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CXCL12 estimula fibroblastos pulmonares a produzir CCL3, CXCL2, LTB4 e LTC4 via p38, MEK1/2, PI-3K e JNK /Danilucci, Taís Marolato. January 2013 (has links)
Orientador: Sandra Helena Penha de Oliveira / Banca: Edson Antunes / Banca: Lucia Helena Faccioli / Resumo: A quimiocina C-X-X motif ligand 12 (CXCL12) e seu receptor de quimiocina 4 (CXCR4) desenvolvem um papel crítico na inflamação das vias aéreas. No entanto, os efeitos da ativação da via CXCL12/CXCR4 sobre fibroblastos pulmonares ainda são desconhecidos. Neste estudo, investigamos o efeito da via CXCL12/CXCR4 sobre a quimiocina (C-C motif) ligante 3 (CCL3) e (C-X-C motif) ligante 2 (CXCL2) e sobre os mediadores lipídicos leucotrienos B4 (LTB4) e C4 (LTC4) por fibroblastos pulmonares e a sinalização intracelular envolvida neste processo. CXCL12 foi capaz de induzir a produção de CCL3, CXCL2, LTB4 e LTC4; a produção de CCL3 não é dependente da produção de CXCL2, mas a produção de CXCL2 é dependente da produção de CCL3. A produção de LTB4 pode ser parcialmente regulada por CXCL2 e CCL3 e a produção de LTC4 é dependente da produção de CCL3 e CXCL2. Fibroblastos pulmonares constitutivamente expressam CXCR4 e a estimulação com CXCL12 induz sua expressão. Análises de Western blot mostraram que CXCL12 aumenta a expressão proteica de CXCR4 e induz a fosforilação da S339 do CXCR4. A expressão gênica constitutiva e induzida de CXCR4 foram inibidas pelo anticorpo anti-CXCL2. No entanto, o anticorpo anti-CCL3 e o inibidor farmacológico MK886 foram capazes de diminuir a expressão gênica induzida de CXCR4. Os fibroblastos pulmonares foram pré-tratados com MK886, dexametasona (Dexa) e/ou loratadina (Lor). MK886 e Lor promoveram a diminuição da produção de LTC4 e LTB4, mas não a de CCL3 e CXCL2. Dexa diminuiu níveis de CCL3, CXCL2, LTB4 e LTC4, e quando associado com Lor esta diminuição foi mais eficaz. Identificamos... / Abstract: C-X-X motif ligand 12 (CXCL12) and its specific receptor Chemokine receptor 4 (CXCR4) play a critical role in airway inflammation. However, the effects of CXCL12/CXCR4 axis on pulmonary fibroblast activation are unknown. In this study, we investigated the effect of CXCL12/CXCR4 axis on chemokine (C-C motif) ligand 3 (CCL3), chemokine (C-X-C motif) ligand 2 (CXCL2), leukotrienes B4 (LTB4) and C4 (LTC4) production by pulmonary fibroblasts and the intracellular signaling involved in the process. CXCL12 induced CCL3, CXCL2, LTB4 and LTC4 production, and CCL3 production is not dependent on CXCL2; but CXCL2 production is dependent on CCL3 production. LTB4 production can be partially down-regulated by CXCL2 and CCL3 production and LTC4 production is dependent on CCL3 and CXCL2 production. Pulmonary fibroblasts constitutively expressed CXCR4, and CXCL12 stimulation up-regulated its expression. Western blot analysis showed that CXCL12 increased protein expression of CXCR4 and induced phosphorylation at S339 of CXCR4. Constitutive CXCR4 expression was decreased by anti-CCL3 antibody or MK 886. Inducible CXCR4 was inhibited by anti-CXCL2 antibody. Indeed pulmonary fibroblasts were pretreated with MK886, dexamethasone (Dexa) and loratadine (Lor). MK886 and loratadine was able to reduced LTB4 and LTC4 production but not CCL3 and CXCL2. Dexa decreased CCL3, CXCL2, LTB4 and LTC4 production, and when associated with Lor this decrease was more effective. We found that PI-3K and JNK intracellular signaling play a role in CCL3 production; p38, MEK1/2, PI-3K and JNK are involved in CXCL2 production and p38 and MEK1/2 pathways are involved in LTB4 production by... / Mestre
