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1.
JCI Insight ; 2(5): e91127, 2017 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-28289717

RESUMO

Retinoic-acid-orphan-receptor-C (RORC) is a master regulator of Th17 cells, which are pathogenic in several autoimmune diseases. Genetic Rorc deficiency in mice, while preventing autoimmunity, causes early lethality due to metastatic thymic T cell lymphomas. We sought to determine whether pharmacological RORC inhibition could be an effective and safe therapy for autoimmune diseases by evaluating its effects on Th17 cell functions and intrathymic T cell development. RORC inhibitors effectively inhibited Th17 differentiation and IL-17A production, and delayed-type hypersensitivity reactions. In vitro, RORC inhibitors induced apoptosis, as well as Bcl2l1 and BCL2L1 mRNA downregulation, in mouse and nonhuman primate thymocytes, respectively. Chronic, 13-week RORC inhibitor treatment in rats caused progressive thymic alterations in all analyzed rats similar to those in Rorc-deficient mice prior to T cell lymphoma development. One rat developed thymic cortical hyperplasia with preneoplastic features, including increased mitosis and reduced IKAROS expression, albeit without skewed T cell clonality. In summary, pharmacological inhibition of RORC not only blocks Th17 cell development and related cytokine production, but also recapitulates thymic aberrations seen in Rorc-deficient mice. While RORC inhibition may offer an effective therapeutic principle for Th17-mediated diseases, T cell lymphoma with chronic therapy remains an apparent risk.


Assuntos
Receptores do Ácido Retinoico/antagonistas & inibidores , Células Th17/citologia , Timo/patologia , Animais , Regulação para Baixo , Feminino , Expressão Gênica , Humanos , Células Jurkat , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Endogâmicos Lew , Ratos Sprague-Dawley , Receptores do Ácido Retinoico/genética , Células Th17/metabolismo
2.
Toxicol Pathol ; 43(5): 694-703, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25630683

RESUMO

Sphingosine-1-phosphate (S1P) lyase is considered as a drug target in autoimmune diseases based on the protective effect of reducing activity of the enzyme in animal models of inflammation. Since S1P lyase deficiency in mice causes a severe, lethal phenotype, it was of interest to investigate any pathological alterations associated with only partially reduced activity of S1P lyase as may be encountered upon pharmacological inhibition. Both genetic reduction of S1P lyase activity in mice and inhibition of S1P lyase with a low-molecular-weight compound in rats consistently resulted in podocyte-based kidney toxicity, which is the most severe finding. In addition, skin irritation and platelet activation were observed in both instances. The similarity of the findings in both the genetic model and the pharmacological study supports the value of analyzing inducible partially target-deficient mice for safety assessment. If the findings described in rodents translate to humans, target-related toxicity, particularly podocyte dysfunction, may limit chronic systemic treatment of autoimmune diseases with S1P lyase inhibitors. Furthermore, partial deficiency or inhibition of S1P lyase appears to provide an in vivo rodent model to enable studies on the mechanism of podocyte dysfunction.


Assuntos
Aldeído Liases/antagonistas & inibidores , Aldeído Liases/metabolismo , Ativação Plaquetária/fisiologia , Podócitos/enzimologia , Proteinúria/enzimologia , Aldeído Liases/genética , Animais , Feminino , Rim/enzimologia , Rim/patologia , Masculino , Camundongos , Proteinúria/sangue , Ratos , Pele/enzimologia , Pele/patologia , Tamoxifeno/farmacologia
3.
Toxicol Sci ; 139(1): 245-56, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24519524

RESUMO

Phototoxic properties of systemically applied pharmaceuticals may be the cause of serious adverse drug reactions. Therefore, a reliable preclinical photosafety assessment strategy, combining in vitro and in vivo approaches in a quantitative manner, is important and has not been described so far. Here, we report the establishment of an optimized modified murine local lymph node assay (LLNA), adapted for phototoxicity assessment of systemically applied compounds, as well as the test results for 34 drug candidates in this in vivo photo-LLNA. The drug candidates were selected based on their ability to absorb ultraviolet/visible light and the photo irritation factors (PIFs) determined in the well-established in vitro 3T3 neutral red uptake phototoxicity test. An in vivo phototoxic potential was identified for 13 of these drug candidates. The use of multiple dose levels in the described murine in vivo phototoxicity studies enabled the establishment of no- and/or lowest-observed-adverse-effect levels (NOAELs/LOAELs), also supporting human photosafety assessment. An in vitro-in vivo correlation demonstrated that a drug candidate classified as "phototoxic" in vitro is not necessarily phototoxic in vivo. However, the probability for a drug candidate to cause phototoxicity in vivo clearly correlated with the magnitude of the phototoxicity identified in vitro.


