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1.
Liver Transpl ; 22(1): 80-90, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26335784

RESUMO

Hepatic ischemia/reperfusion injury (IRI) remains a major clinical problem and involves the innate immune system's recognition of "nonself." Considering the efficient nonself recognition by natural killer (NK) cells, we hypothesize in this study that hepatic IRI associated with liver transplantation (LT) could be augmented in allogeneic rather than in syngeneic (Syn) grafts due to alloantigen recognition by innate immune cells, especially by NK cells. Using green fluorescent protein (GFP)/Sprague-Dawley rats, we tested our hypothesis in a rat LT model with 18 hours of cold storage in University of Wisconsin solution. Hepatic IRI was significantly augmented in allografts with higher alanine transaminase levels, increased necrosis, and vigorous proinflammatory mediator up-regulation compared to Syn grafts. Injury increased in allografts associated with augmented GFP+ host leukocyte infiltration due to significantly increased host CD11b/c+ and RP-1(+) neutrophil recruitment. A large number of liver-resident (donor) mature CD11b/c+ NK cells quickly diminished from allografts, but not from Syn grafts. Depletion of mature NK cells from liver grafts with anti-asialo monosialotetrahexosylganglioside significantly improved hepatic IRI and reduced neutrophil infiltration and proinflammatory mediators. In conclusion, early innate immune responses were more significantly enhanced in allografts than in Syn grafts during hepatic IRI, in part through NK cell recognition of "missing self."


Assuntos
Isoantígenos/fisiologia , Células Matadoras Naturais/fisiologia , Hepatopatias/imunologia , Traumatismo por Reperfusão/imunologia , Animais , Anticorpos/imunologia , Imunidade Inata , Masculino , Infiltração de Neutrófilos , Ratos Endogâmicos Lew
2.
Eye (Lond) ; 29(1): 136-44, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25323855

RESUMO

PURPOSE: To investigate the effect of host immunity (allospecific) and surgical manipulation (non-allospecific) on corneal endothelial cells (CECs) in corneal transplantation. METHODS: Draining lymph nodes and grafted C57BL/6 corneas were harvested from syngeneic recipients, allograft acceptors, and allograft rejectors (BALB/c) 1, 3, and 8 weeks after transplantation. We analyzed CEC apoptosis using an ex vivo cornea-in-the-cup assay, and visualized cell-to-cell junctions using immunohistochemical staining (ZO-1). Automatic cell analysis using Confoscan software was used to measure CEC density as well as changes in CEC morphology by quantifying the coefficient of variation in cell size (polymegethism) and shape (pleomorphism). RESULTS: The cornea-in-the-cup assay showed that allogeneic acceptor T cells and to an even greater extent rejector T cells (but not syngeneic T cells) induced CEC apoptosis. CEC density after corneal transplantation was significantly reduced in allogeneic acceptors compared with syngeneic grafts (P<0.001), and CEC density was even further reduced in the allo-rejector group compared with the allo-acceptor group. Allogeneic grafts showed a greater increase in the coefficient of variation in cell size (polymegethism) when compared with syngeneic grafts 1 week after transplantation (P=P<0.001). However, pleomorphism was not significantly different between syngeneic and allo-acceptor grafts, indicating that polymegethism (but not pleomorphism or cell density) is a sensitive indicator of the effect of alloimmunity on CECs. CONCLUSIONS: Our data demonstrate that host alloimmunity rather than surgical manipulation alone is the major cause of CEC damage in corneal transplantation, and such morphologic changes of CECs can be detected before the clinically visible onset of allograft rejection.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Perda de Células Endoteliais da Córnea/diagnóstico , Transplante de Córnea , Endotélio Corneano/patologia , Imunidade Inata/fisiologia , Isoantígenos/fisiologia , Animais , Apoptose , Contagem de Células , Forma Celular , Tamanho Celular , Perda de Células Endoteliais da Córnea/imunologia , Endotélio Corneano/imunologia , Marcação In Situ das Extremidades Cortadas , Isoenxertos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Proteína da Zônula de Oclusão-1/metabolismo
3.
Artigo em Inglês | MEDLINE | ID: mdl-24077235

RESUMO

AIMS: Mesenchymal stromal cells (MSC) derived from adult bone marrow or adipose tissue offer the potential to open a new frontier in medicine. MSC are involved in modulating immune response and tissue repair in vitro and in vivo. Experimental evidence and preliminary clinical studies have demonstrated that MSC exhibit an important immunomodulatory function in patients with graft versus host disease (GVHD) following allogeneic hematopoietic stem cell transplantation. The immunosuppressive properties of MSC have already been exploited in the clinical setting. However the precise mechanisms are being still investigated. METHODS: We examined the immunosuppressive function of MSC by coculturing them with stimulated HLA incompatible allogeneic lymphocytes in a mixed lymphocyte culture test. The metabolic and proliferative activity of lymphocytes was determined by MTT test. RESULTS: After stimulation with alloantigens the presence of MSC caused significant decrease of absorbance levels by 62% (P<0.01), 26% (P<0.01) and 6% (P=0.0437) in comparison to positive control depending on the MSC/lymphocyte ratio (1:5, 1:50, 1:500). The mitogenic stimulation of lymphocytes with fMLP or PHA was also significantly reduced during MSC cocultivation. The absorbance was reduced by 42% (P<0.001) and 67% (P<0.001). CONCLUSIONS: Allogeneic bone marrow is an ideal source of MSC for clinical application. The experiments confirmed the dose-dependent inhibitory effect of MSC on lymphocyte proliferation triggered by cellular or mitogenic stimulation. The mixed lymphocyte culture test offers a simple method for characterization and verification of the immunosuppressive potential of MSC, being prepared for clinical use.