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CXCL12 estimula fibroblastos pulmonares a produzir CCL3, CXCL2, LTB4 e LTC4 via p38, MEK1/2, PI-3K e JNKDanilucci, Taís Marolato [UNESP] 05 August 2013 (has links) (PDF)
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000749985.pdf: 1413898 bytes, checksum: 7421bc33cc6d75478dcb676de29021bd (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A quimiocina C-X-X motif ligand 12 (CXCL12) e seu receptor de quimiocina 4 (CXCR4) desenvolvem um papel crítico na inflamação das vias aéreas. No entanto, os efeitos da ativação da via CXCL12/CXCR4 sobre fibroblastos pulmonares ainda são desconhecidos. Neste estudo, investigamos o efeito da via CXCL12/CXCR4 sobre a quimiocina (C-C motif) ligante 3 (CCL3) e (C-X-C motif) ligante 2 (CXCL2) e sobre os mediadores lipídicos leucotrienos B4 (LTB4) e C4 (LTC4) por fibroblastos pulmonares e a sinalização intracelular envolvida neste processo. CXCL12 foi capaz de induzir a produção de CCL3, CXCL2, LTB4 e LTC4; a produção de CCL3 não é dependente da produção de CXCL2, mas a produção de CXCL2 é dependente da produção de CCL3. A produção de LTB4 pode ser parcialmente regulada por CXCL2 e CCL3 e a produção de LTC4 é dependente da produção de CCL3 e CXCL2. Fibroblastos pulmonares constitutivamente expressam CXCR4 e a estimulação com CXCL12 induz sua expressão. Análises de Western blot mostraram que CXCL12 aumenta a expressão proteica de CXCR4 e induz a fosforilação da S339 do CXCR4. A expressão gênica constitutiva e induzida de CXCR4 foram inibidas pelo anticorpo anti-CXCL2. No entanto, o anticorpo anti-CCL3 e o inibidor farmacológico MK886 foram capazes de diminuir a expressão gênica induzida de CXCR4. Os fibroblastos pulmonares foram pré-tratados com MK886, dexametasona (Dexa) e/ou loratadina (Lor). MK886 e Lor promoveram a diminuição da produção de LTC4 e LTB4, mas não a de CCL3 e CXCL2. Dexa diminuiu níveis de CCL3, CXCL2, LTB4 e LTC4, e quando associado com Lor esta diminuição foi mais eficaz. Identificamos... / C-X-X motif ligand 12 (CXCL12) and its specific receptor Chemokine receptor 4 (CXCR4) play a critical role in airway inflammation. However, the effects of CXCL12/CXCR4 axis on pulmonary fibroblast activation are unknown. In this study, we investigated the effect of CXCL12/CXCR4 axis on chemokine (C-C motif) ligand 3 (CCL3), chemokine (C-X-C motif) ligand 2 (CXCL2), leukotrienes B4 (LTB4) and C4 (LTC4) production by pulmonary fibroblasts and the intracellular signaling involved in the process. CXCL12 induced CCL3, CXCL2, LTB4 and LTC4 production, and CCL3 production is not dependent on CXCL2; but CXCL2 production is dependent on CCL3 production. LTB4 production can be partially down-regulated by CXCL2 and CCL3 production and LTC4 production is dependent on CCL3 and CXCL2 production. Pulmonary fibroblasts constitutively expressed CXCR4, and CXCL12 stimulation up-regulated its expression. Western blot analysis showed that CXCL12 increased protein expression of CXCR4 and induced phosphorylation at S339 of CXCR4. Constitutive CXCR4 expression was decreased by anti-CCL3 antibody or MK 886. Inducible CXCR4 was inhibited by anti-CXCL2 antibody. Indeed pulmonary fibroblasts were pretreated with MK886, dexamethasone (Dexa) and loratadine (Lor). MK886 and loratadine was able to reduced LTB4 and LTC4 production but not CCL3 and CXCL2. Dexa decreased CCL3, CXCL2, LTB4 and LTC4 production, and when associated with Lor this decrease was more effective. We found that PI-3K and JNK intracellular signaling play a role in CCL3 production; p38, MEK1/2, PI-3K and JNK are involved in CXCL2 production and p38 and MEK1/2 pathways are involved in LTB4 production by...