Assuntos
Dermatite Fototóxica , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Testes de Toxicidade/métodos , Animais , Avaliação Pré-Clínica de Medicamentos , Feminino , Linfonodos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Espectrofotometria Ultravioleta
4.
J Med Chem ; 50(15): 3489-96, 2007 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-17608465

RESUMO

Isoglobotrihexosylceramide (iGb3) is an endogenous antigen of mammalian cells and can stimulate invariant natural killer T (iNKT) cells to evoke autoimmune activities by the release of T helper 1 (Th1) and Th2 cytokines. Th1 cytokines are correlated with the antitumor and antiviral response, while Th2 cytokines are correlated with the amelioration of autoimmune diseases. iGb3 is a very weak agonist compared to the exogenous alpha-galactosylceramide; however, modification of the ceramide moiety has been advocated as one of the approaches to improve its stimulatory activity and to change the bias of release of Th1 and Th2 cytokines. Two analogues of iGb3, 2H-iGb3 and HO-iGb3 with different ceramide moieties, were synthesized. Bioassay results showed that HO-iGb3 was much more effective in stimulating iNKT cells than iGb3 at low concentration. The assay also showed that the CD1d/2H-iGb3 complexes are remarkably efficient in stimulating iNKT cells.


Assuntos
Globosídeos/síntese química , Células Matadoras Naturais/efeitos dos fármacos , Ativação Linfocitária/efeitos dos fármacos , Receptores de Antígenos de Linfócitos T/agonistas , Triexosilceramidas/síntese química , Antígenos CD1/metabolismo , Antígenos CD1d , Sequência de Carboidratos , Linhagem Celular , Globosídeos/química , Globosídeos/farmacologia , Humanos , Interferon gama/biossíntese , Interleucina-4/biossíntese , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Estereoisomerismo , Relação Estrutura-Atividade , Triexosilceramidas/química , Triexosilceramidas/farmacologia
5.
Eur J Immunol ; 37(7): 1724-6, 2007 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-17587196

RESUMO

There is increasing evidence that T cells recognizing lipid antigens contribute to the immunological regulation of different disease conditions including autoimmunity. The best-known subset is CD1d-restricted lipid-reactive T cells characterized by the expression of an invariant TCRalpha chain. Much less is known about the biology of another invariant T cell subset, which is restricted to the MHC class I-like molecule MR1. A beneficial role of MR1-restricted T cells has been suggested in a mouse EAE model. However, the nature of antigens that can be presented by MR1 to this invariant T cell subset remained largely unclear. An article in this issue of the European Journal of Immunology presents strong indications that derivatives of alpha-mannosyl ceramide (alpha-ManCer), i.e. glycolipids, can serve as ligands for MR1-restricted invariant T cells. In addition to that, the structure of the alpha-ManCer sphingosine chain influences the Th1-Th2 polarization of the cytokine response. These important new findings will foster further research on the identity of physiological ligands for MR1-restricted T cells and on their relation with immunoregulation. See accompanying article: (http://dx.doi.org/10.1002/eji.200636689).


Assuntos
Antígenos de Histocompatibilidade Classe I/imunologia , Lipídeos/imunologia , Ativação Linfocitária/imunologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T/imunologia , Animais , Apresentação de Antígeno/imunologia , Humanos , Antígenos de Histocompatibilidade Menor , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia
6.
Eur J Immunol ; 37(6): 1431-41, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17492806

RESUMO

Deficiencies in enzymes of the lysosomal glycosphingolipid degradation pathway or in lysosomal lipid transfer proteins cause an imbalance in lipid metabolism and induce accumulation of certain lipids. A possible impact of such an imbalance on the presentation of lipid antigens to lipid-reactive T cells has only been hypothesized but not extensively studied so far. Here we demonstrate that presentation of lipid antigens to, and development of, lipid-reactive CD1d-restricted NKT cells, are impaired in mice deficient in the lysosomal enzyme beta-galactosidase (betaGal) or the lysosomal lipid transfer protein Niemann-Pick C (NPC) 2. Importantly, the residual populations of NKT cells selected in betaGal-/- and NPC2-/- mice showed differential TCR and CD4 repertoire characteristics, suggesting that differential selecting CD1d:lipid antigen complexes are formed. Furthermore, we provide direct evidence that accumulation of lipids impairs lipid antigen presentation in both cases. However, the mechanisms by which imbalanced lipid metabolism affected lipid antigen presentation were different. Based on these results, the impact of lipid accumulation should be generally considered in the interpretation of immunological deficiencies found in mice suffering from lipid metabolic disorders.


