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MODULATING THE INNATE IMMUNE RESPONSE TO ELECTROSPUN SCAFFOLDS AND POLYMER DEGRADATIVE BYPRODUCTSAbebayehu, Daniel 01 January 2017 (has links)
Implanted biomaterials often induce inflammation that frequently leads to the foreign body response, fibrosis, and the failure of the implant. Thus, it is important to evaluate how cells interact with materials to promote a more regenerative response. It is critical to determine how to modulate the response of tissue resident innate immune cells, as they are among the first cells to interact with implanted materials. Among tissue resident innate immune cells are mast cells, which are inflammatory sentinels that degranulate and orchestrate the fate of other cell populations, such as monocytes/macrophages and lymphocytes. Mast cells have also been reported to play a vital role in the foreign body response of implanted biomaterials as well as angiogenesis. The goal of this study was to determine how to modulate mast cell responses to electrospun scaffolds by altering scaffold architecture and composition to promote anti-inflammatory and regenerative cell-scaffold interactions. Scaffold architecture was manipulated by changing either fiber diameter or pore diameter and mast cell responses were mediated by endogenous and exogenous DAMPs (i.e. IL-33 and LPS, respectively). Particularly in response to IL-33, scaffolds with increased fiber and pore diameter promoted less inflammatory cytokine and chemokine release while increasing angiogenic cytokine release. Additionally, taking scaffolds that promoted increased inflammatory cytokine expression and increasing the pore diameter alone dampened inflammatory cytokine expression. The next question we wanted to answer was how might the degradative byproducts of scaffolds alter mast cell inflammatory responses. Given the widespread use of polylactic acid, we decided to investigate this question using lactic acid as a degradative byproduct. In the presence of physiologically relevant levels of lactic acid, IL-33- and IgE-mediated inflammatory cytokines and chemokines are suppressed, while angiogenic cytokines are enhanced. This response was shown to be pH- and MCT1-dependent and was recapitulated in primary human skin mast cells as well as in vivo. In summary, scaffold architecture and the presence of select polymer degradative byproducts have the potential of selectively suppressing inflammatory cytokines and enhancing angiogenic cytokines.
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The effect of fluvastatin on mast cell function: genotype dependenceKolawole, Elizabeth M 01 January 2014 (has links)
Fluvastatin, the HMG-CoA reductase inhibitor known for its role in the treatment of hypercholesterolemia and cardiovascular disease, has more recently been shown to play a role in the immune response. Given the critical role that mast cells play in allergy and inflammatory diseases such as asthma, which effects one third of America’s population, we assessed the effect of fluvastatin on mast cell and basophils function. We demonstrate that fluvastatin downregulated IgE-mediated cytokine production. Additionally, in vivo studies showed that fluvastatin suppressed IgE-mediated anaphylaxis. Interestingly, the effects of fluvastatin showed dependence on genetic background, as C57BL/6 mast cells were sensitive, while 129/Sv mast cells were resistant to fluvastatin. Characterizing the role of fluvastatin on mast cells may prove to be therapeutically important.
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The Effect of Statins on IL-33 Mediated Mast Cell FunctionTaruselli, Marcela 01 January 2015 (has links)
This study demonstrates original findings of statin effects on IL-33 stimulated mast cells. Statins are a class of drugs used to lower cholesterol production by targeting HMG CoA reductase. These commonly prescribed drugs have been shown to be immunomodulatory. In this study, we have found that pretreatment with statins has a variety of effects on IL-33 stimulated mast cells. Atorvastatin suppresses TNF and IL-6 production, while fluvastatin significantly enhances release of these proinflammatory cytokines in BMMCs. Although they have differing effects on cytokine production, both statins lowered ST2 expression on the cell surface, decreased cell viability, and enhanced expression of the transcription factor KLF2, a negative regulator of NFκB. Blocking isoprenylation by using geranylgeranyl transferase inhibitor, but not farnesyl transferase, mimicked the effects of atorvastatin, while neither mirrored the effect of fluvastatin. Furthermore, fluvastatin effects were not reversed by mevalonic acid, the product of HMG-CoA reductase. These data indicate that fluvastatin effects are distinct from its activities as an HMG CoA reductase inhibitor. Fluvastatin effects required the presence of stem cell factor (SCF), and were enhanced by increasing SCF concentrations. Finally, fluvastatin enhanced IL-33-induced cytokine production and neutrophil recruitment in vivo. Collectively, these data suggest that statins can alter the mast cell response, and that drug choice can have divergent effects on outcome.