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Stem cell factor estimula células da musculatura lisa da traqueia a produzir TGF-β, FGF-2 E CCL3/MIP-1αOliveira, Luis Cezar Farias de [UNESP] 03 August 2012 (has links) (PDF)
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oliveira_lcf_me_araca.pdf: 916072 bytes, checksum: a0dad11c3aa9513daedea50b01cd4ff9 (MD5) / O objetivo deste estudo foi avaliar o mecanismo envolvido na produção de TGF-β, FGF-2 e CCL3/MIP-1α induzida por Stem cell factor (SCF) em células da musculatura lisa de traqueia (CMLT) e as vias de transdução de sinalização ativadas. Traqueias de camundongos normais foram coletadas, fragmentadas e colocadas em garrafas contendo meio de cultura DMEM com 10% de Soro Fetal Bovino. CMLT foram estimuladas com SCF (1, 10 and 100 ng/mL) e avaliadas após 1, 6 e 24 horas. Características fenotípicas das CMLT foram analisadas por imunofluorescência para α-actina de músculo liso (α-AML), α-citoqueratina e α-proteína de ativação de fibroblastos (α-FAP). Ativação de c-kit em CMLT estimuladas por SCF foi avaliada por citometria de fluxo. A expressão de RNAm para TGF-β foi observada pela reação de polimerase em cadeia-transcriptase reversa (RT-PCR) e a produção da proteína, por ensaio de imunoabsorção enzimática (ELISA). A produção de FGF-2 foi avaliada por immunoblot e a produção de CCL3/MIP-1α por ELISA. Em outro conjunto de experimentos, CMLT foram pré-tratadas com inibidores de MAPK p42/44(PD 98059 [PD]), p38(SB 202190[SB]), e JNK (SP 600125 [SP]) por 30 minutos seguidos de estimulação com SCF (10 ng/mL) por 24 horas. Células pré-tratadas com anticorpos específicos não revelaram qualquer marcação para citoqueratina nem para α-FAP, contudo, ocorrendo a marcação para α-AML, indicando a pureza da linhagem celular primária. SCF induziu a expressão de receptores c-kit em CMLT. CMLT estimuladas por 10 ng/mL de SCF expressaram TGF-β mRNA e produziram TGF- β proteína, FGF-2 e CCL3/MIP-1α após 24 horas. O pré-tratamento com SB, PD e SP inibiu estas produções. Estas produções foram mediadas pela ativação das vias p42/44, p38, e JNK. CMLT parecem ser importantes células residentes... / The aim of this study was to evaluate the mechanism involved in SCF-induced TGF-β, FGF-2 and CCL3/MIP-1α production in tracheal smooth muscle cells (tSMC) and the activated signaling transduction pathway. Normal mouse tracheas were collected, fragmented and placed in bottles containing the culture medium DMEM with 10% Fetal Bovine Serum. tSMC primary cultures were stimulated with SCF (1, 10 and 100 ng/mL) and evaluated at 1, 6 and 24 hours. The phenotypic characteristic of tSMC in primary culture was analyzed using immunofluorescence staining for α-smooth muscle actin (α-SMA), α-cytokeratin and α-Fibroblast Activation Protein (α-FAP). c-Kit activation in SCF-stimulated tSMC was evaluated by flow cytometric. The TGF-β mRNA expression was observed by reverse-transcriptase polymerase chain reaction (RT-PCR) and protein production by enzyme-linked immunosorbent assay (ELISA). FGF-2 production was evaluated by immunoblot and CCL3/MIP-1α production by ELISA. In other set of experiment, tSMC were pretreated p42/44 inhibitor (PD 98059 [PD]), p38 inhibitor (SB 202190[SB]), or JNK inhibitor (SP 600125 [SP]) for 30 minutes followed by stimulation with SCF (10 ng/mL) for 24 hour. Cells treated with specific antibodies, showing neither labeling for cytokeratin nor either FAP, however, labeling for α-SMA indicating purity of the primary cell line. SCF induces c-Kit expression on tSMC. SCF-stimulated tSMC express TGF-β mRNA expression and FGF-2 and CCL3/MIP-1α production at 10 ng/mL after 24 hours. SB, PD and SP pre-treatment inhibited these productions. These productions were mediated by activation pathways p42/44, p38, and JNK. tSMC seems to be important resident cells involved in the cell activation and tissue repair, since they are capable of producing growth factors and chemokines and added new... (Complete abstract click electronic access below)
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Histamina induz inflamação das vias aéreas e produção de muco em pulmão de camundongos /Vieira, Letícia Vilma dos Santos. January 2017 (has links)
Orientador: Sandra Helena Penha de Oliveira / Banca: Valeria Marçal Felix de Lima / Banca: Richardt Gama Landgraf / Resumo: Na inflamação alérgica, a histamina desencadeia papel crucial na indução de sintomas, tais como vasodilatação, broncoconstrição e produção de muco. O objetivo deste estudo foi investigar os efeitos da histamina na inflamação e no remodelamento das vias aéreas, avaliando para tanto a produção de muco e colágeno em modelo murino de inflamação pulmonar. Camundongos Balb/c machos de 20-25g foram estimulados, por via intratraqueal, com histamina em diferentes concentrações (10, 50 e 100 µM) e avaliados após 6, 24 e 48 horas. A partir dos dados resultantes do ensaio dose-resposta, foi realizado tratamento farmacológico, onde animais foram separados em 5 grupos (6 camundongos por grupo): Grupo 1 (PBS), controle não estimulado; Grupo 2 (Histamina), controle estimulado; Grupo 3 (Histamina + Loratadina); Grupo 4 (Histamina + Dexametasona) e Grupo 5 (Histamina + Dexametasona e Loratadina). Analisamos a migração de leucócitos no lavado broncoalveolar (LBA) e no tecido pulmonar. As alterações estruturais no tecido pulmonar e na traqueia foram avaliadas por análise histopatológica, bem como a produção de muco e deposição de colágeno usando Alcian blue/Periodic Acid Shiff e Tricômio de Masson, respectivamente. O sobrenadante do LBA e o tecido pulmonar foram coletados para quantificar os níveis SCF e CCL3 por ELISA de captura. A expressão gênica de Muc5ac, Gob-5, Col1a2 e receptores de histamina foi avaliada por RT-PCR em tempo real. Nossos resultados demonstram que a histamina induz infla... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: In allergic inflammation, histamine exerts a crucial role in the induction of symptoms, such as vasodilation, bronchoconstriction and mucus production. The aim of this study was to investigate the effects of histamine in inflammation and airway remodeling, evaluating the mucus production and collagen in a murine model of pulmonary inflammation. Male Balb/c mice of 20-25g were challenged by intratracheal instillation of histamine at different concentrations (10, 50 and 100 μM) and evaluated after 6, 24 and 48 hours. From the data resulting from the dose-response assay, pharmacological treatment was performed, and animals were separated into 5 groups (6 mice per group): Group 1 (PBS), non-challenged control; Group 2 (Histamine), challenged control; Group 3 (Histamine + Loratadine); Group 4 (Histamine + Dexamethasone) and Group 5 (Histamine + Dexamethasone and Loratadine). We analyzed the recruitment of leukocytes in bronchoalveolar lavage (BAL) and lung tissue. Structural changes in lung tissue and trachea were assessed by histopathological analysis, as well as mucus production and collagen deposition using Alcian blue/Periodic Acid Shiff and Masson's Trichrome, respectively. BAL supernatant and lung tissue were collected for SCF and CCL3 levels quantification by capture ELISA. The gene expression of Muc5ac, Gob-5, Col1a2, and histamine receptors was evaluated by RT-PCR real time. Our results demonstrated that histamine induces airway inflammation, with presence of leukocyte infiltrate in BAL and lung tissue. In addition, histamine-challenged animals demonstrated a significant increase in the gene expression of Muc5ac, Gob-5 and Col1a2 in lung tissue, SCF and CCL3 production in BAL and lung, as well as structural changes in the trachea and lung tissue, such as leukocyte infiltrate, presence of goblet cell, mucus production and collagen... / Mestre
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Neuroinflammation & Insulinorésistance : contribution au développement physiopathologique de la maladie d’Alzheimer / Neuroinflammation & Insulinoresistance : involvement in pathophysiological development of Alzheimer DiseaseMarciniak, Elodie 14 December 2015 (has links)
La maladie d’Alzheimer (MA) est une maladie neurodégénérative caractérisée d’un point de vue anatomopathologique par une accumulation extracellulaire de peptides amyloïdes et d’une dégénérescence neurofibrillaire (DNF), résultant de l’agrégation intraneuronale de protéines Tau hyper-et-anormalement phosphorylées. D’autres déterminants sont également associés à la MA dont une neuroinflammation chronique et une insulinorésistance centrale qui contribueraient tous deux au développement des lésions ainsi qu’aux troubles synaptiques et mnésiques associés.La neuroinflammation observée dans la MA est caractérisée par une activation des cellules gliales, une infiltration lymphocytaire ainsi que par la libération de médiateurs inflammatoires solubles dont les chimiokines. Le CCL3 est une chimiokine hautement dérégulée dans le cerveau des patients de MA. Dans notre laboratoire, nous avons montré que dans un modèle de DNF, le CCL3 était le facteur pro-inflammatoire le plus affecté au niveau hippocampique laissant ouverte l’hypothèse d’un rôle dans les dysfonctions mnésiques associées à la pathologie Tau. Pour aborder cette question, nous avons précisément évalué l’impact du CCL3 sur le fonctionnement synaptique hippocampique et sur les fonctions mnésiques. Nos travaux montrent que l’application de CCL3 sur des tranches d’hippocampe entraine une diminution des activités basales ainsi que de la potentialisation à long terme (LTP) sans altérer le fonctionnement présynaptique ni la dépression synaptique à long terme (LTD). Par ailleurs, l’élévation intracérébrale de CCL3 par injections intracérébroventriculaires sub-chroniques affecte également la transmission synaptique basale et la LTP ainsi que la mémoire spatiale à court terme et la mémoire à long terme. La réversion de ces altérations par le Maraviroc permet de conclure que l’effet néfaste du CCL3 est dépendant du récepteur CCR5. Ainsi, ces travaux soulignent l’importance du CCL3 dans la physiopathologie de la MA, notamment en lien avec la DNF.L’insulinorésistance observée dans les cerveaux de patients atteints de la MA est connue pour favoriser le développement des lésions caractéristiques et est suggérée comme participant aux atteintes synaptiques et mnésiques. Cependant