Assuntos
Apresentação de Antígeno/imunologia , Glicolipídeos/imunologia , Metabolismo dos Lipídeos/imunologia , Subpopulações de Linfócitos T/imunologia , 1-Desoxinojirimicina/análogos & derivados , 1-Desoxinojirimicina/farmacologia , Animais , Apresentação de Antígeno/efeitos dos fármacos , Antígenos CD1/análise , Antígenos CD1/metabolismo , Antígenos CD1d , Antígenos CD4/análise , Antígenos CD4/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Inibidores Enzimáticos/farmacologia , Galactosilceramidas/imunologia , Galactosilceramidas/metabolismo , Globosídeos/imunologia , Glicolipídeos/metabolismo , Humanos , Transtornos do Metabolismo dos Lipídeos/genética , Transtornos do Metabolismo dos Lipídeos/imunologia , Fígado/citologia , Fígado/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Baço/citologia , Baço/imunologia , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/metabolismo , Timo/citologia , Timo/imunologia , Triexosilceramidas/imunologia , Proteínas de Transporte Vesicular/genética , beta-Galactosidase/genética
7.
Bioorg Med Chem Lett ; 16(8): 2195-9, 2006 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-16458002

RESUMO

Glycoceramides can activate NKT cells by binding with CD1d to produce IFN-gamma, IL-4, and other cytokines. An efficient synthetic pathway for alpha-galactosylceramide (KRN7000) was established by coupling a protected galactose donor to a properly protected ceramide. During the investigation, it was discovered that when the ceramide was protected with benzyl groups, only beta-galactosylceramide was produced from the glycosylation reaction. In contrast, the ceramide with benzoyl protecting groups produced alpha-galactosylceramide. Isoglobotrihexosylceramide (iGb3) was prepared by glycosylation of Galalpha1-3Galbeta1-4Glc donor with 2-azido-sphingosine in high yield. Biological assays on the synthetic KRN7000 and iGb3 were performed using human and murine iNKT cell clones or hybridomas.


Assuntos
Galactosilceramidas/síntese química , Galactosilceramidas/farmacologia , Globosídeos/síntese química , Globosídeos/farmacologia , Animais , Antígenos CD1/metabolismo , Células Cultivadas , Citocinas/metabolismo , Glicosilação , Humanos , Hibridomas/metabolismo , Interferon gama/metabolismo , Interleucina-4/metabolismo , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Camundongos , Transfecção
8.
J Immunol ; 176(4): 2064-8, 2006 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-16455960

RESUMO

Invariant Valpha14 (Valpha14i) NKT cells are a murine CD1d-dependent regulatory T cell subset characterized by a Valpha14-Jalpha18 rearrangement and expression of mostly Vbeta8.2 and Vbeta7. Whereas the TCR Vbeta domain influences the binding avidity of the Valpha14i TCR for CD1d-alpha-galactosylceramide complexes, with Vbeta8.2 conferring higher avidity binding than Vbeta7, a possible impact of the TCR Vbeta domain on Valpha14i NKT cell selection by endogenous ligands has not been studied. In this study, we show that thymic selection of Vbeta7(+), but not Vbeta8.2(+), Valpha14i NKT cells is favored in situations where endogenous ligand concentration or TCRalpha-chain avidity are suboptimal. Furthermore, thymic Vbeta7(+) Valpha14i NKT cells were preferentially selected in vitro in response to CD1d-dependent presentation of endogenous ligands or exogenously added self ligand isoglobotrihexosylceramide. Collectively, our data demonstrate that the TCR Vbeta domain influences the selection of Valpha14i NKT cells by endogenous ligands, presumably because Vbeta7 confers higher avidity binding.


Assuntos
Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Animais , Antígenos CD1/genética , Antígenos CD1/metabolismo , Antígenos CD1d , Células Cultivadas , Humanos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Estrutura Terciária de Proteína , Linfócitos T Reguladores/metabolismo , Timo/imunologia , Timo/metabolismo
9.
Trends Immunol ; 27(2): 57-9, 2006 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16377244

RESUMO

T cells recognizing lipid antigens presented by CD1 molecules have an important role in the immune response. Several lipid antigens for CD1-restricted T cells have been identified, as have some rules of CD1 loading and CD1-restricted presentation. Little is known, however, about the delivery of lipid antigens from either extracellular compartments or CD1-negative cells to CD1-expressing antigen-presenting cells (APCs). A recent study provides evidence for a role for apolipoprotein E in binding lipid antigens and delivering them to APCs.