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Transforming Growth Factor-β1 (TGF-β1) Induces Mast Cell ApoptosisNorozian, Farnaz 01 January 2006 (has links)
Mast cells are potent effectors of the inflammatory response, playing an important role in atopy, bacterial immunity, and animal models of arthritis, multiple sclerosis, and heart disease. Hence controlling mast cell numbers and responsiveness is essential for preventing inflammatory disease. This work demonstrated that the cytokine TGF-β1 is a potent inducer of mast cell apoptosis, a finding that was consistent for cultured mouse bone marrow-derived mast cells, peritoneal mast cells, and human mast cells. Cell death appeared to be the result of TGF-mediated repression of IL-3 receptor expression and function, leading to mitochondria1 damage and activation of an apoptotic cascade acting via p53 and caspases. While IL-3 receptor expression was reduced within one day of TGF-βl stimulation, apoptosis required at least 3 days to occur. This delay in onset is postulated to allow for protective mast cell effector functions, protecting the host from infection while preventing the establishment of chronic inflammation. These studies support the theory that TGF- β1 is an inhibitor of mast cell survival. Because of the widespread expression of TGF-β1, this cytokine may be an ideal candidate for control of mast cell homeostasis.
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Stains Induce Apoptosis and Autophagy in Primary and Transformed Mast CellsPaez, Patrick A 01 January 2016 (has links)
Statin drugs are widely employed in the clinic to reduce serum low density lipoproteins (LDLs) in patients with hypocholesteremia. In addition to their cholesterol-lowering effects through HMG CoA reductase antagonism, isoprenyl lipids necessary for membrane anchorage and signaling of small G-proteins are abrogated. We previously found that statins suppress mast cell activation in murine and human cells, suggesting these drugs might be useful in treating allergic disease. While mast cell function is critical to allergic inflammation, mast cell hyperplasia and survival also impact these diseases, and were not studied in our previous work. In this study, we describe Fluvastatin-mediated apoptosis in both primary and transformed mast cells. An IC50 was achieved between 1-5μM in both systems, and apoptosis was preceded by mitochondrial dysfunction and caspase release. In addition to apoptosis, our work also uncovered evidence of autophagy, which can serve as a compensatory mechanism during apoptosis. Interestingly, autophagy appeared to be cyto-protective in the primary cells yet cytotoxic in transformed mast cells. These findings offer insight into the mechanisms of mast cell survival and support the possible utility of statins in mast cell-associated allergic and neoplastic diseases.