les causes de cette insulinorésistance centrale sont peu connues. Quelques études montrent que les oligomères d’Aβ, le diabète de type II ou même la neuroinflammation sont susceptibles de conduire à une insulinorésistance centrale. Néanmoins, à ce jour aucune relation n'a été établie avec la protéine Tau. La seconde partie de ce travail s’est attachée à étudier le rôle de la protéine Tau dans la régulation de la réponse centrale à l’insuline. Diverses expériences réalisées in vitro et in vivo montrent que la surexpression de la protéine Tau induit une augmentation de la sensibilité neuronale à l’insuline alors que la délétion de Tau aboutit à l’effet inverse. Cette régulation semble être liée à une interaction entre la protéine Tau et PTEN, une phospholipase inhibitrice de la signalisation de l’insuline. L’interaction entre Tau et PTEN réduirait l’activité de cette dernière, favorisant de ce fait l’action de l’insuline au niveau central. Par ailleurs, des données physiologiques indiquent que cette régulation de la signalisation centrale de l’insuline pourrait avoir une répercussion sur la régulation de l’homéostasie énergétique. En ce sens, la délétion de la protéine Tau induit une prise de poids, une hyperinsulinémie et une glucointolérance périphérique. Ces données proposent donc une nouvelle fonction de la protéine Tau dans la signalisation neuronale et le métabolisme. Cette perte de fonction dans la MA pourrait expliquer les mécanismes d’insulinorésistance centrale liés à la DNF.En conclusion, nos données mettent en évidence deux mécanismes liant la pathologie Tau aux atteintes mnésiques, l’un passant par la production de la chimiokine CCL3, l’autre impliquant une résistance neuronale à l’insuline. / Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by extracellular amyloid deposits and intraneuronal neurofibrillary tangles, made of aggregated and abnormally hyperphosphorylated Tau proteins. Other components are also involved in AD pathophysiology, including chronic neuroinflammation and central insulinoresistance that would contribute both to the development of Alzheimer lesions as well as associated synaptic and memory impairments.Neuroinflammation observed in AD is characterized by glial cell activation, lymphocyte infiltration, and the release of soluble inflammatory mediators including chemokines. CCL3 is a highly upregulated chemokine in the brain of AD patients. In our lab, we have shown, in a mouse model of Tau pathology, that hippocampal CCL3 was largely upregulated and, thus, we made the assumptions that such increase could play a key-role in the memory dysfunctions associated with Tau pathology. To address to this question, we precisely evaluated the impact of CCL3 upon hippocampal synaptic activity and memory function. Our data show that CCL3 application on hippocampal slices induces a significant decrease of basal synaptic activity and long term potentiation (LTP) impairment without affecting presynaptic activity and long term depression (LTD). Further, intracerebral elevation of CCL3 by sub-chronical intracerebroventricular injections was also found to impact hippocampal basal synaptic activity and LTP but also short term spatial memory and long term memory. Reversion of these alterations by Maraviroc finally suggests that CCL3 deleterious effects are CCR5 dependent. Overall, these studies show the important role of CCL3 towards plasticity and memory as well as in AD physiopathology.Besides chronic inflammation, insulinoresistance observed in AD brain is suggested to favor the development of amyloid and Tau lesions but also to participate to synaptic impairments underlying memory loss. However, origins of the brain insulinoresistance described in AD are unclear. Previous studies ascribed central insulin-resistance to Aβ oligomers, type II diabetes or even neuroinflammation. So far, no relationship has been established with Tau protein. The aim of the second part of the present thesis was evaluate the potential role of Tau protein towards the regulation of central insulin sensitivity. Various experiments performed in vitro and in vivo show that Tau favors the neuronal response to insulin, whereas Tau deletion favors insulin-resistance. This regulation seems to be related to an interaction between Tau and PTEN, a phospholipase inhibiting insulin signaling, which results in a reduced PTEN activity, itself favoring insulin pathway activation. Regulation of brain insulin signaling is known to modulate energy homeostasis, food intake and weight gain. In line with the idea that Tau protein modulates insulin signaling, we found that Tau deletion induces weight gain, hyperinsulinemia and glucointolerance. Together, these data provide a new function for Tau in the control of neuronal signaling and peripheral metabolism. These data also highlight that the loss of Tau function in AD might explain at last in part the central insulinoresistance described as “type 3 diabetes”.In conclusion, our data highlight two mechanisms linking Tau pathology and memory deficits, one through the detrimental effect of the chemokine CCL3 and the other involving neuronal insulin resistance.