Assuntos
Lipoproteínas/sangue , Lipoproteínas/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , Antígenos/imunologia , Humanos , Imunoterapia , Lipídeos/imunologia , Vacinação
10.
J Immunol ; 175(11): 7303-10, 2005 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-16301636

RESUMO

CD1d-dependent invariant Valpha14 (Valpha14i) NKT cells are innate T lymphocytes expressing a conserved semi-invariant TCR, consisting, in mice, of the invariant Valpha14-Jalpha18 TCR alpha-chain paired mostly with Vbeta8.2 and Vbeta7. The cellular requirements for thymic positive and negative selection of Valpha14i NKT cells are only partially understood. Therefore, we generated transgenic mice expressing human CD1d (hCD1d) either on thymocytes, mainly CD4+ CD8+ double positive, or on APCs, the cells implicated in the selection of Valpha14i NKT cells. In the absence of the endogenous mouse CD1d (mCD1d), the expression of hCD1d on thymocytes, but not on APCs, was sufficient to select Valpha14i NKT cells that proved functional when activated ex vivo with the Ag alpha-galactosyl ceramide. Valpha14i NKT cells selected by hCD1d on thymocytes, however, attained lower numbers than in control mice and expressed essentially Vbeta8.2. The low number of Vbeta8.2+ Valpha14i NKT cells selected by hCD1d on thymocytes was not reversed by the concomitant expression of mCD1d, which, instead, restored the development of Vbeta7+ Valpha14i NKT cells. Vbeta8.2+, but not Vbeta7+, NKT cell development was impaired in mice expressing both hCD1d on APCs and mCD1d. Taken together, our data reveal that selective CD1d expression by thymocytes is sufficient for positive selection of functional Valpha14i NKT cells and that both thymocytes and APCs may independently mediate negative selection.


Assuntos
Células Apresentadoras de Antígenos/citologia , Células Apresentadoras de Antígenos/imunologia , Antígenos CD1/imunologia , Células Matadoras Naturais/imunologia , Linfócitos T/imunologia , Animais , Células Apresentadoras de Antígenos/metabolismo , Antígenos CD1/genética , Antígenos CD1/metabolismo , Diferenciação Celular/imunologia , Citometria de Fluxo , Regulação da Expressão Gênica/imunologia , Humanos , Células Matadoras Naturais/metabolismo , Ativação Linfocitária/imunologia , Camundongos , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Linfócitos T/metabolismo , Timo/citologia , Timo/imunologia
12.
J Immunol ; 172(5): 3034-41, 2004 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-14978108

RESUMO

Pseudomonas aeruginosa exotoxin A (PEA) causes T cell- and Kupffer cell (KC)-dependent liver injury in mice. TNF-alpha as well as IL-18 and perforin are important mediators of liver damage following PEA injection. In this study, we focus on the role of NK and NKT cells in PEA-induced liver toxicity. Depletion of both NK and NKT cells by injection of anti-NK1.1 Ab as well as depletion of NK cells alone by anti-asialo GM1 Ab protected mice from PEA-induced hepatotoxicity, whereas mice lacking only NKT cells were susceptible. Additionally, we observed infiltration of NK cells, T cells, and neutrophils into liver parenchyma after injection of PEA. The number of NKT cells, however, remained unchanged. The increase in intrahepatic NK cells depended on KCs and the TNF-alpha-dependent up-regulation of the adhesion molecule VCAM-1 in the liver, but not on NKT cells. PEA also augmented the cytotoxicity of hepatic NK cells against typical NK target cells (YAC-1 cells). This effect depended on KCs, but not on TNF-alpha or NKT cells. Furthermore, only weak expression of MHC class I was detected on hepatocytes, which was further down-regulated in PEA-treated mice. This could explain the susceptibility of hepatocytes to NK cell cytolytic activity in this model. Our results demonstrate that NK cells, activated and recruited independently of NKT cells, contribute to PEA-induced T cell-dependent liver injury in mice.


Assuntos
ADP Ribose Transferases/toxicidade , Toxinas Bacterianas/toxicidade , Citotoxicidade Imunológica/imunologia , Exotoxinas/toxicidade , Células Matadoras Naturais/imunologia , Fígado/imunologia , Fígado/microbiologia , Subpopulações de Linfócitos T/imunologia , Fatores de Virulência/toxicidade , Adjuvantes Imunológicos/toxicidade , Animais , Movimento Celular/imunologia , Regulação para Baixo/imunologia , Antígenos de Histocompatibilidade Classe I/biossíntese , Injeções Intravenosas , Células Matadoras Naturais/microbiologia , Células Matadoras Naturais/patologia , Células de Kupffer/imunologia , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infiltração de Neutrófilos/imunologia , Subpopulações de Linfócitos T/patologia , Fator de Necrose Tumoral alfa/fisiologia , Regulação para Cima/imunologia , Molécula 1 de Adesão de Célula Vascular/biossíntese , Exotoxina A de Pseudomonas aeruginosa
13.
J Hepatol ; 39(3): 333-40, 2003 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-12927918

