeal Circulation : Ex vivo Studies." Doctoral thesis, Uppsala University, Department of Medical Sciences, 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-5908.
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<p>Nitric oxide (NO) is expressed in inflammatory tissues. However, NO effects are controversial in inflammation; NO is described as acting in a dose dependent manner and possess both pro-inflammatory and anti-inflammatory properties. </p><p>The present thesis explored the role of NO in relation to white blood cell (WBC) and protein system activation by foreign surfaces in simulated extracorporeal circulation (SECC) using human whole blood from volunteer donors. Three doses of NO, 40 ppm, 80 ppm and 500 ppm, were administered and an array of markers of WBC and protein activation were studied. Neutrophil degranulation was detected with myeloperoxidase (MPO), human neutrophil lipocalin (HNL) and lactoferrin (LF); eosinophil degranulation with eosinophil cationic protein (ECP) and eosinophil peroxidase (EPO); and basophil degranulation with histamine. Furthermore, whole blood and WBC capacity to produce reactive oxygen species (ROS) were studied and cytokine release was measured with IL-1 and IL-10. Complement activation was measured with C3a and C5b-9 complex and contact system activation with FXIIa-C1INH, FXIIa-AT, FXIa-C1INH and FXIa-AT.</p><p>NO increased neutrophil degranulation at all dose levels and 80 ppm NO increased basophil degranulation; whereas, NO exerted no effect on eosinophil degranulation, WBC subset counts, cytokine release or capacity to produce ROS. In addition, while increasing both specific and azurophil degranulation with 40 ppm, 80 ppm and 500 ppm, NO reversed the classical degranulation hierarchy with 500 ppm and azurophil degranulation became predominant. Furthermore, NO effect was greater with 500 ppm than with 80 ppm, indicating a dose response effect. The lack of iNOS mRNA expression in WBC and lack of L-NAME effect on degranulation and nitrite/nitrate production, together with absent increase in nitrite/nitrate in controls, excluded autocrine or paracrine regulation of degranulation. FXIIa-AT and FXIa-AT complexes increased and became predominant during early recirculation, whereas FXIIa-C1INH and FXIa-C1INH complexes were predominant at baseline but remained unaltered, suggesting contact system inhibition predominantly via AT. C3a and C5b-C9 increased. NO had no effect on either contact or complement system activation; however, 500 ppm NO shortened active clotting time.</p><p>In conclusion, the present data suggest that NO has a direct effect on neutrophil and basophil degranulation. Recognition of NO as an enhancer of degranulation may give access to new therapeutic tools for local and systemic inflammatory therapies; whereas, the identification of increased AT mediated inhibition of FXIIa and unchanged C1INH complexes presents new possibilities for therapeutic intervention in conditions such as hereditary angioedema and heart surgery.</p>
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Jelena, Ilić Sabo. "Efekat akutnog izlaganja peroralno unetog akrilamida na histološke strukture želuca pacova soja Wistar." Phd thesis, Univerzitet u Novom Sadu, Medicinski fakultet u Novom Sadu, 2016. http://www.cris.uns.ac.rs/record.jsf?recordId=100354&source=NDLTD&language=en.