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Stem cell factor estimula células da musculatura lisa da traqueia a produzir TGF-β, FGF-2 E CCL3/MIP-1α /Oliveira, Luis Cezar Farias de. January 2012 (has links)
Orientador: Sandra Helena Penha de Oliveira / Banca: Lúcia Helena Faccioli / Banca: Carla Máximo Prado / Resumo: O objetivo deste estudo foi avaliar o mecanismo envolvido na produção de TGF-β, FGF-2 e CCL3/MIP-1α induzida por Stem cell factor (SCF) em células da musculatura lisa de traqueia (CMLT) e as vias de transdução de sinalização ativadas. Traqueias de camundongos normais foram coletadas, fragmentadas e colocadas em garrafas contendo meio de cultura DMEM com 10% de Soro Fetal Bovino. CMLT foram estimuladas com SCF (1, 10 and 100 ng/mL) e avaliadas após 1, 6 e 24 horas. Características fenotípicas das CMLT foram analisadas por imunofluorescência para α-actina de músculo liso (α-AML), α-citoqueratina e α-proteína de ativação de fibroblastos (α-FAP). Ativação de c-kit em CMLT estimuladas por SCF foi avaliada por citometria de fluxo. A expressão de RNAm para TGF-β foi observada pela reação de polimerase em cadeia-transcriptase reversa (RT-PCR) e a produção da proteína, por ensaio de imunoabsorção enzimática (ELISA). A produção de FGF-2 foi avaliada por immunoblot e a produção de CCL3/MIP-1α por ELISA. Em outro conjunto de experimentos, CMLT foram pré-tratadas com inibidores de MAPK p42/44(PD 98059 [PD]), p38(SB 202190[SB]), e JNK (SP 600125 [SP]) por 30 minutos seguidos de estimulação com SCF (10 ng/mL) por 24 horas. Células pré-tratadas com anticorpos específicos não revelaram qualquer marcação para citoqueratina nem para α-FAP, contudo, ocorrendo a marcação para α-AML, indicando a pureza da linhagem celular primária. SCF induziu a expressão de receptores c-kit em CMLT. CMLT estimuladas por 10 ng/mL de SCF expressaram TGF-β mRNA e produziram TGF- β proteína, FGF-2 e CCL3/MIP-1α após 24 horas. O pré-tratamento com SB, PD e SP inibiu estas produções. Estas produções foram mediadas pela ativação das vias p42/44, p38, e JNK. CMLT parecem ser importantes células residentes... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The aim of this study was to evaluate the mechanism involved in SCF-induced TGF-β, FGF-2 and CCL3/MIP-1α production in tracheal smooth muscle cells (tSMC) and the activated signaling transduction pathway. Normal mouse tracheas were collected, fragmented and placed in bottles containing the culture medium DMEM with 10% Fetal Bovine Serum. tSMC primary cultures were stimulated with SCF (1, 10 and 100 ng/mL) and evaluated at 1, 6 and 24 hours. The phenotypic characteristic of tSMC in primary culture was analyzed using immunofluorescence staining for α-smooth muscle actin (α-SMA), α-cytokeratin and α-Fibroblast Activation Protein (α-FAP). c-Kit activation in SCF-stimulated tSMC was evaluated by flow cytometric. The TGF-β mRNA expression was observed by reverse-transcriptase polymerase chain reaction (RT-PCR) and protein production by enzyme-linked immunosorbent assay (ELISA). FGF-2 production was evaluated by immunoblot and CCL3/MIP-1α production by ELISA. In other set of experiment, tSMC were pretreated p42/44 inhibitor (PD 98059 [PD]), p38 inhibitor (SB 202190[SB]), or JNK inhibitor (SP 600125 [SP]) for 30 minutes followed by stimulation with SCF (10 ng/mL) for 24 hour. Cells treated with specific antibodies, showing neither labeling for cytokeratin nor either FAP, however, labeling for α-SMA indicating purity of the primary cell line. SCF induces c-Kit expression on tSMC. SCF-stimulated tSMC express TGF-β mRNA expression and FGF-2 and CCL3/MIP-1α production at 10 ng/mL after 24 hours. SB, PD and SP pre-treatment inhibited these productions. These productions were mediated by activation pathways p42/44, p38, and JNK. tSMC seems to be important resident cells involved in the cell activation and tissue repair, since they are capable of producing growth factors and chemokines and added new... (Complete abstract click electronic access below) / Mestre
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CCL3 Augments Antitumor Responses in CT26 by Enhancing Cellular Trafficking and Interferon-Gamma ExpressionAllen, Frederick, Jr. 02 February 2018 (has links)
No description available.