Assuntos
Tolerância Imunológica/imunologia , Isoantígenos/fisiologia , Linfócitos/imunologia , Células-Tronco Mesenquimais/imunologia , Adulto , Proliferação de Células/fisiologia , Células Cultivadas , Doença Enxerto-Hospedeiro/imunologia , Antígenos HLA/imunologia , Humanos , Técnicas In Vitro , Linfócitos/citologia
4.
Artigo em Inglês | MEDLINE | ID: mdl-24186492

RESUMO

Regulatory T cells (Tregs) are essential to transplantation tolerance and their therapeutic efficacy is well documented in animal models. Moreover, human Tregs can be identified, isolated, and expanded in short-term ex vivo cultures so that a therapeutic product can be manufactured at relevant doses. Treg therapy is being planned at multiple transplant centers around the world. In this article, we review topics critical to effective implementation of Treg therapy in transplantation. We will address issues such as Treg dose, antigen specificity, and adjunct therapies required for transplant tolerance induction. We will summarize technical advances in Treg manufacturing and provide guidelines for identity and purity assurance of Treg products. Clinical trial designs and Treg manufacturing plans that incorporate the most up-to-date scientific understanding in Treg biology will be essential for harnessing the tolerogenic potential of Treg therapy in transplantation.


Assuntos
Terapia Baseada em Transplante de Células e Tecidos/métodos , Rejeição de Enxerto/prevenção & controle , Linfócitos T Reguladores/transplante , Tolerância ao Transplante/fisiologia , Transplante/métodos , Adjuvantes Imunológicos/uso terapêutico , Aloenxertos , Animais , Separação Celular/métodos , Ensaios Clínicos como Assunto , Modelos Animais de Doenças , Humanos , Terapia de Imunossupressão/métodos , Imunossupressores/uso terapêutico , Isoantígenos/fisiologia , Camundongos
5.
Clin Exp Immunol ; 174(1): 179-91, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23750604

RESUMO

Detection and isolation of viable alloreactive T cells at the single-cell level requires a cell surface marker induced specifically upon T cell receptor activation. In this study, a member of the tumour necrosis factor receptor (TNFR)-family, CD137 (4-1BB) was investigated for its potential to identify the total pool of circulating alloreactive T cells. Optimal conditions for sensitive and specific detection of allogeneic-induced CD137 expression on circulating T cells were established. Thereafter, CD137(+) alloreactive T cells were phenotypically and functionally characterized by multi-parameter flow cytometry. Alloantigen-induced CD137 expression identified both alloreactive CD8(+) T cells (mean ± standard error of the mean: 0·21 ± 0·07%) and alloreactive CD4(+) T cells (0·21 ± 0·05%). CD137(+) alloreactive T cells were detected in different T cell subsets, including naive T cells, but were found preferentially in CD28(+) T cells and not in the terminally differentiated T cell subset. Upon allogeneic (re-)stimulation, the cytokine-producing as well as proliferative capacity of T cells resided mainly within the CD137-expressing fraction. About 10% of the CD137(+) alloreactive T cells produced any combination of interferon (IFN)-γ, interleukin (IL)-2 and TNF-α. Polyfunctional alloreactive T cells, defined by multiple cytokine expression, were observed infrequently. In conclusion, activation-induced CD137 expression is a fast assay allowing for detection and functional analysis of the total alloreactive T cell compartment at the single-cell level by multi-parameter flow cytometry.


Assuntos
Citometria de Fluxo/métodos , Isoantígenos/fisiologia , Ativação Linfocitária/imunologia , Subpopulações de Linfócitos T/imunologia , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/biossíntese , Células Apresentadoras de Antígenos/citologia , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Proliferação de Células , Citometria de Fluxo/normas , Humanos , Imunofenotipagem/métodos , Imunofenotipagem/normas , Teste de Cultura Mista de Linfócitos/métodos , Teste de Cultura Mista de Linfócitos/normas , Depleção Linfocítica , Sensibilidade e Especificidade , Células-Tronco/citologia , Células-Tronco/imunologia , Células-Tronco/metabolismo , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/metabolismo , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/genética
6.
Development ; 139(19): 3583-9, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22949614

RESUMO

SPACA1 is a membrane protein that localizes in the equatorial segment of spermatozoa in mammals and is reported to function in sperm-egg fusion. We produced a Spaca1 gene-disrupted mouse line and found that the male mice were infertile. The cause of this sterility was abnormal shaping of the sperm head reminiscent of globozoospermia in humans. Disruption of Spaca1 led to the disappearance of the nuclear plate, a dense lining of the nuclear envelope facing the inner acrosomal membrane. This coincided with the failure of acrosomal expansion during spermiogenesis and resulted in the degeneration and disappearance of the acrosome in mature spermatozoa. Thus, these findings clarify part of the cascade leading to globozoospermia.