RESUMO

BACKGROUND/AIMS: Silibinin is the major pharmacologically active compound of the Silybum marianum fruit extract silymarin. Its well-known hepatoprotective activities are mostly explained by antioxidative properties, inhibition of phosphatidylcholine synthesis or stimulation of hepatic RNA and protein synthesis. Here, we characterized the hepatoprotective potential of silibinin as an immune-response modifier in T cell-dependent hepatitis in vivo. METHODS: Silibinin was tested in the mouse model of concanavalin A (ConA)-induced, T cell-dependent hepatitis. Liver injury was assessed by quantification of plasma transaminase activities and intrahepatic DNA fragmentation. Plasma cytokine concentrations were determined by enzyme-linked immunosorbent assay (ELISA), intrahepatic cytokine and inducible NO synthase (iNOS) mRNA levels by reverse transcriptase polymerase chain reaction, intrahepatic iNOS expression by immunofluorescent staining, and intrahepatic nuclear factor kappa B (NF-kappaB) activation by electrophoretic mobility shift assay. RESULTS: Silibinin significantly inhibited ConA-induced liver disease. Silibinin proved to be an immune-response modifier in vivo, inhibiting intrahepatic expression of tumor necrosis factor, interferon-gamma, interleukin (IL)-4, IL-2, and iNOS, and augmenting synthesis of IL-10. In addition, silibinin inhibited intrahepatic activation of NF-kappaB. CONCLUSIONS: Silibinin, suppressing T cell-dependent liver injury as an immune-response modifier, might be a valuable drug in therapeutic situations in which intrahepatic immunosuppression is required.


Assuntos
Citoproteção , Hepatopatias/etiologia , Hepatopatias/prevenção & controle , Fígado/efeitos dos fármacos , Silimarina/farmacologia , Linfócitos T , Animais , Células Cultivadas , Doença Hepática Induzida por Substâncias e Drogas , Concanavalina A/farmacologia , Citocinas/genética , Citocinas/metabolismo , Indução Enzimática/efeitos dos fármacos , Galactosamina , Leucócitos/efeitos dos fármacos , Leucócitos/metabolismo , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , NF-kappa B/metabolismo , Óxido Nítrico Sintase/biossíntese , Óxido Nítrico Sintase Tipo II , RNA Mensageiro/biossíntese , Proteínas Recombinantes , Silibina , Fator de Necrose Tumoral alfa
14.
J Immunol ; 170(12): 5815-9, 2003 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-12794105

RESUMO

CD1d tetramers loaded with alpha-galactosylceramide (alpha-GalCer) bind selectively to mouse invariant Valpha14 (Valpha14i) NKT cells and their human counterparts. Whereas tetramer binding strictly depends on the expression of a Valpha14-Jalpha18 chain in murine NKT cells, the associated beta-chain (typically expressing Vbeta8.2 or Vbeta7) appears not to influence tetramer binding. In this study, we describe novel alpha-GalCer-loaded mouse and human CD1d-IgG1 dimers, which revealed an unexpected influence of the TCR-beta chain on the avidity of CD1d:alpha-GalCer binding. A subset of Valpha14i NKT cells clearly discriminated alpha-GalCer bound to mouse or human CD1d on the basis of avidity differences conferred by the Vbeta domain of the TCR-beta chain, with Vbeta8.2 conferring higher avidity binding than Vbeta7.


Assuntos
Antígenos CD1/metabolismo , Galactosilceramidas/metabolismo , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Fragmentos de Peptídeos/fisiologia , Receptores de Antígenos de Linfócitos T alfa-beta/fisiologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Animais , Antígenos CD1d , Adesão Celular/imunologia , Dimerização , Relação Dose-Resposta Imunológica , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Terciária de Proteína , Células Tumorais Cultivadas
15.
J Exp Med ; 197(7): 919-25, 2003 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-12682111

RESUMO

In addition to their CD1d-restricted T cell receptor (TCR), natural killer T (NKT) cells express various receptors normally associated with NK cells thought to act, in part, as modulators of TCR signaling. Immunoreceptor-tyrosine activation (ITAM) and inhibition (ITIM) motifs associated with NK receptors may augment or attenuate perceived TCR signals respectively, potentially influencing NKT cell development and function. ITIM-containing Ly49 family receptors expressed by NKT cells are proposed to play a role in their development and function. We have produced mice transgenic for the ITAM-associated Ly49D and ITIM-containing Ly49A receptors and their common ligand H2-Dd to determine the importance of these signaling interplays in NKT cell development. Ly49D/H2-Dd transgenic mice had selectively and severely reduced numbers of thymic and peripheral NKT cells, whereas both ligand and Ly49D transgenics had normal numbers of NKT cells. CD1d tetramer staining revealed a blockade of NKT cell development at an early precursor stage. Coexpression of a Ly49A transgene partially rescued NKT cell development in Ly49D/H2-Dd transgenics, presumably due to attenuation of ITAM signaling. Thus, Ly49D-induced ITAM signaling is incompatible with the early development of cells expressing semi-invariant CD1d-restricted TCRs and appropriately harmonized ITIM-ITAM signaling is likely to play an important role in the developmental program of NKT cells.