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Akrilamid je toksična hemijska supstanca koja ima vrlo široku primenu u hemijskoj industriji, a 2002. godine otkriveno je njegovo prisustvo u namirnicima bogatim skrobom koje se pripremaju na visokim temperaturama. U poslednjh desetak godina primećen je veliki porast gastrointestinalnih tegoba u ljudskoj populaciji. Cilj istraživanja bio je ispitati patohistološke promene u tkivu želuca pacova soja Wistar izazvanih peroralnim aplikovanjem akrilamida i na taj način povući paralelu sa mogućim gastrointestinalnim tegobama nastalim kao posledica konzumiranja hrane bogate akrilamidom. U istraživanju je ispitivano 6 grupa od po 5 eksperimentalnih životinja (pacovi soja Wistar). Dve kontrolne grupe kojima je peroralno aplikovana destilovana voda i koje su žrtvovane posle 24h i 72h; dve eksperimentalne kojima je peroralno aplikovan akrilamid u dnevnoj dozi od 25 mg/kg i koje su žrtvovane posle 24h i 72h; dve eksperimentalne grupe kojima je peroralno aplikovan akrilamid u dnevnoj dozi od 50 mg/kg i koje su žrtvovane posle 24h i 72h. Na histološkom materijalu tkiva želuca primenjena je kvalitativna histološka analiza pod svetlosnim mikroskopom, semikvantitativna procena tipa mucina u epitelnim ćelijama sluznice želuca, prisustvo limfocita i granulocita u sluznici želuca, stereološka merenja pojedinih kompartmana zida želuca, linearna merenja broja i veličine ganglijskih ćelija u Maissner-ovom i Auerbach-ovom nervnom pleksusu, kao i broj mastocita u lamini propriji sluznice i podsluznici želuca. Dobijene vrednosti merenih parametara su potom statistički obrađene. Nastale promene na tkivu želuca pacova soja Wistar se ogledaju u vidu blagog direktnog oštećenja površnog epitela sa propratnom blagom inflamatornom reakcijom i blagom degranulacijom mastocita. U Maissner-ovom i Auerbach-ovom nervnom pleksusu su smanjene volumenske gustine nervnih vlakana i ganglijskih ćelija, kao i broj i veličina ganglijskih ćelija. Direktno toksično delovanje na epitel dovodi do posledične obnove epitela, te je potvrđeno prisustvo nezrelijih oblika mukoproduktivnih ćelija koje sadrže kisele, AB pozitivne mucine. Ispitani inflamatorni i degenerativni parametri pokazuju pozitivnu korelaciju u odnosu na dozu i/ili dužinu ekspozicije akrilamidu. Primena akrilamida peroralno pokazala je da postoje patohistološke promene na tkivu želuca u vidu direktnog toksičnog oštećenja epitela, inflamatorne reakcije i oštećenja nervnih pleksusa. Poznavanjem mehanizma delovanja ove toksične materije moguće je primeniti adekvatnu prevenciju u ishrani i izvršiti odgovarajući izbor terapijskih metoda.<br>Acrylamide is a toxic chemical substance with wide implementation in chemical industry. In 2002 it was discovered the presence of acrylamide in foods rich in starch which are prepared at high temperatures. In the last ten years there is a large increase in gastrointestinal illnesses in human population. The aim of this study was to investigate the histopathological changes in the gastric tissue in Wistar rats induced with injection of oral acrylamide and thus draw a parallel with possible gastrointestinal problems arising as a result of the consumption of foods rich in acrylamide. The research was carried out 6 groups of 5 experimental animals (Wistar rats). Two control groups that are orally concomitant application of distilled water and which were sacrificed after 24h and 72h; two experimental groups which are orally administrated acrylamide in a daily dose of 25 mg / kg and that were sacrificed after 24h and 72h; two experimental groups which were orally administrated acrylamide in a daily dose of 50 mg / kg and that were sacrificed after 24h and 72h. On histological gastric tissue material is applied qualitative histological analysis by light microscopy, semi-quantitative assessment of the type of mucin in epithelial cells of the stomach lining, the presence of lymphocytes and granulocytes in gastric mucosa, stereological measurements of individual compartments of the stomach wall, linear measuring the number and size of ganglion cells in the Maissner and Auerbach's nerve plexus, and the number of mast cells in the lamina propria of the mucosa and in the submucosis of the stomach. Obtained values of measured parameters were statistically processed. Histological changes in the stomach tissue of Wistar rats are seen as a direct slight damage of the surface epithelium, with accompanynig mild inflammatory reaction and the degranulation of mast cells. The Meissner's and Auerbach's nerve plexus decreased volume density of nerve fibers and ganglion cells, as well as the number and size of the ganglion cells. Directly toxic effect on epithelium leads to the result of the reconstruction of the epithelium, which is confirmed by the presence of immature form of mucoproductive cells which contain acid, AB positive mucins. Examined inflammatory and degenerative parameters show a positive correlation with respect to dose and / or a time of exposition to acrylamide. Acrylamide oral application revealed that there are histologic changes in the stomach tissue in the form of a direct toxic damage to the epithelium, inflammatory reaction and damage to the nerve plexus. Knowing the mechanism of action of these toxic substances allows to apply adequate prevention in nutrition and make an appropriate choice of therapeutic methods.