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Rôle des mastocytes dans le développement de la rectite radique in vivo et la réponse endothéliale à l’irradiation in vitro / Role of mast cells in radiation proctitis development in vivo and endothelial response to radiation exposure in vitro.Blirando, Karl 27 January 2011 (has links)
La radiothérapie est utilisée seule ou en association avec la chimiothérapie dans le traitement de plus de 50% des cancers. En dépit des progrès techniques dans la balistique, l'irradiation des tissus sains entourant la tumeur et les effets secondaires qui lui sont associés sont une limite à la dose d'irradiation utilisée. Ces effets secondaires, lorsqu'ils concernent le tube digestif, ont un retentissement important sur la qualité de vie des patients et peuvent même engager leur pronostic vital. La compréhension des mécanismes impliqués dans le développement de ces lésions est donc un enjeu majeur dans l'identification de cibles thérapeutiques permettant leur prévention et leur traitement. Durant ma thèse nous avons étudié le rôle des mastocytes dans le développement de la rectite radique in vivo et dans la réponse endothéliale à l'irradiation in vitro. Nos résultats suggèrent un rôle délétère des mastocytes dans le développement de la rectite radique humaine et murine, notamment par l'influence de certains de leurs médiateurs tels que l'histamine et les protéases sur le phénotype des cellules musculaires lisses de la muscularis propria. Le ciblage de certains médiateurs mastocytaires pourrait représenter une nouvelle stratégie thérapeutique pour prévenir et/ou limiter les atteintes radiques digestives. D'autre part, nos travaux montrent que des médiateurs mastocytaires comme l'histamine peuvent exacerber la réponse inflammatoire de l'endothélium à l'irradiation par des mécanismes de signalisation impliquant l'activation de la voie p38 MAPKinase et du facteur de transcription NF-B. L'étude approfondie des voies de signalisation activées lors du développement des lésions radiques pourrait offrir de nouvelles possibilités thérapeutiques dans la gestion des dommages radiques aux tissus sains. / Radiation therapy is used alone or in combination with chemotherapy in more than 50% of cancer treatments. Despite recent advances in treatment delivery such as dose-sculpting techniques, irradiation of healthy tissues surrounding the tumor and the associated side effects limit the radiation amount used. Those side effects when concerning the gastrointestinal tract, have profound repercussions on patient's quality of life and may even engage their vital prognosis. The comprehension of the mechanisms implicated in the development of these lesions is thus a major stake in the identification of therapeutic targets allowing their prevention and treatment. During my PhD, we studied the role of mast cells in the development of radiation proctitis in vivo and in the endothelial response to irradiation in vitro. Our results suggest that mast cells have a deleterious role in the development of human and murine radiation proctitis, in particular by the influence of some of its mediators such as histamine and proteases on the phenotype of the smooth muscle cells of the muscularis propria. Targeting mast cells'mediators may represent new therapeutic tools to prevent and/or limit digestive radiation damage. Other shares our work shows that mast cells mediators such as histamine can exacerbate the endothelial inflammatory response to irradiation by mechanisms involving the activation of the p38MAPKinase pathway and the transcription factor NF-B. The study of intracellular signaling pathways activated during radiation damage development may offer new therapeutic possibilities in the management of healthy tissues radiation damage.
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O papel de gangliosídeos específicos como moduladores da liberação de mediadores de mastócitos / The role of mast cell specific gangliosides in modulating mediator releaseFreitas Filho, Edismauro Garcia 30 March 2015 (has links)
