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1.
J Leukoc Biol ; 99(3): 467-73, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26428677

RESUMO

Although neutrophils are typically the first immune cells attracted to an infection site, little is known about how neutrophils dynamically interact with invading pathogens in vivo. Here, with the use of intravital microscopy, we demonstrate that neutrophils migrate to the arrested Cryptococcus neoformans, a leading agent to cause meningoencephalitis, in the brain microvasculature. Following interactions with C. neoformans, neutrophils were seen to internalize the organism and then circulate back into the bloodstream, resulting in a direct removal of the organism from the endothelial surface before its transmigration into the brain parenchyma. C. neoformans infection led to enhanced expression of adhesion molecules macrophage 1 antigen on neutrophils and ICAM-1 on brain endothelial cells. Depletion of neutrophils enhanced the brain fungal burden. Complement C3 was critically involved in the recognition of C. neoformans by neutrophils and subsequent clearance of the organism from the brain. Together, our finding of the direct removal of C. neoformans by neutrophils from its arrested site may represent a novel mechanism of host defense in the brain, in addition to the known, direct killing of microorganisms at the infection sites. These data are the first to characterize directly the dynamic interactions of leukocytes with a microbe in the brain of a living animal.


Assuntos
Encéfalo/microbiologia , Encéfalo/parasitologia , Cryptococcus neoformans/imunologia , Endotélio Vascular/microbiologia , Endotélio Vascular/parasitologia , Neutrófilos/imunologia , Animais , Encéfalo/irrigação sanguínea , Complemento C3/imunologia , Molécula 1 de Adesão Intercelular/análise , Antígeno de Macrófago 1/análise , Camundongos Endogâmicos C57BL , Monócitos/imunologia
2.
J Immunol Methods ; 426: 120-7, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26342259

RESUMO

ß2 integrins play critical roles in migration of immune cells and in the interaction with other cells, pathogens, and the extracellular matrix. Among the ß2 integrins, Mac-1 (Macrophage antigen-1), composed of CD11b and CD18, is mainly expressed in innate immune cells and plays a major role in cell migration and trafficking. In order to image Mac-1-expressing cells both in live cells and mouse, we generated a knock-in (KI) mouse strain expressing CD11b conjugated with monomeric yellow fluorescent protein (mYFP). Expression of CD11b-mYFP protein was confirmed by Western blot and silver staining of CD11b-immunoprecipitates and total cell lysates from the mouse splenocytes. Mac-1-mediated functions of the KI neutrophils were comparable with those in WT cells. The fluorescence intensity of CD11b-mYFP was sufficient to image CD11b expressing cells in live mice using intravital two-photon microscopy. In vitro, dynamic changes in the intracellular localization of CD11b molecules could be measured by epifluorescent microscopy. Finally, CD11b-expressing immune cells from tissue were easily detected by flow cytometry without anti-CD11b antibody staining.


Assuntos
Antígeno CD11b/imunologia , Antígeno de Macrófago 1/análise , Antígeno de Macrófago 1/imunologia , Neutrófilos/imunologia , Baço/citologia , Animais , Proteínas de Bactérias/genética , Antígenos CD18/imunologia , Adesão Celular , Movimento Celular/imunologia , Matriz Extracelular/imunologia , Citometria de Fluxo , Técnicas de Introdução de Genes , Molécula 1 de Adesão Intercelular/metabolismo , Proteínas Luminescentes/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia de Fluorescência , Fagocitose/imunologia , Baço/imunologia , Coloração e Rotulagem/métodos
3.
Cell Transplant ; 23(3): 345-54, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23394628

RESUMO

We have previously shown that interspecies incompatibility of CD47 plays an important role in triggering rejection of xenogeneic hematopoietic cells by macrophages. However, whether CD47 incompatibility also induces rejection of nonhematopoietic cellular xenografts remains unknown. Herein, we have addressed this question in a mouse model of hepatocyte transplantation in which CD47(-/-) hepatocytes were used to resemble xenografts for CD47 incompatibility. We show that intrasplenic transplantation of CD47(-/-), but not wild-type (WT) hepatocytes, into partially hepatectomized syngeneic WT mice resulted in a rapid increase in Mac-1(+) cells with an activation phenotype (i.e., Mac-1(+)CD14(+) and Mac-1(+)CD16/32(high)), compared to nontransplant controls. In addition, CD47(-/-) hepatocytes were more severely damaged than WT hepatocytes as indicated by the greater AST and ALT serum levels in these mice. Furthermore, long-term donor hepatocyte survival and liver repopulation were observed in mice receiving WT hepatocytes, whereas CD47(-/-) hepatocytes were completely rejected within 2 weeks. These results suggest that CD47 on donor hepatocytes prevents recipient myeloid innate immune cell activation, hence aiding in graft survival after hepatocyte transplantation. Thus, CD47 incompatibility is likely to present an additional barrier to hepatocyte xenotransplantation.