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Misumi, Denise Shimbo. "Validação do Teste de ativação de basófilos no diagnóstico de reações de hipersensibilidade a anti-inflamatórios não esteroidais." Universidade de São Paulo, 2013. http://www.teses.usp.br/teses/disponiveis/5/5146/tde-24062013-152145/.
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Introdução: Atualmente, o diagnóstico das reações de hipersensibilidade a anti-inflamatórios não esteroidais (AINEs) baseia-se na história relatada pelo paciente e, em determinados casos, é realizado o Teste de Provocação. Todavia, este teste pode expor os pacientes a riscos graves, inclusive anafilaxia. Em busca de ferramenta mais segura, tem-se estudado o Teste de Ativação de Basófilos (BAT). Trata-se de um teste in vitro, no qual é possível testar diversos estímulos em uma única amostra de sangue, avaliando a ativação dos basófilos (indicativo de reação de hipersensibilidade), através do aumento da expressão de moléculas na superfície desses leucócitos, como o CD63. Objetivo: Padronizar e validar o BAT para ácido acetilsalicílico (AAS), diclofenaco, dipirona e paracetamol em pacientes com hipersensibilidade a AINEs. Metodologia: Participaram 20 (testados com os quatro AINEs) + 33 (testados somente com AAS) pacientes atendidos no Serviço de Imunologia Clínica e Alergia do HCFMUSP, que apresentaram manifestações cutâneas em até 24 horas após exposição a um ou múltiplos AINEs, bem como 13 (quatro AINEs) + 26 (AAS) controles. A técnica consistiu em incubar sangue total com os AINEs já mencionados e, depois, marcar as amostras com anticorpos monoclonais (CD45, anti-IgE e CD63) para posterior leitura por citometria de fluxo. Os resultados obtidos foram comparados com as histórias clínicas e os testes de provocação oral, quando realizados. Resultados: Utilizando os critérios de positividade do BAT empregados na literatura (isto é, porcentagem de CD45+IgE+highCD63+ e índice de estimulação), a sensibilidade e a especificidade variaram de acordo com o AINE: para ácido acetilsalicílico foram 75,0% e 16,7%, respectivamente, diclofenaco, 100% e 0%, dipirona, 23,5% e 66,7%, paracetamol, 40,0% e 42,9%. Após a realização de curvas dose-resposta e tempo-resposta somente com AAS, foi encontrado novo critério de positividade: média de intensidade de fluorescência (MFI) menor do que 6575 representava BAT positivo; com isso, os valores de sensibilidade e especificidade foram: 84,4% e 34,6%, respectivamente. O BAT foi mais sensível em pacientes cuja última reação ocorreu há menos de um ano da data de execução do BAT (93,7%). Conclusão: Devido aos baixos valores de sensibilidade e/ou especificidade, não foi possível padronizar e, por conseguinte, validar o BAT para ácido acetilsalicílico, diclofenaco, dipirona e paracetamol.<br>Introduction: Currently, the diagnosis of nonsteroidal antiinflammatory drugs (NSAIDs) hypersensivitity is based on patients´ clinical history and drug provocation tests, which are done in selected cases. Nevertheless, this test may expose patients to severe risks, including anaphylaxis. Looking for a safer tool, Basophil Activation Test (BAT) for allergy diagnosis has been studied in the last years. It is an in vitro method where a wide variety of stimuli can be tested, incubating them with the patient\'s blood sample, and observing basophil activation (indication of hypersensitivity) through upregulation of CD63 (or other basophil activation markers) on this leucocyte\'s membrane. Objective: To standardize and validate BAT stimulated with acetylsalicylic acid (ASA), diclophenac, dipyrone and paracetamol in NSAID hypersensitive patients. Methods: Patients which reported immediate reactions (less than 24 hours) after