Os mastócitos são células multifuncionais do sistema imunológico que participam em diversos processos biológicos. As funções dos mastócitos estão diretamente relacionados com a sua ativação e, subsequente, liberação de mediadores químicos. Os eventos iniciais da ativação dos mastócitos e da transdução de sinais ocorrem em microdomínios lipídicos (lipid rafts) da membrana plasmática. Os gangliosídeos derivados do GD1b são constituintes dos lipid rafts de mastócitos de roedores. O intercruzamento destes gangliosídeos pelo mAb AA4, resulta na formação de agregados (caps) na superfície celular e promove uma ativação parcial dos mastócitos, sem que ocorra a desgranulação. A ativação é semelhante a observada quando os FcRIs são intercruzados por antígenos multivalentes ligados a IgEs, mas neste caso ocorre a desgranulação. O presente estudo tem como objetivo caracterizar o papel dos gangliosídeos derivados do GD1b na liberação de mediadores de mastócitos da linhagem RBL-2H3. O intercruzamento dos gangliosídeos derivados do GD1b resulta na ativação dos fatores de transcrição NFAT e NFB e esta ativação é mediada pela proteína quinase Syk. A ativação destes fatores de transcrição resulta na liberação de mediadores neo-sintetizados, tais como: TNF-, interleucina (IL)-4. Por outro lado, o intercruzamento dos gangliosídeos derivados de GD1b não induz a liberação dos mediadores neoformados como o leucotrieno B4 (LTB4) e o leucotrieno C4 (LTC4). A agregação dos gangliosídeos derivados do GD1b resulta na desorganização dos lipid rafts e na redistribuição de seus componentes, como demostrado pela análise proteômica. Estes dados mostraram proteínas capazes de desencadear uma ativação parcial dos mastócitos e proteínas reguladoras negativas da desgranulação estão up reguladas, enquanto que proteínas críticas para a transdução do sinal estão down reguladas. Os resultados obtidos neste trabalho demonstram que os gangliosídeos derivados do GD1b desempenham papel crucial na integridade dos lipid rafts modulando a ativação e liberação de mediadores de mastócitos. / Mast cells are immunoregulatory cells that participate in diverse biological events. The action of mast cells is directly related to their activation and subsequent mediator release. Early signal transduction events occur in lipid rafts in the plasma membrane. GD1b-derived gangliosides are known constituents of lipid rafts in rodent mast cells. The cross-linking of these gangliosides by mAb AA4 results in a partial activation of mast cells similar to that observed when FcRIs are cross-linked, but does not result in the mast cell degranulation. With time, the gangliosides bound to mAb AA4 cap on the cell surface. The present study aims to characterize the role of the rodent mast cell specific gangliosides derived from GD1b in mediator release from RBL-2H3 mast cells. Cross-linking the GD1b-derived gangliosides activated the transcription factors NFAT and NFB and this activation was mediated by Syk. The activation of theses transcription factors by cross-linked GD1b-derived gangliosides results in the release of the neo-synthesized mediators TNF- and interleukin (IL)-4. However, cross-linking GD1b-derived gangliosides did not stimulate release of the newly formed mediators leukotriene B4 (LTB4) and leukotriene C4 (LTC4). Capping of GD1b-derived gangliosides disorganized lipid rafts and resulted in a redistribution of lipid raft components. Proteomic analysis showed that proteins that trigger mast cell activation and negative regulatory proteins of degranulation are up regulated, whereas proteins critical for signal transduction are down regulated in mast cells where the gangliosides are capped. The results of this work demonstrate that the mast cell-specific GD1b-derived gangliosides are crucial in maintaining the functional integrity of the lipid rafts and modulate cell activation and subsequent mediator release from mast cells.
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Expressão e caracterização das quimases recombinantes específicas de mastócitos de camundongos (mMCP4 e 5) / Expression and characterization of recombinant mouse mast cell chymases (mMCP4 e 5)Santana, Ana Carolina 31 October 2014 (has links)