Assuntos
Antígeno CD47/genética , Deleção de Genes , Rejeição de Enxerto/genética , Hepatócitos/transplante , Animais , Antígeno CD47/imunologia , Células Cultivadas , Rejeição de Enxerto/imunologia , Hepatócitos/imunologia , Hepatócitos/metabolismo , Imunidade Inata , Antígeno de Macrófago 1/análise , Antígeno de Macrófago 1/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células Mieloides/citologia , Células Mieloides/imunologia
4.
World J Gastroenterol ; 19(37): 6258-64, 2013 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-24115824

RESUMO

AIM: To investigate the stress-induced apoptosis of natural killer (NK) cells and the changes in their killing activity in mouse livers. METHODS: A restraint stress model was established in mice. Flow cytometry was employed to measure the percentage of NK cells and the changes in their absolute number in mouse liver. The cytotoxicity of hepatic and splenic NK cells was assessed against YAC-1 target cells via a 4 h 51Cr-release assay. RESULTS: The restraint stress stimulation induced the apoptosis of NK cells in the liver and the spleen, which decreased the cell number. The number and percentage of NK cells in the spleen decreased. However, the number of NK cells in the liver decreased, whereas the percentage of NK cells was significantly increased. The apoptosis of NK cells increased gradually with prolonged stress time, and the macrophage-1 (Mac-1)(+) NK cells were more susceptible to apoptosis than Mac-1(-) NK cells. Large numbers of Mac-1(-) NK cells in the liver, which are more resistant to stress-induced apoptosis, were observed than the Mac-1(-) NK cells in the spleen. The stress stimulation diminished the killing activity of NK cells in the spleen was significantly decreased, but the retention of numerous Mac-1(-) NK cells in the liver maintained the killing ability. CONCLUSION: Significant stress-induced apoptosis was observed among Mac-1(+) NK cells, but not Mac-1(-) NK cells in the mouse liver. Stress stimulation markedly decreased the killing activity of NK cells in the spleen but remained unchanged in the liver.


Assuntos
Apoptose , Células Matadoras Naturais/patologia , Fígado/patologia , Restrição Física/psicologia , Baço/patologia , Estresse Psicológico/patologia , Animais , Biomarcadores/análise , Células Matadoras Naturais/imunologia , Fígado/imunologia , Antígeno de Macrófago 1/análise , Camundongos , Camundongos Endogâmicos C57BL , Baço/imunologia , Estresse Psicológico/imunologia
5.
Cell Physiol Biochem ; 30(4): 853-62, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22907484

RESUMO

TFF2 is one of the members of the trefoil factor family, known for its role in protection of gastrointestinal epithelia upon injury; however, recent studies suggest that TFF2 could also play an important role in the immune system. In the present study Tff2 deficient and wild type mice were infected by Y. enterocolitica which resulted in a lethal outcome in all Tff2 deficient mice, but not in WT animals. Yersinia invaded Peyer's patches more efficiently as shown by high bacterial titers in the KO mice while wild type mice displayed lower titers and a visible bacterial accumulation in the intestine. Bacterial accumulation in Peyer's patches of Tff2 deficient mice was accompanied by increased recruitment of macrophages. While an increased level of MAC-1 positive cells was observed in the spleens of both Tff2 deficient and WT mice at third day post infection, bacterial dissemination to liver, lung and kidneys was observed only in Tff2 knock-out mice. Analysis of the cellular composition of spleen did not reveal any substantial alteration to WT animals, suggesting possible disregulation of hemopoietic cells involved in immune response to Y. enterocolitica. These new data indicate that Tff2 plays an important role in immune response by protecting the organism from consequences of infection and that Tff2 knock-out mice react adversely to bacterial infections, in this case specifically to Y. enterocolitica.


Assuntos
Técnicas de Inativação de Genes , Intestinos/microbiologia , Mucinas/genética , Proteínas Musculares/genética , Peptídeos/genética , Nódulos Linfáticos Agregados/microbiologia , Yersiniose/genética , Yersiniose/transmissão , Yersinia enterocolitica/patogenicidade , Animais , Feminino , Imunidade Inata , Mucosa Intestinal/metabolismo , Intestinos/imunologia , Intestinos/patologia , Antígeno de Macrófago 1/análise , Antígeno de Macrófago 1/imunologia , Camundongos , Camundongos Knockout , Mucinas/imunologia , Proteínas Musculares/imunologia , Peptídeos/imunologia , Nódulos Linfáticos Agregados/imunologia , Nódulos Linfáticos Agregados/metabolismo , Nódulos Linfáticos Agregados/patologia , Baço/imunologia , Baço/metabolismo , Baço/microbiologia , Baço/patologia , Fator Trefoil-2 , Yersiniose/imunologia , Yersiniose/patologia , Yersinia enterocolitica/imunologia
6.
Cell Immunol ; 272(1): 71-8, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-22019129

RESUMO

Natural killer (NK) cells are important effectors in resistance to viral infections. The role of NK cells in the acute response to human immunodeficiency virus 1 (HIV-1) infected cells was investigated in a mouse model based on a HIV-1/murine leukemia virus (MuLV) pseudovirus. Splenocytes infected with HIV-1/MuLV were injected intraperitoneally and local immunologic responses and persistence of infected cells were investigated. In vivo depletion with an anti-NK1.1 antibody showed that NK cells are important in resistance to virus infected cells. Moreover, NK cell frequency in the peritoneal cavity increased in response to infected cells and these NK cells had a more mature phenotype, as determined by CD27 and Mac-1 expression. Interestingly, after injection of HIV-1/MuLV infected cells, but not MuLV infected cells, peritoneal NK cells had an increased cytotoxic activity. In conclusion, NK cells play a role in the early control of HIV-1/MuLV infected cells in vivo.