exposure to one or multiple NSAIDs, with cutaneous symptoms were enrolled from Clinical Immunology and Allergy outpatient clinic from HC-FMUSP. BAT with the four NSAIDs was tested on 20 patients and 13 controls and BAT with ASA only, on 33 patients and 26 controls. BAT consisted of incubating whole blood with NSAIDs, then triple-labeled with monoclonal antibodies (CD45, anti-IgE, CD63) for analysis by flow cytometry. BAT results were compared to clinical history and oral provocation tests, when available. Results: According to literature\'s positivity criteria (percentage of CD45+IgE+highCD63+ and stimulation index), sensitivity and specificity varied according to the NSAID tested: for ASA was 75.0% and 16.7% respectively, diclophenac, 100.0% and 0.0%, dipyrone, 23.5% and 66.7%, paracetamol, 40.0% and 42.9%. A new positivity criterion was possible to be defined after further dose-response and time-response curves only for ASA: Mean Fluorescence Intensity lower than 6575 (positive BAT). Accordingly, new sensitivity and specificity for BAT in ASA hypersensitivity were 84,4% and 34,6%. Patients that presented the last reaction in the last year were more likely to present a positive BAT (93.7%). Conclusion: Due to low values for sensitivity and/or specificity, it was not possible to standardize and validate BAT for ASA, diclophenac, dipyrone and paracetamol.
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Mould, Arne Wyndham. "Mechanisms of eosinophil recruitment and degranulation." Phd thesis, 1998. http://hdl.handle.net/1885/144700.
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"The effects of beta-adrenoceptor agonists on mast cell degranulation." Chinese University of Hong Kong, 1993. http://library.cuhk.edu.hk/record=b5887775.
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Pui Lan Wong.<br>Thesis (M.Phil.)--Chinese University of Hong Kong, 1993.<br>Includes bibliographical references (Leaves 109-122).<br>Abstract --- p.i<br>Acknowledgements --- p.iii<br>Chapter Chapter1 --- Introduction<br>Chapter 1.1 --- A general introduction on mast cells --- p.1<br>Chapter 1.2 --- Activation of mast cells --- p.6<br>Chapter 1.3 --- Mediators of mast cells --- p.18<br>Chapter 1.4 --- Usage of β-adrenoceptor agonists in asthma therapy --- p.26<br>Chapter 1.5 --- Aim of this study --- p.32<br>Chapter Chapter2 --- Materials and methods<br>Chapter 2.1 --- Chemicals --- p.42<br>Chapter 2.2 --- Buffers and stock solutions --- p.43<br>Chapter 2.3 --- Source of mast cells --- p.45<br>Chapter 2.4 --- Animal sensitization --- p.45<br>Chapter 2.5 --- Isolation of mast cells --- p.46<br>Chapter 2.6 --- Procedure for the investigation of the effects of adrenoceptor agonists on histamine release from mast cells --- p.48<br>Chapter 2.7 --- Procedure for the investigation of propranolol antagonism --- p.49<br>Chapter 2.8 --- Histamine assay --- p.50<br>Chapter 2.9 --- Data analysis --- p.50<br>Chapter Chapter3 --- Results<br>Chapter 3.1 --- Establishment of experimental conditions --- p.53<br>Chapter 3.2 --- The effects of β-agonists on immunologically induced histamine release from guinea pig lung mast cells --- p.54<br>Chapter 3.3 --- The effects of β-agonists and two anti-allergic drugs on immunologically induced histamine release from guinea pig lung mast cells --- p.56<br>Chapter 3.4 --- The effects of β2-agonists on histamine release induced by non-immunological agents from guinea pig lung mast cells --- p.56<br>Chapter 3.5 --- Antagonism by propranolol on the effects of β2-agonists on histamine release from guinea pig lung mast cells --- p.57<br>Chapter 3.6 --- The effects of β2-agonists on immunologically induced histamine release from rat peritoneal mast cells --- p.58<br>Chapter 