Assuntos
Infertilidade Masculina/genética , Isoantígenos/genética , Proteínas de Plasma Seminal/genética , Cabeça do Espermatozoide/patologia , Espermatozoides/anormalidades , Acrossomo/metabolismo , Acrossomo/fisiologia , Animais , Forma Celular/genética , Expressão Gênica , Infertilidade Masculina/patologia , Isoantígenos/metabolismo , Isoantígenos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Knockout , Proteínas de Plasma Seminal/metabolismo , Proteínas de Plasma Seminal/fisiologia , Espermatogênese/genética , Espermatogênese/fisiologia , Espermatozoides/ultraestrutura , Distribuição Tecidual
7.
J Immunol ; 189(8): 3848-58, 2012 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-22962681

RESUMO

Hepatic stellate cells (HSCs) are critical for hepatic wound repair and tissue remodeling. They also produce cytokines and chemokines that may contribute to the maintenance of hepatic immune homeostasis and the inherent tolerogenicity of the liver. The functional relationship between HSCs and the professional migratory APCs in the liver, that is, dendritic cells (DCs), has not been evaluated. In this article, we report that murine liver DCs colocalize with HSCs in vivo under normal, steady-state conditions, and cluster with HSCs in vitro. In vitro, HSCs secrete high levels of DC chemoattractants, such as MΙP-1α and MCP-1, as well as cytokines that modulate DC activation, including TNF-α, IL-6, and IL-1ß. Culture of HSCs with conventional liver myeloid (m) DCs resulted in increased IL-6 and IL-10 secretion compared with that of either cell population alone. Coculture also resulted in enhanced expression of costimulatory (CD80, CD86) and coinhibitory (B7-H1) molecules on mDCs. HSC-induced mDC maturation required cell-cell contact and could be blocked, in part, by neutralizing MΙP-1α or MCP-1. HSC-induced mDC maturation was dependent on activation of STAT3 in mDCs and, in part, on HSC-secreted IL-6. Despite upregulation of costimulatory molecules, mDCs conditioned by HSCs demonstrated impaired ability to induce allogeneic T cell proliferation, which was independent of B7-H1, but dependent upon HSC-induced STAT3 activation and subsequent upregulation of IDO. In conclusion, by promoting IDO expression, HSCs may act as potent regulators of liver mDCs and function to maintain hepatic homeostasis and tolerogenicity.


Assuntos
Células Dendríticas/imunologia , Regulação para Baixo/imunologia , Células Estreladas do Fígado/imunologia , Indolamina-Pirrol 2,3,-Dioxigenase/biossíntese , Fígado/imunologia , Células Mieloides/imunologia , Fator de Transcrição STAT3/fisiologia , Animais , Células Cultivadas , Técnicas de Cocultura , Indução Enzimática/genética , Indução Enzimática/imunologia , Células Estreladas do Fígado/enzimologia , Células Estreladas do Fígado/metabolismo , Imunofenotipagem , Isoantígenos/genética , Isoantígenos/fisiologia , Fígado/citologia , Fígado/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos
8.
J Immunol ; 188(5): 2419-26, 2012 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-22266279

RESUMO

Neutrophil transmigration requires the localization of neutrophils to endothelial cell junctions, in which receptor-ligand interactions and the action of serine proteases promote leukocyte diapedesis. NB1 (CD177) is a neutrophil-expressed surface molecule that has been reported to bind proteinase 3 (PR3), a serine protease released from activated neutrophils. PR3 has demonstrated proteolytic activity on a number of substrates, including extracellular matrix proteins, although its role in neutrophil transmigration is unknown. Recently, NB1 has been shown to be a heterophilic binding partner for the endothelial cell junctional protein, PECAM-1. Disrupting the interaction between NB1 and PECAM-1 significantly inhibits neutrophil transendothelial cell migration on endothelial cell monolayers. Because NB1 interacts with endothelial cell PECAM-1 at cell junctions where transmigration occurs, we considered that NB1-PR3 interactions may play a role in aiding neutrophil diapedesis. Blocking Abs targeting the heterophilic binding domain of PECAM-1 significantly inhibited transmigration of NB1-positive neutrophils through IL-1ß-stimulated endothelial cell monolayers. PR3 expression and activity were significantly increased on NB1-positive neutrophils following transmigration, whereas neutrophils lacking NB1 demonstrated no increase in PR3. Finally, using selective serine protease inhibitors, we determined that PR3 activity facilitated transmigration of NB1-positive neutrophils under both static and flow conditions. These data demonstrate that PR3 contributes in the selective recruitment of the NB1-positive neutrophil population.


Assuntos
Isoantígenos/biossíntese , Mieloblastina/fisiologia , Neutrófilos/enzimologia , Neutrófilos/imunologia , Receptores de Superfície Celular/biossíntese , Migração Transendotelial e Transepitelial/imunologia , Ativação Enzimática/genética , Ativação Enzimática/imunologia , Proteínas Ligadas por GPI/biossíntese , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/fisiologia , Regulação da Expressão Gênica/imunologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Isoantígenos/genética , Isoantígenos/fisiologia , Mieloblastina/biossíntese , Mieloblastina/genética , Neutrófilos/citologia , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/fisiologia
9.
J Immunol ; 186(5): 2809-18, 2011 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-21270412