Assuntos
Anticorpos Neutralizantes/farmacologia , Infecções por HIV/imunologia , HIV-1/imunologia , Imunidade Inata , Células Matadoras Naturais , Vírus da Leucemia Murina/imunologia , Vírus Reordenados/imunologia , Animais , Anticorpos Neutralizantes/efeitos adversos , Citotoxicidade Imunológica/efeitos dos fármacos , Citometria de Fluxo , Infecções por HIV/patologia , Infecções por HIV/virologia , HIV-1/genética , HIV-1/metabolismo , Humanos , Injeções Intraperitoneais , Células Matadoras Naturais/citologia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/virologia , Vírus da Leucemia Murina/genética , Vírus da Leucemia Murina/metabolismo , Ativação Linfocitária , Depleção Linfocítica , Antígeno de Macrófago 1/análise , Antígeno de Macrófago 1/biossíntese , Macrófagos/citologia , Macrófagos/imunologia , Macrófagos/virologia , Camundongos , Camundongos Transgênicos , Monócitos/citologia , Monócitos/imunologia , Monócitos/virologia , Vírus Reordenados/genética , Vírus Reordenados/metabolismo , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/análise , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/biossíntese , Carga Viral/efeitos dos fármacos , Carga Viral/imunologia
7.
J Virol ; 85(19): 10201-12, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21775455

RESUMO

Plasmid DNA vaccines serve in a wide array of applications ranging from prophylactic vaccines to potential therapeutic tools against infectious diseases and cancer. In this study, we analyzed the mechanisms underlying the activation of natural killer (NK) cells and their potential role in adaptive immunity during DNA-based immunization against hepatitis B virus surface antigen in mice. We observed that the mature Mac-1(+) CD27(-) NK cell subset increased in the liver of mice early after DNA injection, whereas the number of the less mature Mac-1(+) CD27(+) NK cells in the liver and spleen was significantly reduced. This effect was attributed to bacterial sequences present in the plasmid backbone rather than to the encoded antigen and was not observed in immunized MyD88-deficient mice. The activation of NK cells by plasmid-DNA injection was associated with an increase in their effector functions that depended on the expressed antigen. Maturation of NK cells was abrogated in the absence of T cells, suggesting that cross talk exists between NK cells and antigen-specific T cells. Taken together, our data unravel the mechanics of plasmid vector-induced maturation of NK cells and plasmid-encoded antigen-dependent activation of NK cells required for a crucial role of NK cells in DNA vaccine-induced immunogenicity.


Assuntos
Antígenos de Superfície da Hepatite B/imunologia , Vacinas contra Hepatite B/imunologia , Imunização/métodos , Células Matadoras Naturais/imunologia , Ativação Linfocitária , Vacinas de DNA/imunologia , Animais , Antígenos de Superfície da Hepatite B/genética , Vacinas contra Hepatite B/genética , Células Matadoras Naturais/química , Fígado/imunologia , Antígeno de Macrófago 1/análise , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/deficiência , Plasmídeos , Baço/imunologia , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/análise , Vacinas de DNA/genética
8.
Int J Cardiol ; 140(1): 82-7, 2010 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-19042046

RESUMO

BACKGROUND: The inflammatory response to percutaneous coronary intervention (PCI) contributes to restenosis. However, it is not known if advances in PCI have attenuated this response. This study sought to determine the prevalence of systemic inflammation immediately after contemporary PCI, and to identify the predictors of the acute proinflammatory response to PCI. METHODS: Twenty seven consecutive eligible patients undergoing uncomplicated single lesion PCI were recruited. Clinical and procedural characteristics were collected. Neutrophil Mac-1 and plasma matrix metalloproteinase-9 (MMP-9) levels were measured by flow cytometry and ELISA. RESULTS: Overall, neutrophils were de-activated post-procedure [median (IQR) Mac-1: 329(277-555) versus 423 (273-533) MFI, p=0.011] but MMP-9 was unchanged [2.6 (1.8-5.1) versus 2.0 (1.5-3.8) ng/ml, p=ns]. There was a heterogeneous inflammatory response: Neutrophils were activated in 6 (22%) patients, whilst plasma MMP-9 rose in 10 (37%) patients. Twelve (44%) patients had either neutrophil activation or increased MMP-9 level post-procedure. There was no relationship between these two biomarkers. Lesion length predicted both neutrophil activation (OR, 95%CI: 19.0, 2.0-178.0, p=0.010) and increased MMP-9 (16.0, 1.5-17.2, p=0.022), and lesion complexity predicted the latter (9.6, 1.5-62.2, p=0.018). Presentation with an acute coronary syndrome, diabetes mellitus, receipt of drug-eluting stent, and stent diameter were not associated with an acute inflammatory response to PCI. CONCLUSIONS: In contrast to the balloon angioplasty era, widespread inflammation is absent in most patients after contemporary PCI. Lesion length and complexity predicted an inflammatory reaction, suggesting it to be primarily a response to vascular injury.


Assuntos
Angioplastia Coronária com Balão , Reestenose Coronária/epidemiologia , Inflamação/sangue , Idoso , Reestenose Coronária/sangue , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Humanos , Inflamação/fisiopatologia , Antígeno de Macrófago 1/análise , Masculino , Metaloproteinase 9 da Matriz/sangue , Pessoa de Meia-Idade , Ativação de Neutrófilo/fisiologia
9.
J Neurol ; 256(8): 1296-302, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19353220

RESUMO

Leukocyte recruitment and inflammatory response play an important patho-physiologic role after cerebral ischemia. This study aimed to evaluate whether leukocyte adhesion molecules can predict clinical outcome in patients after ischemic stroke. We prospectively examined serial changes in p-selectin glycoprotein ligand-1 (PSGL-1), macrophage antigen-1 (Mac-1), and lymphocyte function-associated antigen-1 (LFA-1) expression by leukocyte subsets using flow cytometry at various time points in 65 acute ischemic stroke patients and 60 controls. PSGL-1 expression on neutrophils and monocytes was significantly higher from day 1 to 90 after stroke as compared with control subjects (p < 0.05). The expression of monocyte Mac-1, LFA-1, and neutrophil Mac-1 were also significantly increased on days 1 and 7 after stroke than in control subjects (p < 0.05). Neutrophil PSGL-1 expression on day 1 was significantly higher in patients with early neurologic deterioration (END) (p < 0.01). Monocyte Mac-1 expression positively correlated with National Institutes of Health Stroke Scale (NIHSS) scores on admission (p = 0.013, gamma = 0.318). Underlying disease of diabetes mellitus and NIHSS score on admission were independently associated with 3-month outcome. The expressions of leukocyte adhesion molecules on admission are significantly increased in patients with acute ischemic stroke. This study shows that higher neutrophil PSGL-1 expression on admission may imply a higher risk for END and that monocyte Mac-1 expression on admission reflects the severity of ischemic stroke on admission.