3.7 --- The effects of β2-agonists on immunologically induced histamine release from human lung mast cells --- p.58<br>Chapter 3.8 --- "Comparison of the effects of β2-agonists on immunologically induced histamine release from mast cells isolated from the rat peritoneum, the guinea pig lung and the human lung" --- p.59<br>Chapter Chapter4 --- Discussion<br>Chapter 4.1 --- The effects of β-agonists on immunologically induced histamine release from guinea pig lung mast cells --- p.89<br>Chapter 4.2 --- The effects of β2-agonists and two anti-allergic drugs on immunologically induced histamine release from guinea pig lung mast cells --- p.97<br>Chapter 4.3 --- The effects of novel β2-agonists on histamine release induced by non-immunological agents from guinea pig lung mast cells --- p.99<br>Chapter 4.4 --- The study of propranolol --- p.100<br>Chapter 4.5 --- The heterogeneity of mast cells --- p.103<br>Chapter Chapter5 --- General conclusion --- p.107<br>References --- p.109
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Sun, Mu-Jie, and 孫睦傑. "Dengue Virus Induces Chemokine Release and Degranulation of Human Neutrophils." Thesis, 2003. http://ndltd.ncl.edu.tw/handle/24252589093757474969.
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碩士<br>國立成功大學<br>醫事技術學系<br>91<br>Dengue viruses (DV) are lipid-enveloped RNA viruses, which are transmitted by the mosquito vectors, Aedes aegypti and Aedes albopictus. DV are the most important flaviviruses in tropical and subtropical countries. Infection with DV may cause mild dengue fever or life threatening diseases such as dengue hemorrhagic fever and dengue shock syndrome. Vascular leakage, hemorrhage, neutropenia and complement activation are the hallmarks of these diseases. Polymorphonuclear leukocytes (PMNs) or neutrophils are critical effectors cells of the innate immune system, which can protect hosts by migrating to inflammatory sites and killing pathogen. However, the role of PMNs in the pathogenesis of DV infection is unclear. In this study, human neutrophils were incubated with DV to understand the effect of DV on the biological activities of neutrophils. Live but not UV inactivated DV (UV-DV) decreased the viability of neutrophils and increased the release of LDH and chemokines such as IL-8, and macrophage inflammatory protein (MIP-1a) from neutrophils. However, the granule enzyme, myeloperoxidase (MPO), was released from both live and UV-DV-stimulated neutrophils. In addition, the levels of IL-8 and MPO in the sera of dengue patients were also increased. Using fluorescent confocal microscopy, we found that DV was phagocytosed into neutrophils and co-localized with FITC-beads in neutrophils. Furthermore, DV-stimulation also increased the expression of CD11b neutrophils and ICAM-1 of HMEC-1 endothelial cell line and increased the binding of neutrophils to endothelial cell. In addition, the signal transduction molecule Toll-like receptor 4 (TLR4) was also increased in DV stimulated neutrophils. Taken together, neutrophils can uptake DV through phagocytosis and kill the virus. In this process, adherence of neutrophils to endothelial cells and necrosis of neutrophils were increased along with the release of chemokines, granule enzymes, and the expression of surface molecule. Therefore neutrophils may play important roles in the innate immune response against DV infection and may also contribute to the pathogenesis of dengue virus infection.
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Zankl, Claudia [Verfasser]. "Stereoselektive Synthese von Sphingolipiden zur Inhibierung der Degranulation von Mastzellen / von Claudia Zankl." 2009. http://d-nb.info/1007530677/34.
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