Assuntos
Antígenos CD1/fisiologia , Antígenos Ly/fisiologia , Antígenos H-2/fisiologia , Células Matadoras Naturais/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Motivos de Aminoácidos , Animais , Antígenos CD1d , Antígenos Ly/química , Antígeno de Histocompatibilidade H-2D , Lectinas Tipo C , Ligantes , Proteínas de Membrana , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Transgênicos , Subfamília A de Receptores Semelhantes a Lectina de Células NK , Receptores de Antígenos de Linfócitos T alfa-beta/fisiologia , Receptores Imunológicos/fisiologia , Receptores Semelhantes a Lectina de Células NK
16.
J Immunol ; 170(5): 2390-8, 2003 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-12594262

RESUMO

A sizable fraction of T cells expressing the NK cell marker NK1.1 (NKT cells) bear a very conserved TCR, characterized by homologous invariant (inv.) TCR V alpha 24-J alpha Q and V alpha 14-J alpha 18 rearrangements in humans and mice, respectively, and are thus defined as inv. NKT cells. Because human inv. NKT cells recognize mouse CD1d in vitro, we wondered whether a human inv. V alpha 24 TCR could be selected in vivo by mouse ligands presented by CD1d, thereby supporting the development of inv. NKT cells in mice. Therefore, we generated transgenic (Tg) mice expressing the human inv. V alpha 24-J alpha Q TCR chain in all T cells. The expression of the human inv. V alpha 24 TCR in TCR C alpha(-/-) mice indeed rescues the development of inv. NKT cells, which home preferentially to the liver and respond to the CD1d-restricted ligand alpha-galactosylceramide (alpha-GalCer). However, unlike inv. NKT cells from non-Tg mice, the majority of NKT cells in V alpha 24 Tg mice display a double-negative phenotype, as well as a significant increase in TCR V beta 7 and a corresponding decrease in TCR V beta 8.2 use. Despite the forced expression of the human CD1d-restricted TCR in C alpha(-/-) mice, staining with mCD1d-alpha-GalCer tetramers reveals that the absolute numbers of peripheral CD1d-dependent T lymphocytes increase at most by 2-fold. This increase is accounted for mainly by an increased fraction of NK1.1(-) T cells that bind CD1d-alpha-GalCer tetramers. These findings indicate that human inv. V alpha 24 TCR supports the development of CD1d-dependent lymphocytes in mice, and argue for a tight homeostatic control on the total number of inv. NKT cells. Thus, human inv. V alpha 24 TCR-expressing mice are a valuable model to study different aspects of the inv. NKT cell subset.


Assuntos
Antígenos CD1/fisiologia , Antígenos de Diferenciação de Linfócitos B/fisiologia , Antígenos/biossíntese , Antígenos de Histocompatibilidade Classe II/fisiologia , Células Matadoras Naturais/imunologia , Camundongos Transgênicos/imunologia , Biossíntese de Proteínas , Proteínas , Receptores de Antígenos de Linfócitos T alfa-beta/fisiologia , Subpopulações de Linfócitos T/imunologia , Animais , Antígenos CD1d , Antígenos de Diferenciação de Linfócitos B/biossíntese , Antígenos de Diferenciação de Linfócitos B/genética , Antígenos Ly , Antígenos de Superfície , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Células Cultivadas , Epitopos de Linfócito T/imunologia , Galactosilceramidas/imunologia , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Rearranjo Gênico da Cadeia beta dos Receptores de Antígenos dos Linfócitos T/genética , Genes Codificadores da Cadeia alfa de Receptores de Linfócitos T/genética , Genes Codificadores da Cadeia alfa de Receptores de Linfócitos T/fisiologia , Antígenos de Histocompatibilidade Classe II/biossíntese , Antígenos de Histocompatibilidade Classe II/genética , Humanos , Regiões Constantes de Imunoglobulina/genética , Imunofenotipagem , Células Matadoras Naturais/citologia , Células Matadoras Naturais/metabolismo , Lectinas Tipo C , Contagem de Linfócitos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Knockout , Subfamília B de Receptores Semelhantes a Lectina de Células NK , Receptores de Antígenos de Linfócitos T alfa-beta/biossíntese , Receptores de Antígenos de Linfócitos T alfa-beta/deficiência , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/metabolismo
17.
J Immunol ; 170(4): 2129-37, 2003 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-12574385