RESUMO

Rapamycin (Rapa), an immunosuppressive drug that acts through mammalian target of Rapa inhibition, broadly synergizes with tolerogenic agents in animal models of transplantation and autoimmunity. Rapa preferentially inhibits conventional CD4(+) Foxp3(-) T cells (Tconv) and promotes outgrowth of CD4(+)Foxp3(+) regulatory T cells (Treg) during in vitro expansion. Moreover, Rapa is widely perceived as augmenting both expansion and conversion of Treg in vivo. However, most quantitative studies were performed in lymphopenic hosts or in graft-versus-host disease models. We show in this study that in replete wild-type mice, Rapa significantly inhibits both homeostatic and alloantigen-induced proliferation of Treg, and promotes their apoptosis. Together, these lead to significant Treg depletion. Tconv undergo depletion to a similar degree, resulting in no change in the percent of Treg among CD4 cells. Moreover, in this setting, there was no evidence of conversion of Tconv into Treg. However, after withdrawal of Rapa, Treg recover Ag-induced proliferation more quickly than Tconv, leading to recovery to baseline numbers and an increase in the percent of Treg compared with Tconv. These findings suggest that the effects of Rapa on Treg survival, homeostasis, and induction, depend heavily on the cellular milieu and degree of activation. In vivo, the resistance of Treg to mammalian target of Rapa inhibition is relative and results from lymphopenic and graft-versus-host disease models cannot be directly extrapolated to settings more typical of solid organ transplantation or autoimmunity. Moreover, these results have important implications for the timing of Rapa therapy with tolerogenic agents designed to increase the number of Treg in vivo.


Assuntos
Proliferação de Células , Homeostase/imunologia , Isoantígenos/fisiologia , Transdução de Sinais/imunologia , Linfócitos T Reguladores/imunologia , Serina-Treonina Quinases TOR/fisiologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/imunologia , Técnicas de Introdução de Genes , Homeostase/efeitos dos fármacos , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Contagem de Linfócitos , Depleção Linfocítica , Tecido Linfoide/citologia , Tecido Linfoide/efeitos dos fármacos , Tecido Linfoide/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Transdução de Sinais/efeitos dos fármacos , Sirolimo/administração & dosagem , Transplante de Pele/imunologia , Transplante de Pele/patologia , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/transplante
10.
Clin Exp Immunol ; 161(1): 89-97, 2010 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-20491791

RESUMO

Proteinase 3 (PR3) is a major autoantigen in anti-neutrophil cytoplasmic antibodies (ANCA)-associated systemic vasculitis (AASV), and the proportion of neutrophils expressing PR3 on their membrane (mPR3+) is increased in AASV. We have shown recently that mPR3 and CD177 are expressed on the same cells in healthy individuals. In this study we try to elucidate mechanisms behind the increased mPR3 expression in AASV and its relationship to CD177. All neutrophils in all individuals were either double-positive or double-negative for mPR3 and CD177. The proportion of double-positive neutrophils was increased significantly in AASV and systemic lupus erythematosus patients. The proportion of mPR3+/CD177+ cells was not correlated to general inflammation, renal function, age, sex, drug treatment and levels of circulating PR3. AASV patients had normal levels of granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor. Pro-PR3 was found to constitute 10% of circulating PR3 but none of the mPR3. We found increased mRNA levels of both PR3 and CD177 in AASV, but they did not correlate with the proportion of double-positive cells. In cells sorted based on membrane expression, CD177-mRNA was several-fold higher in mPR3+ cells. When exogenous PR3 was added to CD177-transfected U937 cells, only CD177+ cells bound PR3 to their membrane. In conclusion, the increased membrane expression of PR3 found in AASV is not linked directly to circulating PR3 or PR3 gene transcription, but is dependent upon CD177 expression and correlated with the transcription of the CD177 gene.


Assuntos
Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/enzimologia , Membrana Celular/metabolismo , Isoantígenos/fisiologia , Glicoproteínas de Membrana/fisiologia , Mieloblastina/biossíntese , Neutrófilos/enzimologia , Receptores de Superfície Celular/fisiologia , Adulto , Idoso , Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/sangue , Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/imunologia , Artrite Reumatoide/sangue , Artrite Reumatoide/enzimologia , Artrite Reumatoide/imunologia , Doadores de Sangue , Indução Enzimática , Feminino , Proteínas Ligadas por GPI , Regulação da Expressão Gênica , Fator Estimulador de Colônias de Granulócitos/sangue , Fator Estimulador de Colônias de Granulócitos e Macrófagos/sangue , Hemoglobinúria Paroxística/sangue , Hemoglobinúria Paroxística/enzimologia , Hemoglobinúria Paroxística/imunologia , Humanos , Isoantígenos/biossíntese , Isoantígenos/genética , Transplante de Rim , Lúpus Eritematoso Sistêmico/sangue , Lúpus Eritematoso Sistêmico/enzimologia , Lúpus Eritematoso Sistêmico/imunologia , Masculino , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/genética , Pessoa de Meia-Idade , Mieloblastina/genética , Mieloblastina/farmacologia , Neutrófilos/imunologia , RNA Mensageiro/biossíntese , RNA Mensageiro/sangue , Receptores de Superfície Celular/biossíntese , Receptores de Superfície Celular/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Transcrição Gênica , Células U937/efeitos dos fármacos , Células U937/enzimologia
11.
J Immunol ; 184(9): 4966-72, 2010 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-20348423