Assuntos
Isquemia Encefálica/complicações , Isquemia Encefálica/imunologia , Moléculas de Adesão Celular/metabolismo , Quimiotaxia de Leucócito/imunologia , Encefalite/complicações , Encefalite/imunologia , Idoso , Biomarcadores/análise , Biomarcadores/metabolismo , Isquemia Encefálica/fisiopatologia , Adesão Celular/imunologia , Moléculas de Adesão Celular/análise , Progressão da Doença , Encefalite/fisiopatologia , Feminino , Citometria de Fluxo , Humanos , Leucócitos/citologia , Leucócitos/metabolismo , Antígeno-1 Associado à Função Linfocitária/análise , Antígeno-1 Associado à Função Linfocitária/metabolismo , Antígeno de Macrófago 1/análise , Antígeno de Macrófago 1/metabolismo , Masculino , Glicoproteínas de Membrana/análise , Glicoproteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Prognóstico , Estudos Prospectivos , Acidente Vascular Cerebral/complicações , Acidente Vascular Cerebral/imunologia , Acidente Vascular Cerebral/fisiopatologia , Regulação para Cima/imunologia
10.
Bioorg Med Chem ; 17(2): 558-68, 2009 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-19097798

RESUMO

Three series of non-steroidal anti-inflammatory drugs (NSAIDs) inhibiting the cyclooxygenase/5-lipoxygenase (COX/5-LOX) pathways as such as formation of hydroxyl radicals and adhesion were prepared: 4,5-diaryl isothiazoles, 4,5-diaryl 3H-1,2-dithiole-3-thiones and 4,5-diaryl 3H-1,2-dithiole-3-ones. The aim of the present study was to develop substances which can intervene into the inflammatory processes via different mechanisms of action as multiple target non-steroidal anti-inflammatory drugs (MTNSAIDs) with increased anti-inflammatory potential. The current lead 11a was evaluated in COX-1/2, 5-LOX and (*)OH scavenging in vitro assays and in a static adhesion assay where it proved to inhibit adhesion. Moreover, 11a treatment attenuated expression of macrophage adhesion molecule-1 (Mac-1) on extravasated polymorphonuclear leukocytes (PMNs) which indicates that the activation was reduced. The assays used are predictive for the in vivo efficacy of test compounds as shown for 11a in a peritonitis model of acute inflammation in mice. Thus, the novel 5-LOX/COX and (*)OH inhibitor 11a possesses anti-inflammatory activity that, in addition to COX/5-LOX inhibition, implicates effects on leukocyte-endothelial interactions.


Assuntos
Anti-Inflamatórios não Esteroides/química , Inibidores de Ciclo-Oxigenase/química , Inibidores de Lipoxigenase , Compostos de Sulfidrila/química , Tiazóis/química , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Adesão Celular/efeitos dos fármacos , Ciclo-Oxigenase 1 , Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase/farmacologia , Depuradores de Radicais Livres , Radical Hidroxila , Antígeno de Macrófago 1/análise , Antígeno de Macrófago 1/efeitos dos fármacos , Camundongos , Neutrófilos/efeitos dos fármacos , Peritonite/tratamento farmacológico , Compostos de Sulfidrila/farmacologia , Tiazóis/farmacologia
11.
J Vasc Surg ; 48(5): 1146-55, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18829234

RESUMO

BACKGROUND: Carotid artery lesions from symptomatic patients are characterized by inflammation and neovascularization. The adipokine leptin promotes angiogenesis and activates inflammatory cells, and the leptin receptor (ob gene-encoded receptor), ObR, is expressed in advanced atherosclerotic lesions. The present study quantitatively analyzed ObR messenger RNA (mRNA) expression and immunoreactivity in carotid artery plaques from symptomatic and asymptomatic persons. Plaque angiogenesis, gene expression of vascular endothelial growth factor (VEGF), and macrophage density were also analyzed. METHODS: Carotid endarterectomy specimens were collected from 26 patients undergoing surgery for hemispheric cerebrovascular symptoms (n = 13) or progressive asymptomatic internal carotid stenosis (n = 13). A representative sample, including part of the most active site, was collected from each lesion and evaluated by real-time polymerase chain reaction analysis for ObR(long) and ObR(common) isoforms, VEGF(165), and macrophage adhesion molecule-1 (Mac-1) mRNA, and by immunohistochemistry for ObR, von Willebrand factor (vWF), and CD68 antigen expression. RESULTS: All plaques exhibited advanced atherosclerosis (American Heart Association class IV through VI). Transcript levels were preferentially elevated in symptomatic plaques for ObR(long) (P = .0006) and ObR(common) (P = .033), with a simultaneous upregulation of VEGF(165) (P = .001) and Mac-1 mRNA expression (P = .003). Immunohistochemical analysis confirmed a significant increase of ObR antigen levels (P = .011) and CD68-positive inflammatory cells (P = .049) in symptomatic plaques, whereas neovascularization, evident in all plaques, was similar in both groups (P = .7). CONCLUSION: The ObR(long) and ObR(common) genes are upregulated and their protein preferentially synthesized in clinically symptomatic carotid plaques. Moreover, ObR expression is positively correlated with augmentation of gene transcripts related to macrophage density and neovascularization. These data suggest that ObR(long) and ObR(common) may be linked with histologic features of carotid plaque instability, which are associated with cerebral ischemic symptoms.