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Histamina induz inflamação das vias aéreas e produção de muco em pulmão de camundongos / Histamine induces airway inflammation and mucus production in lung tissue of miceVieira, Letícia Vilma dos Santos [UNESP] 07 August 2017 (has links)
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Previous issue date: 2017-08-07 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Na inflamação alérgica, a histamina desencadeia papel crucial na indução de sintomas, tais como vasodilatação, broncoconstrição e produção de muco. O objetivo deste estudo foi investigar os efeitos da histamina na inflamação e no remodelamento das vias aéreas, avaliando para tanto a produção de muco e colágeno em modelo murino de inflamação pulmonar. Camundongos Balb/c machos de 20-25g foram estimulados, por via intratraqueal, com histamina em diferentes concentrações (10, 50 e 100 µM) e avaliados após 6, 24 e 48 horas. A partir dos dados resultantes do ensaio dose-resposta, foi realizado tratamento farmacológico, onde animais foram separados em 5 grupos (6 camundongos por grupo): Grupo 1 (PBS), controle não estimulado; Grupo 2 (Histamina), controle estimulado; Grupo 3 (Histamina + Loratadina); Grupo 4 (Histamina + Dexametasona) e Grupo 5 (Histamina + Dexametasona e Loratadina). Analisamos a migração de leucócitos no lavado broncoalveolar (LBA) e no tecido pulmonar. As alterações estruturais no tecido pulmonar e na traqueia foram avaliadas por análise histopatológica, bem como a produção de muco e deposição de colágeno usando Alcian blue/Periodic Acid Shiff e Tricômio de Masson, respectivamente. O sobrenadante do LBA e o tecido pulmonar foram coletados para quantificar os níveis SCF e CCL3 por ELISA de captura. A expressão gênica de Muc5ac, Gob-5, Col1a2 e receptores de histamina foi avaliada por RT-PCR em tempo real. Nossos resultados demonstram que a histamina induz inflamação das vias aéreas, com presença de infiltrado leucocitário no LBA e no tecido pulmonar. Além disso, os animais estimulados com histamina demonstraram aumento significativo na expressão gênica de Muc5ac, Gob-5 e Col1a2 no tecido pulmonar, produção SCF e CCL3 no LBA e pulmão, bem como apresentaram alterações estruturais na traqueia e no tecido pulmonar, tais como infiltrado leucocitário, presença de células caliciformes, produção de muco e depósito de colágeno. O pré-tratamento com dexametasona (DEX) inibiu a migração leucocitária no LBA e tecido pulmonar, e também diminuiu a expressão gênica de Muc5ac no tecido pulmonar, diminuiu a produção de SCF e CCL3 no LBA e pulmão, bem como reduziu a produção de muco e a deposição de colágeno no pulmão. Já o pré-tratamento com loratadina (LOR), bloqueador de H1, inibiu parcialmente a migração celular no LBA, mas não no tecido pulmonar, enquanto que a associação farmacológica entre DEX e LOR diminuiu significativamente a migração leucocitária tanto no LBA como no tecido pulmonar. Com base nos dados acima, sugere-se que a histamina induz inflamação das vias aéreas, promovendo a migração de leucócitos possivelmente através da produção de SCF e CCL3, bem como provoca alterações estruturais na traqueia e no tecido pulmonar, tais como produção de muco, deposição de colágeno, e esses efeitos tem a participação dos receptores H1 e H4. / In allergic inflammation, histamine exerts a crucial role in the induction of symptoms, such as vasodilation, bronchoconstriction and mucus production. The aim of this study was to investigate the effects of histamine in inflammation and airway remodeling, evaluating the mucus production and collagen in a murine model of pulmonary inflammation. Male Balb/c mice of 20-25g were challenged by intratracheal instillation of histamine at different concentrations (10, 50 and 100 μM) and evaluated after 6, 24 and 48 hours. From the data resulting from the dose-response assay, pharmacological treatment was performed, and animals were separated into 5 groups (6 mice per group): Group 1 (PBS), non-challenged control; Group 2 (Histamine), challenged control; Group 3 (Histamine + Loratadine); Group 4 (Histamine + Dexamethasone) and Group 5 (Histamine + Dexamethasone and Loratadine). We analyzed the recruitment of leukocytes in bronchoalveolar lavage (BAL) and lung tissue. Structural changes in lung tissue and trachea were assessed by histopathological analysis, as well as mucus production and collagen deposition using Alcian