RESUMO

Most allergens exist in several variants (isoallergens), each of which may be recognized differently by patient IgE. We have previously shown that several properties of the IgE repertoire, including IgE affinity and IgE clonality, are important factors determining degranulation responses of effector cells involved in type I allergic reactions. However, less is known about how the repertoire of naturally occurring isoallergens may affect this response. Thus, in this study, we investigated how individual rIgE Ab clones derived from a human subject are able to distinguish among variants of Der p 2 isoallergens and assessed the impact on basophil degranulation. Biacore analyses showed that individual rIgE clones cloned from an individual allergic to house dust mites recognized Der p 2 with binding affinities varying up to 100-fold between different Der p 2 isoforms. In a well-defined biological system consisting of human basophils sensitized with low rIgE clonality, degranulation responses were directly related to rIgE affinity toward particular rDer p 2 isoallergens. However, basophils sensitized with polyclonal patients' sera showed no differences in degranulation responses toward the different rDer p 2 isoallergens. In conclusion, our study shows that individual IgE Abs are able to bind single allergens with a broad range of affinities due to natural isoallergen variations, contributing further to the overall complexity of IgE-allergen interactions at the effector cell surface, which is, however, blurred by the polyclonal nature of patients' IgE repertoires.


Assuntos
Alérgenos/genética , Antígenos de Dermatophagoides/imunologia , Basófilos/imunologia , Sítios de Ligação de Anticorpos/imunologia , Degranulação Celular/imunologia , Imunoglobulina E/metabolismo , Isoantígenos/genética , Polimorfismo Genético/imunologia , Alérgenos/imunologia , Alérgenos/fisiologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/metabolismo , Especificidade de Anticorpos , Antígenos de Dermatophagoides/metabolismo , Proteínas de Artrópodes , Basófilos/metabolismo , Sítios de Ligação de Anticorpos/genética , Degranulação Celular/genética , Linhagem Celular , Dermatophagoides pteronyssinus/imunologia , Poeira/imunologia , Humanos , Imunoglobulina E/genética , Imunoglobulina E/fisiologia , Isoantígenos/imunologia , Isoantígenos/fisiologia , Dados de Sequência Molecular
12.
An Acad Bras Cienc ; 81(3): 559-69, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19722024

RESUMO

Neutrophil alloantigens are involved in a variety of clinical conditions including immune neutropenias, transfusion-related acute lung injury (TRALI), refractoriness to granulocyte transfusions and febrile transfusion reactions. In the last decade, considerable progress has been made in the characterization of the implicated antigens. Currently, seven antigens are assigned to five human neutrophil antigen (HNA) systems. The HNA-1a, HNA-1b and HNA-1c antigens have been identified as polymorphic forms of the neutrophil Fcgamma receptor IIIb (CD16b), encoded by three alleles. Recently, the primary structure of the HNA-2a antigen was elucidated and the HNA-2a-bearing glycoprotein was identified as a member of the Ly-6/uPAR superfamily, which has been clustered as CD177. The HNA-3a antigen is located on a 70-95 kDa glycoprotein; however, its molecular basis is still unknown. Finally, the HNA-4a and HNA-5a antigens were found to be caused by single nucleotide mutations in the alphaM (CD11b) and alphaL (CD11a) subunits of the leucocyte adhesion molecules (beta2 integrins). Molecular and biochemical characterization of neutrophil antigenshave expanded our diagnostic tools by the introduction of genotyping techniques and immunoassays for antibody identification. Further studies in the field of neutrophil immunology will facilitate the prevention and management of transfusion reactions and immune diseases caused by neutrophil antibodies.


Assuntos
Isoantígenos/genética , Neutrófilos/imunologia , Autoanticorpos/imunologia , Genótipo , Humanos , Isoantígenos/imunologia , Isoantígenos/fisiologia , Fenótipo
13.
An. acad. bras. ciênc ; 81(3): 559-569, Sept. 2009. ilus, tab
Artigo em Inglês | LILACS | ID: lil-523981

RESUMO

Neutrophil alloantigens are involved in a variety of clinical conditions including immune neutropenias, transfusion-related acute lung injury (TRALI), refractoriness to granulocyte transfusions and febrile transfusion reactions. In the last decade, considerable progress has been made in the characterization of the implicated antigens. Currently, seven antigens are assigned to five human neutrophil antigen (HNA) systems. The HNA-1a, HNA-1b and HNA-1c antigens have been identified as polymorphic forms of the neutrophil Fcγ receptor IIIb (CD16b), encoded by three alleles. Recently, the primary structure of the HNA-2a antigen was elucidated and the HNA-2a-bearing glycoprotein was identified as a member of the Ly-6/uPAR superfamily, which has been clustered as CD177. The HNA-3a antigen is located on a 70-95 kDa glycoprotein; however, its molecular basis is still unknown. Finally, the HNA-4a and HNA-5a antigens were found to be caused by single nucleotide mutations in the αM (CD11b) and αL (CD11a) subunits of the leucocyte adhesion molecules (β2 integrins). Molecular and biochemical characterization of neutrophil antigenshave expanded our diagnostic tools by the introduction of genotyping techniques and immunoassays for antibody identification. Further studies in the field of neutrophil immunology will facilitate the prevention and management of transfusion reactions and immune diseases caused by neutrophil antibodies.