Os mastócitos, em associação com outras células inflamatórias se acumulam em locais de tumor. Entretanto, pouco é conhecido sobre o papel exato dos mastócitos e de seus mediadores na progressão tumoral e angiogênese. Resultados recentes do nosso laboratório mostraram que a expressão das triptases específicas de mastócitos está correlacionada com a progressão tumoral (de Souza, Jr, PLosOne 7(7): e40790). Além do mais, existe uma associação entre mastócitos e a angiogênese tumoral. Então, tornou-se interessante investigar o papel das quimases específicas de mastócitos (mMCP-4 e -5) neste processo. Visto que estas quimases não são disponíveis comercialmente, a primeira etapa deste estudo foi produzir as quimases mMCP-4 e -5 recombinantes. Assim, as sequências dos genes respectivos para mMCP-4 e -5, além das sequências para seis resíduos de His e do sítio suscetível a enteroquinase foram clonadas no vetor de expressão pPIC9. A cepa GS115 de Pichia pastoris foi transformada com o respectivo vetor para mMCP-4 ou -5. Os clones transformados foram crescidos em meio BMMY e induzidos com várias concentrações de metanol para a produção de mMCP-4 ou -5 recombinantes. Depois de 96 horas, o meio foi centrifugado e as quimases mMCP-4 ou -5 foram purificadas do sobrenadante usando resina de níquel. A identidade das proteínas foi confirmada por Western Blotting usando o anticorpo anti-his, assim como os anticorpos anti-mMCP-4 e -5. As proteases foram ativadas pela clivagem do sítio suscetível a enteroquinase por cinco horas, a 22°C. A ativação das enzimas foi confirmada através da degradação de seus substratos específicos. Estes resultados mostram que P. pastoris é um organismo eficiente para a expressão de ambas as proteases. Estas proteases recombinantes se constituem em ferramentas importantes para se elucidar o papel da mMCP-4 ou -5 na angiogênese. / Mast cells, in association with other inflammatory cells, are known to accumulate at tumor sites. However, little is known about the exact role that mast cells and their mediators play in tumor progression and angiogenesis. Recent results from our laboratory have shown that expression of mast cell specific tryptases correlates with tumor progression (de Souza, Jr, PLosOne 7(7): e40790). Furthermore, there is an association between mast cells and tumor angiogenesis. It then became of interest to investigate the role of mast cell specific chymases (mMCP-4 and -5) in this process. Since these chymases are not commercially available, the first step in this study was to produce recombinant mMCP-4 and -5. The gene sequences for these chymases along with the sequence for six His residues and an enterokinase susceptible peptide were cloned into the expression vector pPIC9. The GS115 strain of Pichia pastoris was transformed with the respective vector for either rmMCP-4 or -5. The transformed clones were grown in BMMY media and induced to produce rmMCP-4 or -5 with various concentrations of methanol. After 96 hours, the medium was centrifuged and rmMCP-4 or -5 was purified from the supernatant using nickel-nitrilotriacetic acid agarose beads. The identity of the proteins was confirmed by Western blotting using an anti-his antibody as well as anti-mMCP-4 and -5. These results show that P. pastoris is an efficient organism in which to express both proteases.
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Estudo comparativo dos efeitos da dexametasona e do diclofenaco sódico sobre o processo reparativo de feridas induzidas no ventre lingual de ratos / Comparative study of the effects of dexamethasone and diclofenac sodium on the repair process of induced wounds in the ventral tongue of ratsArtes, Gisele Ebling 18 September 2012 (has links)
A inflamação é uma resposta protetora para livrar o organismo da causa inicial da lesão celular e suas consequências, porem se excessiva e não modulada, pode provocar a destruição progressiva do tecido. Este estudo pretende contribuir para o esclarecimento da influência de dois anti-inflamatórios, a dexametasona e o diclofenaco sódico, sobre a fase inflamatória do processo reparativo tecidual, bem como sobre o número de mastócitos nas diferentes fases do reparo. Foram utilizadas 60 ratas Wistar, divididas em 3 grupos (1, 2 e 3), medicadas 30 minutos antes de uma intervenção cirúrgica (lesão de 3mm de diâmetro no ventre lingual), com injeção intramuscular de 0,235mg/kg de dexametasona; 4,45mg/kg de diclofenaco sódico ou solução salina estéril, respectivamente. Os 3 grupos foram subdivididos em a, b, c, d, de acordo com o tempo de sacrifício: 6, 24, 48 e 120 horas após a cirurgia. As lesões foram fotografadas logo no momento cirúrgico e no sacrifício e as fotos utilizadas para análise clínica do processo de reparo e morfometria. As línguas foram extirpadas e enviadas para processamento histológico, os cortes foram direcionados para coloração em hematoxilina-eosina e em Azul de Toluidina para evidenciação e contagem do número de mastócitos. As características