Assuntos
Estenose das Carótidas/metabolismo , Transtornos Cerebrovasculares/etiologia , Macrófagos/patologia , Receptores para Leptina/análise , Idoso , Biomarcadores/análise , Biomarcadores/sangue , Proteína C-Reativa/análise , Estenose das Carótidas/complicações , Estenose das Carótidas/patologia , Transtornos Cerebrovasculares/metabolismo , Transtornos Cerebrovasculares/patologia , Feminino , Humanos , Imuno-Histoquímica , Interleucina-6/sangue , Leptina/sangue , Antígeno de Macrófago 1/análise , Antígeno de Macrófago 1/genética , Macrófagos/imunologia , Masculino , Pessoa de Meia-Idade , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Reação em Cadeia da Polimerase , Isoformas de Proteínas , RNA Mensageiro/análise , Receptores para Leptina/sangue , Receptores para Leptina/genética , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular/análise , Fator A de Crescimento do Endotélio Vascular/genética
12.
Hepatogastroenterology ; 55(84): 836-41, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18705278

RESUMO

BACKGROUND/AIMS: The aim of the present study was to investigate the participation of the adhesion molecules and their ligands in the inflammatory process in secondary cholangitis. METHODOLOGY: Biopsy specimens were collected from the common bile duct from 29 patients with extrahepatic bile obstruction. Immunohistochemistry was used to study the expression of adhesion molecules (ICAM-1, VCAM-1, E-selectin, LFA-1, PECAM-1, Mac-1, and VLA-4). The patients were categorized into 2 groups - chronic exacerbated cholangitis and chronic sclerotic cholangitis. RESULTS: An increased ICAM-1 expression was demonstrated and also de novo VCAM-1 and E-selectin appearance on the endothelium of microvessels in chronic exacerbated cholangitis. The inflammatory cells were strongly LFA-1-, Mac-1- positive. In chronic sclerotic cholangitis the E-selectin expression persisted on vascular endothelium in the fibrous tissue and reduced inflammatory infiltrate showed LFA-1 and VLA-4 positivity. The newly formed vessels in the fibrous connective tissue were PECAM-1-positive. CONCLUSIONS: From these results, it could be concluded that in chronic exacerbated cholangitis with complete bile obstruction the firm adhesion is mediated by ICAM-1/LFA-1 and ICAM-1/Mac-1 and less by VCAM-1/VLA-4 pathways. In chronic sclerotic cholangitis, caused by incomplete obstruction, the firm adhesion was maintained by ICAM-1/LFA-1 and less by VCAM-1/VLA-4 pathways. It seems likely that PECAM-1 and VCAM-1/VLA-4 play additional roles in neutrophil recruitment.


Assuntos
Moléculas de Adesão Celular/análise , Colangite/patologia , Colestase Extra-Hepática/patologia , Doenças do Ducto Colédoco/patologia , Adulto , Idoso , Ducto Colédoco/patologia , Selectina E/análise , Feminino , Humanos , Técnicas Imunoenzimáticas , Integrina alfa4beta1 , Molécula 1 de Adesão Intercelular/análise , Testes de Função Hepática , Antígeno-1 Associado à Função Linfocitária/análise , Antígeno de Macrófago 1/análise , Masculino , Pessoa de Meia-Idade , Molécula-1 de Adesão Celular Endotelial a Plaquetas/análise , Molécula 1 de Adesão de Célula Vascular/análise
13.
Proc Natl Acad Sci U S A ; 105(16): 6057-62, 2008 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-18424555

RESUMO

Earlier work has shown that the transcription factor C/EBPalpha induced a transdifferentiation of committed lymphoid precursors into macrophages in a process requiring endogenous PU.1. Here we have examined the effects of PU.1 and C/EBPalpha on fibroblasts, a cell type distantly related to blood cells and akin to myoblasts, adipocytes, osteoblasts, and chondroblasts. The combination of the two factors, as well as PU.1 and C/EBPbeta, induced the up-regulation of macrophage/hematopoietic cell surface markers in a large proportion of NIH 3T3 cells. They also up-regulated these markers in mouse embryo- and adult skin-derived fibroblasts. Based on cell morphology, activation of macrophage-associated genes, and extinction of fibroblast-associated genes, cell lines containing an attenuated form of PU.1 and C/EBPalpha acquired a macrophage-like phenotype. The lines also display macrophage functions: They phagocytose small particles and bacteria, mount a partial inflammatory response, and exhibit strict CSF-1 dependence for growth. The myeloid conversion is primarily induced by PU.1, with C/EBPalpha acting as a modulator of macrophage-specific gene expression. Our data suggest that it might become possible to induce the transdifferentiation of skin-derived fibroblasts into cell types desirable for tissue regeneration.


Assuntos
Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Transdiferenciação Celular , Fibroblastos/citologia , Macrófagos/citologia , Proteínas Proto-Oncogênicas/metabolismo , Transativadores/metabolismo , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Animais , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Proteína beta Intensificadora de Ligação a CCAAT/genética , Transdiferenciação Celular/genética , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Humanos , Antígenos Comuns de Leucócito/análise , Antígenos Comuns de Leucócito/metabolismo , Fator Estimulador de Colônias de Macrófagos/farmacologia , Antígeno de Macrófago 1/análise , Antígeno de Macrófago 1/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Células NIH 3T3 , Fagocitose/genética , Proteínas Proto-Oncogênicas/genética , Retroviridae/genética , Transativadores/genética , Transfecção , Regulação para Cima
14.
Arq Bras Cardiol ; 90(1): 54-63, 2008 Jan.
Artigo em Inglês, Português | MEDLINE | ID: mdl-18317641