Os aloantígenos de neutrófilos estão associados a várias condições clínicas como neutropenias imunes, insuficiência pulmonar relacionada à transfusão (TRALI), refratariedade à transfusão de granulócitos, e reações transfusionais febris. Na última década, foi observado considerável progresso na caracterização dos aloantígenos envolvidos nestas condições clínicas. Atualmente sete antígenos estão incluídos em cinco sistemas de antígenos de neutrófilo humano (HNA). Os antígenos HNA-1a, HNA-1b e HNA-1c foram identificados como formas polimórficas do receptor Fcγ RIIIb (CD16b), codificados por três alelos. Recentemente, a estrutura primária do antígeno HNA-2a foi elucidada e a glicoproteína carreadora do antígeno foi identificada como um membro da superfamília Ly-6/uPARe designada como CD177. O antígeno HNA-3a está localizadoem uma glicoproteína de 70-90 kDa, entretanto sua base molecular ainda é desconhecida. Finalmente, os antígenos HNA-4ae HNA-5a são resultantes de mutações de um único nucleotídeo nas subunidades αM (CD11b) and αL (CD11a) das moléculas de adesão de leucócitos (β2 integrinas). A caracterização molecular e bioquímica dos antígenos neutrofílicos permitiu a expansão das ferramentas diagnósticas pela introdução de técnicas de genotipagem e imunoensaios para a identificação de anticorpos. Novos estudos envolvendo a imunologia de granulócitos serão de grande valor para a prevenção e tratamento de reações transfusionais e doenças imunes causadas por aloanticorpos de neutrófilos.


Assuntos
Humanos , Isoantígenos/genética , Neutrófilos/imunologia , Autoanticorpos/imunologia , Genótipo , Isoantígenos/imunologia , Isoantígenos/fisiologia , Fenótipo
14.
Leuk Res ; 32(5): 811-9, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-17980909

RESUMO

Prv-1 is a hematopoietic cell surface receptor that has been shown to be overexpressed in patients diagnosed with polycythemia vera (PV) and essential thrombocythemia (ET), yet its cellular function remains unclear. In this study, we assessed the role of Prv-1 in thrombopoietin (Tpo)/Mpl signaling with the goal of identifying molecular mechanisms which augment Tpo-induced proliferation. By engineering the cytokine-dependent hematopoietic cell line BaF3 to express both Prv-1 and wild-type or mutant forms of Mpl, we were able to follow the time course of Tpo-dependent proliferation. We report that the overexpression of Prv-1 increased Tpo as well as IL-3-induced proliferation of BaF3/Mpl and BaF3 cells. Cells co-expressing Prv-1 and an Mpl receptor containing a Box 1 motif mutation, which fails to activate Jak2, was completely deficient in Tpo-dependent proliferation. In addition, BaF3 and BaF3/Prv-1 cells stimulated with IL-3 in the presence of the Jak2 inhibitor, AG490, abrogated the proliferative response, indicating that Prv-1 requires a functional Jak2 for its signaling activities. Western blot analysis showed an increase in Tpo and IL-3-induced Stat3 and Stat5 tyrosine phosphorylation in BaF3/Mpl and BaF3 cells expressing Prv-1. These results indicate a novel function for Prv-1 as a signaling molecule in cytokine signaling cascades and may lead to a greater understanding of the mechanism of overexpression of Prv-1 in myeloproliferative disorders.


Assuntos
Glicosilfosfatidilinositóis/metabolismo , Interleucina-3/farmacologia , Isoantígenos/fisiologia , Glicoproteínas de Membrana/fisiologia , Receptores de Superfície Celular/fisiologia , Trombopoetina/farmacologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Proteínas Ligadas por GPI , Sistema Hematopoético/citologia , Humanos , Fosforilação , Policitemia Vera/etiologia , Receptores de Trombopoetina/fisiologia , Fatores de Transcrição STAT/metabolismo , Trombocitemia Essencial/etiologia
15.
Eur J Immunol ; 37(10): 2983-90, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17899549

RESUMO

Programmed cell death-1 (PD-1, CD279) and its widely expressed, inducible ligand, PD-L1 (CD274), together dampen T cell activation, but whether they are essential for allograft tolerance is unknown. We show, using gene-deficient mice and blocking mAbs in wild-type mice, that costimulation blockade is ineffectual in PD-1(-/-) or PD-L1(-/-) allograft recipients, or in wild-type allograft recipients treated with anti-PD-1 or anti-PD-L1 mAb. Alloreactive PD-1(-/-) CD4 and CD8 T cells had enhanced proliferation and cytokine production compared to wild-type controls, and anergy could not be induced in PD-1-deficient CD4 T cells. We conclude that without inhibitory signals from PD-1 ligation, alloantigen-induced T cell proliferation and expansion cannot be regulated by costimulation blockade, and peripheral tolerance induction cannot occur.