RESUMO

TNF-alpha is a central mediator of T cell activation-induced hepatitis in mice, e.g., induced by Pseudomonas exotoxin A (PEA). In this in vivo mouse model of T cell-dependent hepatitis, liver injury depends on both TNFRs. Whereas TNFR1 can directly mediate hepatocyte death, the in vivo functions of TNFR2 in pathophysiology remained unclear. TNFR2 has been implicated in deleterious leukocyte activation in a transgenic mouse model and in enhancement of TNFR1-mediated cell death in cell lines. In this study, we clarify the role of hepatocyte- vs leukocyte-expressed TNFR2 in T cell-dependent liver injury in vivo, using the PEA-induced hepatitis model. Several types of TNFR2-expressing leukocytes, especially neutrophils and NK cells, accumulated within the liver throughout the pathogenic process. Surprisingly, only parenchymal TNFR2 expression, but not the TNFR2 expression on leukocytes, contributed to PEA-induced hepatitis, as shown by analysis of wild-type --> tnfr2 degrees and the reciprocal mouse bone marrow chimeras. Furthermore, PEA induced NF-kappaB activation and cytokine production in the livers of both wild-type and tnfr2 degrees mice, whereas only primary mouse hepatocytes from wild-type, but not from tnfr2 degrees, mice were susceptible to cell death induced by a combination of agonistic anti-TNFR1 and anti-TNFR2 Abs. Our results suggest that parenchymal, but not leukocyte, TNFR2 mediates T cell-dependent hepatitis in vivo. The activation of leukocytes does not appear to be disturbed by the absence of TNFR2.


Assuntos
Antígenos CD/fisiologia , Doença Hepática Induzida por Substâncias e Drogas/imunologia , Doença Hepática Induzida por Substâncias e Drogas/patologia , Hepatócitos/imunologia , Hepatócitos/metabolismo , Leucócitos/imunologia , Leucócitos/metabolismo , Receptores do Fator de Necrose Tumoral/fisiologia , Subpopulações de Linfócitos T/imunologia , Animais , Antígenos CD/biossíntese , Antígenos CD/sangue , Antígenos CD/efeitos da radiação , Proteínas de Bactérias/toxicidade , Células da Medula Óssea/imunologia , Células da Medula Óssea/efeitos da radiação , Transplante de Medula Óssea , Morte Celular/imunologia , Células Cultivadas , Doença Hepática Induzida por Substâncias e Drogas/terapia , Injeções Intravenosas , Interleucina-6/biossíntese , Células de Kupffer/imunologia , Células de Kupffer/metabolismo , Transfusão de Leucócitos , Leucócitos/efeitos da radiação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , NF-kappa B/metabolismo , Quimera por Radiação , Receptores do Fator de Necrose Tumoral/biossíntese , Receptores do Fator de Necrose Tumoral/sangue , Receptores do Fator de Necrose Tumoral/efeitos da radiação , Receptores Tipo II do Fator de Necrose Tumoral , Transdução de Sinais/imunologia , Fator de Necrose Tumoral alfa/biossíntese
18.
Am J Physiol Cell Physiol ; 284(2): C439-46, 2003 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-12388069

RESUMO

Hypoxic/ischemic conditions provoke activation of the transcription factor hypoxia-inducible factor-1 (HIF-1). HIF-1 is composed of HIF-1alpha (subjected to protein stability regulation) and constitutively expressed HIF-1beta. Besides hypoxia, diverse agonists are identified that stabilize HIF-1alpha during normoxia. Here we used a coculture system of RAW 264.7 macrophage cells and tubular LLC-PK(1) cells to establish that lipopolysaccharide- and interferon-gamma-stimulated but not resting macrophages elicited HIF-1alpha accumulation in LLC-PK(1) cells. Via pharmacological interventions such as blockade of nitric oxide (NO) production in macrophages, scavenging of NO with the use of 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, or application of tumor necrosis factor-alpha (TNF-alpha)-neutralizing antibodies, we identified NO and TNF-alpha as signaling molecules. Working in concert, NO and TNF-alpha have a stronger response when allowed direct cell-to-cell contact instead of contact with only the cell supernatant of activated macrophages. We show that signal transmission by NO with TNF-alpha in LLC-PK(1) cells is mediated via the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway, because it is blocked by wortmannin or dominant-negative forms of PI3-K as well as protein kinase B. We conclude that NO and TNF-alpha, derived from activated macrophages, provoke HIF-1alpha stabilization in LLC-PK(1) cells under normoxic conditions, which underscores HIF-1alpha stabilization due to intercellular regulation.