RESUMO

OBJECTIVE: To assess the importance of the interaction between leukocyte integrin Mac-1 (a Mb 2) and platelet glycoprotein (GP) Ib-a for leukocyte recruitment after vascular injury and the effect of the neutralization of the Mac-1-GPIba interaction on cell proliferation and the neointimal hyperplasia triggered by the vascular injury. METHODS: A peptide called M2 or anti-M2 antibody was developed to block the Mac-1-GPIba interaction. This peptide was injected and compared to a control-peptide in C57B1/6J mice submitted to vascular injury of the femoral artery with a guide wire. One, five or 28 days after the vascular injury, the femoral arteries were removed for morphometric and immunohistochemical analyses. RESULTS: The blocking of the Mac-1-GPIba interaction promoted a statistically significant reduction in the number of leukocytes in the neointimal layer on the first day after the vascular injury (control: 7.9+/-5.0% of the cell total versus anti-M2: 2.0+/-1.6%, p=0.021), as well as determined a statistically significant decrease in leukocyte accumulation in the neointimal layer on days 5 and 28 (control: 42.3+/-12.9% versus anti-M2: 24.6+/-10.8%, p=0.047 and control: 7.9+/-3.0% versus anti-M2: 3.3+/-1.3%, p=0.012; respectively). Cell proliferation in the neointimal layer of the vessel five days post-injury was reduced with the blocking of the Mac-1-GPIba interaction (control: 5.0+/-2.9% of the cell total versus anti-M2: 1.8+/-0.5%; p=0.043), along with a significant decrease in cell proliferation in the vessel neointimal layer 28 days post-injury (control: 3.8+/-1.7% versus anti-M2: 2.0+/-1.2%; p=0.047). The blocking of the Mac-1-GPIba interaction also determined a statistically significant decrease of the intimal thickening 28 days post-injury (control: 10,395+/-3,549 microm(2) versus anti-M2: 4,561+/-4,915 microm(2); p=0.012). CONCLUSION: Leukocyte recruitment after a vascular injury depends on the Mac-1-GPIba interaction and the neutralization of this interaction inhibits cell proliferation and neointimal formation.


Assuntos
Anticorpos Monoclonais/administração & dosagem , Artéria Femoral/lesões , Leucócitos/fisiologia , Antígeno de Macrófago 1/fisiologia , Peptídeos/administração & dosagem , Complexo Glicoproteico GPIb-IX de Plaquetas/efeitos dos fármacos , Complexo Glicoproteico GPIb-IX de Plaquetas/fisiologia , Animais , Anticorpos Monoclonais/imunologia , Plaquetas/metabolismo , Proliferação de Células , Artéria Femoral/metabolismo , Imunoglobulina G/administração & dosagem , Inflamação/metabolismo , Antígeno de Macrófago 1/análise , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Peptídeos/imunologia , Adesividade Plaquetária/fisiologia , Coelhos , Estatísticas não Paramétricas , Túnica Íntima/imunologia , Túnica Íntima/patologia
15.
Biochem Biophys Res Commun ; 369(4): 1139-43, 2008 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-18339305

RESUMO

Natural killer (NK) cells are the cells critical for inhibition of repopulation of allogenic bone marrow cells. However, it is not well known if NK cells affect autologous lymphopoiesis. Here, we observed that NK cells could inhibit pre-B cell proliferation in vitro driven by interleukin (IL)-7 in a manner dependent on IL-15. Interestingly, the great majority of expanding NK cells were Mac-1(+)B220(+), a recently identified potent interferon (IFN)-gamma producer. Indeed, IFN-gamma was produced in those cultures, and pre-B cells lacking IFN-gamma receptors, but not those lacking type I IFN receptors, were resistant to such an inhibition. Furthermore, even NK cells from mice lacking beta2-microglobulin, which were known to be functionally dampened, inhibited pre-B cell proliferation as well. Thus, activated NK cells, which were expanded selectively by IL-15, could potentially regulate B lymphopoiesis through IFN-gamma beyond the selection imposed upon self-recognition.


Assuntos
Interferon gama/imunologia , Interleucina-15/imunologia , Células Matadoras Naturais/imunologia , Linfopoese , Células Precursoras de Linfócitos B/imunologia , Animais , Proliferação de Células/efeitos dos fármacos , Interleucina-15/farmacologia , Células Matadoras Naturais/efeitos dos fármacos , Antígenos Comuns de Leucócito/análise , Ativação Linfocitária/efeitos dos fármacos , Antígeno de Macrófago 1/análise , Camundongos , Camundongos Mutantes , Microglobulina beta-2/genética
16.
Arq. bras. cardiol ; 90(1): 54-63, jan. 2008. ilus, graf, tab
Artigo em Inglês, Português | LILACS | ID: lil-476046