Assuntos
Antígenos de Superfície/fisiologia , Proteínas Reguladoras de Apoptose/fisiologia , Antígeno B7-1/fisiologia , Glicoproteínas de Membrana/fisiologia , Peptídeos/fisiologia , Tolerância ao Transplante/imunologia , Animais , Antígenos de Superfície/genética , Antígenos de Superfície/metabolismo , Proteínas Reguladoras de Apoptose/deficiência , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Antígeno B7-1/metabolismo , Antígeno B7-H1 , Proliferação de Células , Células Cultivadas , Anergia Clonal/genética , Anergia Clonal/imunologia , Sobrevivência de Enxerto/genética , Sobrevivência de Enxerto/imunologia , Transplante de Coração/imunologia , Isoantígenos/fisiologia , Ligantes , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Knockout , Peptídeos/metabolismo , Receptor de Morte Celular Programada 1 , Linfócitos T/citologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Tolerância ao Transplante/genética , Transplante Homólogo
16.
J Biol Chem ; 282(32): 23603-12, 2007 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-17580308

RESUMO

Human neutrophil-specific CD177 (NB1 and PRV-1) has been reported to be up-regulated in a number of inflammatory settings, including bacterial infection and granulocyte-colony-stimulating factor application. Little is known about its function. By flow cytometry and immunoprecipitation studies, we identified platelet endothelial cell adhesion molecule-1 (PECAM-1) as a binding partner of CD177. Real-time protein-protein analysis using surface plasmon resonance confirmed a cation-dependent, specific interaction between CD177 and the heterophilic domains of PECAM-1. Monoclonal antibodies against CD177 and against PECAM-1 domain 6 inhibited adhesion of U937 cells stably expressing CD177 to immobilized PECAM-1. Transendothelial migration of human neutrophils was also inhibited by these antibodies. Our findings provide direct evidence that neutrophil-specific CD177 is a heterophilic binding partner of PECAM-1. This interaction may constitute a new pathway that participates in neutrophil transmigration.


Assuntos
Isoantígenos/biossíntese , Isoantígenos/fisiologia , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/fisiologia , Neutrófilos/metabolismo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Receptores de Superfície Celular/biossíntese , Receptores de Superfície Celular/fisiologia , Anticorpos Monoclonais/química , Movimento Celular , Células Endoteliais/metabolismo , Proteínas Ligadas por GPI , Humanos , Leucócitos/citologia , Modelos Biológicos , Monócitos/metabolismo , Ligação Proteica , Ressonância de Plasmônio de Superfície , Transfecção , Células U937
17.
Blood ; 109(10): 4487-93, 2007 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-17244676

RESUMO

Antineutrophil cytoplasmic antibodies (ANCAs) with specificity for proteinase 3 (PR3) are central to a form of ANCA-associated vasculitis. Membrane PR3 (mPR3) is expressed only on a subset of neutrophils. The aim of this study was to determine the mechanism of PR3 surface expression on human neutrophils. Neutrophils were isolated from patients and healthy controls, and hematopoietic stem cells from cord blood served as a model of neutrophil differentiation. Surface expression was analyzed by flow cytometry and confocal microscopy, and proteins were analyzed by Western blot experiments. Neutrophil subsets were separated by magnetic cell sorting. Transfection experiments were carried out in HEK293 and HL60 cell lines. Using neutrophils from healthy donors, patients with vasculitis, and neutrophilic differentiated stem cells we found that mPR3 display was restricted to cells expressing neutrophil glycoprotein NB1, a glycosylphosphatidylinositol (GPI)-linked surface receptor. mPR3 expression was decreased by enzymatic removal of GPI anchors from cell membranes and was absent in a patient with paroxysmal nocturnal hemoglobinuria. PR3 and NB1 coimmunoprecipitated from and colocalized on the neutrophil plasma membrane. Transfection with NB1 resulted in specific PR3 surface binding in different cell types. We conclude that PR3 membrane expression on neutrophils is mediated by the NB1 receptor.


Assuntos
Anticorpos Anticitoplasma de Neutrófilos/imunologia , Antígenos de Superfície/metabolismo , Isoantígenos/fisiologia , Glicoproteínas de Membrana/fisiologia , Mieloblastina/metabolismo , Neutrófilos/enzimologia , Receptores de Superfície Celular/fisiologia , Membrana Celular/metabolismo , Células Cultivadas , Sangue Fetal/citologia , Proteínas Ligadas por GPI , Glicosilfosfatidilinositóis/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Humanos , Imunoprecipitação , Técnicas In Vitro , Mieloblastina/imunologia , Neutrófilos/metabolismo , Transfecção
18.
Am J Pathol ; 169(6): 2127-36, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17148675

RESUMO

Dendritic cells (DCs) can release microvesicles, but the latter's numbers, size, and fate are unclear. Fluorescently labeled DCs were visualized by laser-scanning microscopy. Using a Surpass algorithm, we were able to identify and quantify per cell several hundred microvesicles released from the surface of stimulated DCs. We show that most of these microvesicles are not of endocytic origin but result from budding of the plasma membrane, hence their name, exovesicle. Using a double vital staining, we show that exovesicles isolated from activated DCs can fuse with the membrane of resting DCs, thereby allowing them to present alloantigens to lymphocytes. We concluded that, within a few hours from their release, exovesicles may amplify local or distant adaptive immunological response.