blue/Periodic Acid Shiff and Masson's Trichrome, respectively. BAL supernatant and lung tissue were collected for SCF and CCL3 levels quantification by capture ELISA. The gene expression of Muc5ac, Gob-5, Col1a2, and histamine receptors was evaluated by RT-PCR real time. Our results demonstrated that histamine induces airway inflammation, with presence of leukocyte infiltrate in BAL and lung tissue. In addition, histamine-challenged animals demonstrated a significant increase in the gene expression of Muc5ac, Gob-5 and Col1a2 in lung tissue, SCF and CCL3 production in BAL and lung, as well as structural changes in the trachea and lung tissue, such as leukocyte infiltrate, presence of goblet cell, mucus production and collagen deposition around the airways. Dexamethasone pre-treatment (DEX) inhibited leukocyte recruitment in BAL and lung tissue, decreased gene expression of Muc5ac in lung tissue, decreases SCF and CCL3 production in BAL and lung, as well as mucus production and collagen deposition in the lung. Already pre-treatment with loratadine (LOR), an H1 blocker, partially inhibited cell migration in BAL, but not in lung tissue, whereas the pharmacological association between DEX and LOR significantly reduced leukocyte recruitment in both the BAL and the lung tissue. Putting the above data together, we suggest that histamine induces airway inflammation, promoting the recruitment of leukocytes possibly through the production of SCF and CCL3, as well as causes structural changes in the trachea and lung tissue, such as mucus production, collagen deposition, and these effects might be mediated by H1 and H4 receptors.
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A Novel Motif in HIV-1 Nef that Regulates MIP-1β Chemokine Release in Macrophages: A DissertationDai, Lue 17 June 2010 (has links)
Nef is an accessory protein encoded by human and simian immunodeficiency viruses (HIV and SIV), and is critical for viral pathogenicity in vivo.The structure of Nef has been resolved and the major cellular activities of Nef are generally described as down-regulation of cell surface molecules, enhancement of virus infectivity and regulation of cell signaling and activation. Macrophages represent a key target of HIV-1 infection and may contribute significantly to viral pathogenesis by facilitating viral propagation, maintaining a viral reservoir and regulating viral replication. During HIV-1 infection, various cytokines and chemokines are induced for viral advantages more than for host defense.
We have previously demonstrated that HIV-1 Nef regulates the release of chemokines, MIP-1α and MIP-1ß, from infected macrophages and have proposed that this may enhance conditions for viral replication by promoting recruitment of substrate lymphocytes to sites of infection (1). However, the molecular basis for this Nef activity remains to be defined. The main goals of this thesis are to identify the functional motif in Nef that is responsible for chemokine induction in macrophages and to elucidate the relevance of this motif to other Nef functions. Using a mutagenesis approach, we have eventually identified a novel motif (KEK) that regulates chemokine production in infected macrophages after we excluded several previously described Nef motifs. This motif is conserved in both HIV-1 and SIV Nef proteins. Mutations in this domain abrogated MIP-1ß induction as well as the Nef-dependent release of other secretory factors by macrophages. However, disruption of this motif did not affect other Nef-ascribed activities such as CD4 and MHC-I down-regulation. In addition, we have determined the involvement of viral Env proteins in Nef-induced chemokine production. Distinct signaling pathways that regulate chemokine release in macrophage will also be described. Finally, several possible roles of the KEK motif are proposed and some preliminary results of co-immunoprecipitation experiments will be presented which aim to characterize cellular proteins involved in chemokine regulation by Nef. Collectively, our studies reveal a specific determinant within Nef that is critical for chemokine release by Nef. Identification of this motif paves the way for future studies to explore the molecular machanisms of Nef-regulated cell signaling pathways. Such knowledge may point to new therapeutic strategies that interrupt Nef function and limit the course of HIV-1 infection.
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