RESUMO

OBJETIVO: Avaliar a importância da interação entre a integrina Mac-1 dos leucócitos (a Mb 2) e a glicoproteína (GP) Iba das plaquetas para o recrutamento de leucócitos após a lesão vascular e o efeito da neutralização da interação Mac-1-GPIba sobre a proliferação celular e a hiperplasia neointimal desencadeadas por lesão vascular. MÉTODOS: Um peptídeo denominado M2 ou anticorpo anti-M2 foi desenvolvido para bloquear a interação Mac-1-GPIba . Esse peptídeo foi injetado e comparado com anticorpo-controle em camundongos C57B1/6J submetidos a lesão vascular da artéria femoral com corda-guia. Um, cinco ou 28 dias após a lesão vascular, as artérias femorais foram retiradas para a realização de morfometria e imuno-histoquímica. RESULTADOS: O bloqueio da interação Mac-1-GPIba promoveu uma redução estatisticamente significativa do número de leucócitos na camada média no primeiro dia após a lesão vascular (controle: 7,9±5,0 por cento do total de células versus anti-M2: 2,0±1,6 por cento, p=0,021), bem como determinou uma diminuição estatisticamente significativa do acúmulo de leucócitos na neoíntima em cinco e 28 dias (controle: 42,3±12,9 por cento versus anti-M2: 24,6±10,8 por cento, p=0,047 e controle: 7,9±3,0 por cento versus anti-M2: 3,3±1,3 por cento, p=0,012; respectivamente). A proliferação celular na camada média do vaso em cinco dias pós-lesão foi reduzida com o bloqueio da interação Mac-1-GPIba (controle: 5,0±2,9 por cento do total de células versus anti-M2: 1,8±0,5 por cento; p=0,043), assim como houve diminuição significativa da proliferação celular na camada íntima do vaso em 28 dias (controle: 3,8±1,7 por cento versus anti-M2: 2,0±1,2 por cento; p=0,047). O bloqueio da interação Mac-1-GPIba também determinou uma redução estatisticamente significativa do espessamento intimal em 28 dias pós-lesão (controle: 10.395±3.549 µm² versus anti-M2: 4.561±4.915 ...


OBJECTIVE: To assess the importance of the interaction between leukocyte integrin Mac-1 (a Mb 2) and platelet glycoprotein (GP) Ib-a for leukocyte recruitment after vascular injury and the effect of the neutralization of the Mac-1-GPIba interaction on cell proliferation and the neointimal hyperplasia triggered by the vascular injury. METHODS: A peptide called M2 or anti-M2 antibody was developed to block the Mac-1-GPIba interaction. This peptide was injected and compared to a control-peptide in C57B1/6J mice submitted to vascular injury of the femoral artery with a guide wire. One, five or 28 days after the vascular injury, the femoral arteries were removed for morphometric and immunohistochemical analyses. RESULTS: The blocking of the Mac-1-GPIba interaction promoted a statistically significant reduction in the number of leukocytes in the neointimal layer on the first day after the vascular injury (control: 7.9±5.0 percent of the cell total versus anti-M2: 2.0±1.6 percent, p=0.021), as well as determined a statistically significant decrease in leukocyte accumulation in the neointimal layer on days 5 and 28 (control: 42.3±12.9 percent versus anti-M2: 24.6±10.8 percent, p=0.047 and control: 7.9±3.0 percent versus anti-M2: 3.3±1.3 percent, p=0.012; respectively). Cell proliferation in the neointimal layer of the vessel five days post-injury was reduced with the blocking of the Mac-1-GPIba interaction (control: 5.0±2.9 percent of the cell total versus anti-M2: 1.8±0.5 percent; p=0.043), along with a significant decrease in cell proliferation in the vessel neointimal layer 28 days post-injury (control: 3.8±1.7 percent versus anti-M2: 2.0±1.2 percent; p=0.047). The blocking of the Mac-1-GPIba interaction also determined a statistically significant decrease of the intimal thickening 28 days post-injury (control: 10,395±3,549 µm² versus anti-M2: 4,561±4,915 µm²; ...


Assuntos
Animais , Masculino , Camundongos , Coelhos , Anticorpos Monoclonais/administração & dosagem , Artéria Femoral/lesões , Leucócitos/fisiologia , Antígeno de Macrófago 1/fisiologia , Peptídeos/administração & dosagem , Complexo Glicoproteico GPIb-IX de Plaquetas/efeitos dos fármacos , Complexo Glicoproteico GPIb-IX de Plaquetas/fisiologia , Anticorpos Monoclonais/imunologia , Plaquetas/metabolismo , Proliferação de Células , Artéria Femoral/metabolismo , Imunoglobulina G/administração & dosagem , Inflamação/metabolismo , Modelos Animais , Antígeno de Macrófago 1/análise , Peptídeos/imunologia , Adesividade Plaquetária/fisiologia , Estatísticas não Paramétricas , Túnica Íntima/imunologia , Túnica Íntima/patologia
17.
Biochem Pharmacol ; 75(3): 688-97, 2008 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-17988652

RESUMO

This study investigated the mechanism underlying the inhibiting effect of (2R,3R)-2-(3',4'-dihydroxybenzyl)-3-(3'',4''-dimethoxybenzyl) butyrolactone (PP-6), a lignan from Piper philippinum, on superoxide anion production induced by the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (fMLP) in human neutrophils. Human neutrophils were stimulated with fMLP (1 microM), PMA (100 nM) or leukotriene B(4) (LTB(4); 1 microM) and induced superoxide anion release. PP-6 specifically inhibited fMLP-induced superoxide anion production in a concentration-dependent manner with an IC(50) value of 0.3+/-0.1 microM. Intracellular signaling caused by fMLP, PMA or LTB(4) were evaluated. PP-6 specifically inhibited fMLP-induced intracellular calcium mobilization and ERK (p42/p44), Akt and p38 phosphorylation. Moreover, PP-6 specifically inhibited fMLP-induced Mac-1 expression without affecting this caused by LTB(4) or PMA. PP-6 did not increase cAMP level in human neutrophils. PP-6 did not inhibit superoxide anion production by NaF (20 mM), a direct activator of G-protein, the target of the inhibitory action of PP-6 appears to be a component of the signal transduction pathway upstream of G-protein. PP-6 inhibited FITC-fMLP binding to neutrophils in a concentration-dependent manner with an IC(50) of 1.5+/-0.2 microM. PP-6 did not bring a parallel shift in the concentration response of fMLP-induced superoxide anion. Additionally, the inhibiting effect of PP-6 on fMLP-induced superoxide anion was reversed when PP-6 was washed out. These experimental results suggest that PP-6 exerts non-competitive and reversible antagonistic effect on fMLP receptor.