Assuntos
Apresentação de Antígeno , Vesículas Citoplasmáticas/imunologia , Células Dendríticas/imunologia , Isoantígenos/fisiologia , Microscopia Confocal/métodos , Diferenciação Celular , Membrana Celular/fisiologia , Técnicas de Cocultura , Vesículas Citoplasmáticas/fisiologia , Células Dendríticas/ultraestrutura , Humanos , Lipopolissacarídeos/farmacologia , Monócitos/fisiologia
19.
J Heart Lung Transplant ; 25(9): 1109-16, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16962474

RESUMO

BACKGROUND: CD26 is a T-cell co-stimulator, and interacts with adenosine deaminase, human immunodeficiency virus (HIV) Tat-1 protein and extracellular matrix. It possesses dipeptidylpeptidase IV (DPP IV) catalytic activity, which is linked to its co-stimulatory efficacy. We investigated the effect of specific DPP IV systemic activity inhibition on acute pulmonary rejection. METHODS: Rat single-lung transplantation (Tx) was performed (LBNF1/LEW donor/recipient) in two groups (n = 12). Group I (n = 6) received daily treatment with a Pro-Pro-diphenylphosphonate derivative (AB197), and Group II served as an untreated control. At Day 5 post-Tx, ventilatory parameters, cytotoxicity and mixed lymphocyte reaction were analyzed and staining for ISHLT rejection grade and proliferating cell nuclear antigen (PCNA) was performed. RESULTS: Treatment with AB192 abrogated acute rejection and preserved pulmonary function up to Day 5 post-Tx for PO2 (Group II: 24.9 +/- 6.9 mm Hg; Group I: 149.5 +/- 24.3 mm Hg; p < 0.001), PCO2 (Group II: 53.3 +/- 13.6 mm Hg; Group I: 39.0 +/- 9.8 mm Hg; p < 0.05) and peak airway pressure (Group II: 50.7 +/- 17.2 mm Hg; Group I: 20.2 +/- 10.0 mm Hg; p < 0.01). Controls showed moderate/severe rejection (ISHLT Grade A2 or 3), grafts from inhibited hosts revealed no/mild rejection (Grade A0 to 2: Group II: 2.8 +/- 0.3; Group I: 1.25 +/- 1.0; p < 0.005). Proliferating cell nuclear antigen (PCNA) staining of rejection-associated cellular infiltrates showed a significant reduction in positivity in perivascular infiltrates (34 +/- 11.5%; p < 0.05) and bronchial surface epithelium (31.7 +/- 10.6%; p < 0.05) in Group I vs Group II (55.9 +/- 8.4% and 57.2 +/- 4.5%). CONCLUSIONS: Irreversible enzymatic inhibition of DPP IV has been shown to abrogate acute pulmonary rejection, maintain pulmonary function, and preserve histomorphologic architecture. These results extend earlier findings and illustrate the role of CD26/DPP IV in alloantigen-mediated immune responses.


Assuntos
Dipeptidil Peptidase 4/fisiologia , Inibidores Enzimáticos/uso terapêutico , Rejeição de Enxerto/tratamento farmacológico , Rejeição de Enxerto/enzimologia , Transplante de Pulmão/fisiologia , Animais , Proliferação de Células/efeitos dos fármacos , Dipeptídeos/farmacologia , Dipeptidil Peptidase 4/sangue , Inibidores Enzimáticos/farmacologia , Rejeição de Enxerto/prevenção & controle , Isoantígenos/fisiologia , Pulmão/enzimologia , Pulmão/patologia , Pulmão/fisiopatologia , Transplante de Pulmão/imunologia , Transplante de Pulmão/patologia , Masculino , Ratos , Ratos Endogâmicos
20.
J Immunol ; 177(2): 1078-84, 2006 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-16818764

RESUMO

Intense interest has centered around the role of a subset of regulatory T cells, CD4+CD25+ Treg, in controlling the development of autoimmune disorders, allograft rejection, infection, malignancy, and allergy. We previously reported that MD1, a molecule known to be important in regulation of expression of RP105, also was important in regulating alloimmunity, and that blockade of expression of MD1 diminished graft rejection in vivo. One mechanism by which an MD1-RP105 complex exerts an effect on immune responses is through interference with an LPS-derived signal delivered through the CD14-MD-2-TLR4 complex. We show below that LPS signaling for Treg induction occurs at higher LPS thresholds that for effector T cell responses. In addition, blockade of MD1 functional activity in dendritic cells (using anti-MD1 mAbs, MD1 antisense deoxyoligonucleotides, or responder cells from mice with deletion of the MD1 gene), resulted in elevated Treg induction in response to allogeneic stimulation (in vivo or in vitro) in the presence of LPS. These data offer one mechanistic explanation for the augmented immunosuppression described following anti-MD1 treatment.


Assuntos
Antígenos de Superfície/biossíntese , Diferenciação Celular/imunologia , Glicoproteínas de Membrana/biossíntese , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/imunologia , Animais , Anticorpos Monoclonais/fisiologia , Antígenos de Superfície/imunologia , Linhagem Celular Tumoral , Células Cultivadas , Ilhas de CpG/imunologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Feminino , Sobrevivência de Enxerto/genética , Sobrevivência de Enxerto/imunologia , Inibidores do Crescimento/fisiologia , Isoantígenos/fisiologia , Lipopolissacarídeos/administração & dosagem , Lipopolissacarídeos/imunologia , Ativação Linfocitária/imunologia , Teste de Cultura Mista de Linfócitos , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oligodesoxirribonucleotídeos/administração & dosagem , Oligodesoxirribonucleotídeos/farmacologia , Transplante de Pele/imunologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Reguladores/metabolismo , Regulação para Cima/imunologia
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