do processo reparativo foram descritas por meio de avaliação qualitativa dos seguintes componentes: extensão de áreas necróticas; intensidade de edema intersticial; tipo e intensidade do infiltrado inflamatório; graus de reepitelização, tecido de granulação e neovascularização. Os cortes corados em HE também tiveram seus campos digitalizados e analisados morfometricamente, para quantificação da reepitelização, mensurações do tecido de granulação, celularidade e edema. Os resultados da análise histológica semiquantitativa evidenciaram que o diclofenaco e a dexametasona acarretaram menor infiltrado inflamatório em relação ao controle na fase inflamatória do reparo, porém na fase produtiva a dexametasona exibiu menor intensidade de reepitelização e de neovascularização em relação aos demais grupos. Os resultados da análise histomorfométrica mostraram significativamente menor edema no grupo do diclofenaco em 6h (p=0,0041) e em 24h (p=0,0429), bem como menor porcentagem da celularidade em 6 horas no grupo da dexametasona (p<0,0001). O grupo diclofenaco ainda exibiu menor área de lesão em 120h do que os demais grupos (p=0,0060), indicando maior eficiência de reparo. Quanto à quantidade de mastócitos em 24, 48 e 120 horas, o grupo controle exibiu significativamente os maiores valores (p<0,0001). Com base nesses resultados, conclui-se que o grupo da dexametasona apresentou pior desempenho em relação à reparação; que o diclofenaco sódico apresentou um melhor efeito sobre o fechamento da ferida cirúrgica e redução mais intensa do infiltrado inflamatório, e que ambos provocam redução de mastócitos na área lesionada. / Inflammation is a protective response to rid the body of the initial cause of cell damage and its consequences, but when excessive and unmodulated, may cause progressive destruction of tissue. The aim of this study is to clarify the influence of two anti-inflammatory, diclofenac sodium and dexamethasone on the inflammatory phase of tissue repair process, as well as on the number of mast cells in different stages of repair. It was used 60 Wistar rats, divided into 3 groups (1, 2 and 3), medicated 30 minutes before surgery (lesion 3 mm in diameter in the ventral tongue), with intramuscular injection of 0.235 mg / kg dexamethasone, 4, 45mg/Kg of diclofenac sodium or sterile saline, respectively. The 3 groups were subdivided into a, b, c, d, according to the time of sacrifice, 6, 24, 48 and 120 hours after surgery. The lesions were photographed immediately at surgical time and at sacrifice, the photos were used for clinical analysis of the repair process and morphometry. The tongues were excised and sent for histological processing, the slices were directed for staining with hematoxylin-eosin and toluidine blue, for disclosure and count of the number of mast cells. The characteristics of the repair process were described through qualitative evaluation of the following components: extension of necrotic areas; intensity of interstitial edema, type and intensity of inflammatory infiltrate; degrees of reepithelialization, granulation tissue and neovascularization. Slices stained with HE also had their fields scanned and analyzed morphometrically to quantify reepithelialization, measurements of the granulation tissue, cellularity and edema. The results of semiquantitative histological analysis showed that diclofenac and dexamethasone led to lower inflammatory infiltrate compared to control in the inflammatory phase of repair, although in the productive phase dexamethasone showed less intensity of reepithelialization and neovascularization compared to the other groups. The results of the histomorphometric analysis showed significantly less edema in the diclofenac group at 6h (p = 0.0041) and 24 (p = 0.0429), as well as a lower percentage of cellularity in 6 hours in the dexamethasone group (p <0.0001). The diclofenac group also exhibited lower lesion area at 120h than the other groups (p = 0.0060), indicating greater efficiency of repair. Regarding the quantity of mast cells 24, 48 and 120 hours, the control group exhibited significantly higher values (p <0.0001). Based on these results, we conclude that the dexamethasone group showed the worst performance in relation to repair; that diclofenac sodium showed a better effect on surgical wound closure and greater reduction of inflammatory infiltration, and that both cause a reduction of mast cells in the injured area.