Assuntos
Células Epiteliais/metabolismo , Túbulos Renais/metabolismo , Macrófagos/metabolismo , Óxido Nítrico/metabolismo , Proteínas Serina-Treonina Quinases , Fatores de Transcrição/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Comunicação Celular/fisiologia , Quimiotaxia de Leucócito/efeitos dos fármacos , Quimiotaxia de Leucócito/fisiologia , Técnicas de Cocultura , Inibidores Enzimáticos/farmacologia , Células Epiteliais/efeitos dos fármacos , Sequestradores de Radicais Livres/farmacologia , Hipóxia/metabolismo , Hipóxia/fisiopatologia , Subunidade alfa do Fator 1 Induzível por Hipóxia , Inflamação/metabolismo , Inflamação/fisiopatologia , Mediadores da Inflamação/metabolismo , Túbulos Renais/efeitos dos fármacos , Células LLC-PK1 , Macrófagos/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Transdução de Sinais/fisiologia , Suínos
19.
Cytokine ; 19(3): 115-20, 2002 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-12242077

RESUMO

The activation of T-cells and macrophages and subsequent induction of cytokines are critical factors in the development of hepatitis. Up-regulation of pro-inflammatory cytokines, e.g. TNF has been shown to induce liver injury while counter regulation by anti-inflammatory cytokines, e.g. IL-10 is protective. We compared the induction of liver injury and the expression pattern of a variety of cytokines in T-cell- versus non-T-cell-dependent mouse models of liver injury. TNF, IFNgamma, IL-2, IL-4, IL-6, IL-10 and IL-12 were measured in plasma and liver tissue after either Concanavalin A (Con A), D-galactosamine/lipopolysaccharide (GalN/LPS) or high dose LPS induced liver injury. Additionally, the intra-hepatic expression of the putative pathogenicity factor high mobility group 1 protein (HMG-1) was compared in all three models.


Assuntos
Citocinas/biossíntese , Citocinas/sangue , Hepatite/sangue , Animais , Concanavalina A/farmacologia , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Galactosamina/farmacologia , Interferon gama/biossíntese , Interleucina-10/biossíntese , Interleucina-12/biossíntese , Interleucina-2/biossíntese , Interleucina-4/biossíntese , Interleucina-6/biossíntese , Cinética , Lipopolissacarídeos/farmacologia , Fígado/lesões , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Fator de Necrose Tumoral alfa/biossíntese , Regulação para Cima
20.
J Virol Methods ; 103(1): 101-7, 2002 May.
Artigo em Inglês | MEDLINE | ID: mdl-11906737

RESUMO

A nucleic acid sequence based (NASBA) assay for the generic detection of enterovirus RNA in cerebrospinal fluid (CSF) samples was developed and compared with an established reverse transcription/nested polymerase chain reaction (PCR) protocol. The sensitivity of NASBA followed by detection of amplicons with a biotinylated oligonucleotide probe was < or = ten copies of enterovirus RNA, indicating that enterovirus NASBA achieves a similar sensitivity as nested PCR. Moreover, NASBA detected a panel of 22 different serotypes of the species poliovirus, human enterovirus A, human enterovirus B and human enterovirus C completely. For evaluating NASBA as a diagnostic tool, 61 CSF samples of patients suffering from aseptic meningitis were tested in parallel with NASBA and nested PCR. NASBA detected enterovirus RNA in four CSF samples, two of these were also positive by nested PCR and two other CSF samples were positive only by nested PCR (in total six positive samples). All other 55 of 61 CSF samples were concordantly enterovirus negative by both methods. In conclusion, the more simple to handle 'one step' NASBA is as sensitive as nested PCR and may be used as an alternative method for the detection of enterovirus RNA in CSF samples. Enterovirus NASBA is a 'one step' RNA amplification procedure that is less prone to cross-contamination compared to a three step nested PCR.


Assuntos
Infecções por Enterovirus/líquido cefalorraquidiano , Enterovirus/isolamento & purificação , Meningite Asséptica/líquido cefalorraquidiano , Meningoencefalite/líquido cefalorraquidiano , RNA Viral/líquido cefalorraquidiano , Replicação de Sequência Autossustentável , Sequência de Bases , Biotinilação , Infecções por Coxsackievirus/líquido cefalorraquidiano , Infecções por Coxsackievirus/virologia , Infecções por Echovirus/líquido cefalorraquidiano , Infecções por Echovirus/virologia , Enterovirus/genética , Enterovirus Humano A/genética , Enterovirus Humano A/isolamento & purificação , Enterovirus Humano B/genética , Enterovirus Humano B/isolamento & purificação , Enterovirus Humano C/genética , Enterovirus Humano C/isolamento & purificação , Infecções por Enterovirus/diagnóstico , Infecções por Enterovirus/virologia , Humanos , Meningite Asséptica/virologia , Meningoencefalite/virologia , Dados de Sequência Molecular , Poliovirus/genética , Poliovirus/isolamento & purificação , Reação em Cadeia da Polimerase , Sensibilidade e Especificidade , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico
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