Assuntos
4-Butirolactona/análogos & derivados , Lignanas/farmacologia , N-Formilmetionina Leucil-Fenilalanina/antagonistas & inibidores , Piper/química , Receptores de Formil Peptídeo/antagonistas & inibidores , Superóxidos/metabolismo , 4-Butirolactona/farmacologia , Adulto , Cálcio/metabolismo , AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Humanos , Antígeno de Macrófago 1/análise , Masculino , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Fosforilação , Receptores de Formil Peptídeo/metabolismo
18.
Pediatr Allergy Immunol ; 19(1): 25-32, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17651379

RESUMO

Leukocyte adhesion deficiency type I (LAD I) is characterized by recurrent and fatal bacterial infections, and caused by the mutation of the CD18 gene. A 9-month-old infant whose umbilical cord separated at day 10 of life had sepsis, complicated otitis media and neutrophilia. Molecular analysis showed homozygous intron 7 (+1) g > a in the CD18 gene, resulting in three splicing transcriptions that inserted 64, 298 (5' end of intron 7), and 1157 (whole intron 7) nucleotides into the 300th amino acid of Ile and stopped at the 326th (inserted 64 and 1157 nucleotides) and the 344th (inserted 64 nucleotides), respectively. The two truncated mutations lost cysteine-rich, transmembrane, and cytoplasma domains. Increased susceptibility to infections correlated to polymorphonuclear cell dysfunction, including absent expression of adhesion molecule (CD11b/CD18), impaired chemotaxis, and decreased phagocytosis. Both his heterozygous parents revealed non-random skewing only to the wild type. The skewing pattern and severe phenotype make stem cell transplantation an optimal option.


Assuntos
Antígenos CD18/genética , Síndrome da Aderência Leucocítica Deficitária/genética , Síndrome da Aderência Leucocítica Deficitária/imunologia , Neutrófilos/fisiologia , Antígenos CD18/análise , Antígenos CD18/química , Quimiotaxia de Leucócito , Humanos , Peróxido de Hidrogênio/metabolismo , Lactente , Antígenos CD15/análise , Antígeno de Macrófago 1/análise , Masculino , Mutação , Fagocitose , Cordão Umbilical
19.
J Immunol ; 178(8): 4764-70, 2007 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-17404256

RESUMO

NK cells are important for the clearance of tumors, parasites, and virus-infected cells. Thus, factors that control NK cell numbers and function are critical for the innate immune response. A subset of NK cells express the inhibitory killer cell lectin-like receptor G1 (KLRG1). In this study, we identify that KLRG1 expression is acquired during periods of NK cell division such as development and homeostatic proliferation. KLRG1(+) NK cells are mature in phenotype, and we show for the first time that these cells have a slower in vivo turnover rate, reduced proliferative response to IL-15, and poorer homeostatic expansion potential compared with mature NK cells lacking KLRG1. Transfer into lymphopenic recipients indicate that KLRG1(-) NK cells are precursors of KLRG1(+) NK cells and KLRG1 expression accumulates following cell division. Furthermore, KLRG1(+) NK cells represent a significantly greater proportion of NK cells in mice with enhanced NK cell numbers such as Cd45(-/-) mice. These data indicate that NK cells acquire KLRG1 on their surface during development, and this expression correlates with functional distinctions from other peripheral NK cells in vivo.


Assuntos
Células Matadoras Naturais/fisiologia , Receptores Imunológicos/biossíntese , Animais , Homeostase , Interleucina-12/farmacologia , Interleucina-18/farmacologia , Células Matadoras Naturais/imunologia , Antígenos Comuns de Leucócito/análise , Ativação Linfocitária , Antígeno de Macrófago 1/análise , Camundongos , Camundongos Endogâmicos C57BL , Receptores Imunológicos/análise , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/análise , Regulação para Cima
20.
Kidney Int ; 71(2): 167-72, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17136029

RESUMO

Oxidative stress and inflammation are common features and major mediators of atherosclerosis in end-stage renal disease (ESRD). Available evidence for oxidative stress in ESRD is indirect and based on accumulation of byproducts of interactions of reactive oxygen species (ROS) with various molecules. Inflammation is a major cause of oxidative stress. To explore the direct link between oxidative stress and inflammation in ESRD, we studied leukocyte integrin expression and ROS production in 18 ESRD patients and 18 controls. ESRD patients showed elevated plasma malondialdehyde (MDA) and increased superoxide and hydrogen peroxide (H(2)O(2)) production by granulocytes and monocytes before dialysis. Hemodialysis resulted in a further rise in plasma MDA and H(2)O(2) production by granulocytes and monocytes. Surface expression of Mac-1 (CD11b and CD18) on granulocytes and monocytes was significantly increased (denoting cell activation) in ESRD patients. Granularity of granulocytes was significantly reduced before dialysis and declined further after dialysis. The magnitude of ROS production by granulocytes and monocytes was directly related with CD11b expression as well as plasma ferritin and parathyroid hormone levels and was inversely related to protein catabolic rate. Thus, this study provides direct evidence of spontaneous leukocyte activation and increased ROS generation (hence the link between oxidative stress and inflammation) in ESRD patients.


Assuntos
Radicais Livres/metabolismo , Falência Renal Crônica/etiologia , Leucócitos/imunologia , Estresse Oxidativo , Feminino , Ferritinas/sangue , Radicais Livres/análise , Granulócitos/química , Humanos , Peróxido de Hidrogênio/sangue , Integrina alfaXbeta2/análise , Falência Renal Crônica/imunologia , Falência Renal Crônica/metabolismo , Antígeno de Macrófago 1/análise , Masculino , Malondialdeído/sangue , Pessoa de Meia-Idade , Hormônio Paratireóideo/sangue , Diálise Renal
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