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Estudo comparativo da região Fc de anticorpos IgG1 murinos anafiláticos e não-anafiláticos / Comparative study of the Fc region from murine IgG1 anaphylactic and non anaphylactic antibodiesSilva, Sandriana dos Ramos 15 April 2010 (has links)
Está estabelecido que o processo de glicosilação é essencial para a conformação estrutural e função efetora dos anticorpos. Entretanto, não está completamente claro como diferenças nos carboidratos ligados aos anticorpos podem interferir na sua atividade biológica. Foi previamente descrito que anticorpos IgG1 murinos podem ser divididos em anafiláticos ou não-anafiláticos, de acordo com a sua capacidade de induzir in vivo reação de anafilaxia. Somado a isso, foi verificado que a cadeia de oligossacarídeos N-ligada à molécula de IgG1 é fundamental para a manutenção da sua função efetora. O objetivo do presente trabalho é estudar diferenças estruturais entre os subtipos de IgG murinos que poderiam determinar a sua atividade biológica. O seqüenciamento dos nucleotídeos que codificam os domínios CH2 e CH3 dos dois subtipos de IgG1 permitiu constatar homologia de 100% dessas regiões nas duas moléculas estudadas. Entretanto, ao analisar o padrão de carboidratos N-ligados aos anticorpos IgG1 foi observado maior conteúdo de ácido siálico e fucose na cadeia N-ligada ao anticorpo anafilático em relação à do não-anafilático. Contudo, a remoção de resíduos de ácido siálico por tratamento enzimático do anticorpo IgG1 anafilático resultou na perda da capacidade desta molécula de induzir desgranulação celular in vitro e reação anafilática in vivo, semelhante ao anticorpo IgG1 deglicosilado. Em contraste, a remoção de fucose não afetou a sua função anafilática. A análise por PCR em tempo real da expressão dos genes das enzimas envolvidas no processo de glicosilação das proteínas revelou menor expressão gênica de algumas glicosidases, principalmente as sialiltransferases, no hibridoma e linfócitos B secretores do subtipo IgG1 não-anafilático em relação ao obtido no hibridoma e linfócitos B que secretam a IgG1 anafilática. Além disto, foi observada menor atividade enzimática das sialiltransferases obtidas do hibridoma produtor da IgG1 não-anafilática em relação à do hibridoma que produz a IgG1 anafilática. Em conjunto, estes resultados comprovam que a capacidade de anticorpos IgG1 murinos de induzir anafilaxia é diretamente dependente do conteúdo de ácido siálico presente na cadeia de oligossacarídeos ligada à região Fc do anticorpo, além disso sugerem fortemente que essa maior sialilação observada no tipo anafilático seja resultante da maior expressão gênica destas enzimas e assim da sua atividade enzimática no momento da síntese dos anticorpos. / It is well established that the glycosylation process is essential for the structural conformation and effector function of the antibodies. However, it is quite clear how differences in the carbohydrates attached to the antibodies may interfere with their biological activities. It was previously reported that murine IgG1 antibodies can be divided into anaphylactic or nonanaphylactic according to their ability to induce anaphylaxis. Furthermore, it was demonstrated that the oligosaccharide chain N-linked to the IgG1 is essential for its conformation and biological activity. The objective of this work is to study structural differences between these subtypes of murine IgG1 that could determine their biological activity. The sequencing of the nucleotides encoding the CH2 and CH3 domains of these two subtypes of IgG1 showed 100% of homology in the Fc regions of these molecules. In contrast, the analysis of the carbohydrates N-linked to the IgG1 antibodies demonstrated higher sialic acid and fucose contents in the chain attached to the anaphylactic antibody than in the nonanaphylactic IgG1. However, the removal of sialic acid residues by enzymatic treatment of anaphylactic IgG1 antibody resulted in the abrogation of its ability to induce mast cells degranulation in vitro and anaphylactic reaction in vivo as observed to deglycosylated IgG1 antibody. On the other hand, the removal of fucose did not change the anaphylactic activity. The analysis by real time PCR of the gene expression of enzymes that are involved in the protein glycosylation showed lower gene expression of some glycosyltransferases, mainly sialyltransferases, in the hybridoma and B lymphocytes that produce the non-anaphylactic IgG1 compared to those verified in the hybridoma and B cells producer of the anaphylactic IgG1. Furthermore, it was verified lower enzymatic activity of sialyltransferases purified from the hybridoma producer of the non-anaphylactic IgG1 in relation to the hybridoma producer of the anaphylactic antibody. Together, these results prove that the ability of murine IgG1 to induce anaphylaxis is directly dependent of the sialic acid content in the carbohydrate core attached to the antibody Fc region. It is also strongly suggested that this higher sialylation observed in the anaphylactic IgG1 may be resultant of the higher gene expression and enzymatic activity of some sialyltransferases during the antibody synthesis.
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