Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 11 de 11
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Bioconjug Chem ; 25(1): 52-62, 2014 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-24341642

RESUMO

IL-4 signaling into a cell occurs via assembly of a receptor complex that consists of a high-affinity IL-4Rα chain and a low affinity chain, where the low-affinity chain is either γc or IL-13Rα1. It has been previously shown that mutational disruption of the low affinity interface in the IL-4DM (double mutein) yields an antagonist that inhibits IL-4 as well as IL-13-dependent responses. The present study reveals that new types of IL-4 antagonists can be generated by site-specific chemical modification. The chemically modified IL-4 analogues consist of (1) mixed disulfides created by refolding IL-4 cysteine muteins in the presence of different thiol compounds or (2) maleimide conjugates created by modifying cysteine muteins with maleimide derivatives. IL-4 analogues chemically modified at position 121 retain marginal binding affinity to γc or IL-13Rα1 receptor ectodomains during SPR interaction analysis. The biological activity of the analogues is strongly reduced in HEK-Blue IL-4/IL-13 cells as well as in Jurkat cells. Since the IL-4 analogues modified at position 121 have the ability to inhibit γc (IL-4)- and IL13Rα1 (IL-4/IL-13)-dependent responses in Jurkat and HEK-Blue cell lines, they effectively act as IL-4 antagonists. The results of our IL-4 study provide the first example of a cytokine that is transformed into a competitive inhibitor by site-specific chemical modification.


Assuntos
Interleucina-13/antagonistas & inibidores , Interleucina-4/análogos & derivados , Interleucina-4/antagonistas & inibidores , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Interleucina-4/química , Células Jurkat , Modelos Moleculares , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade
2.
Cytokine ; 64(1): 112-21, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23972727

RESUMO

Interleukin-4 (IL-4) is a potent antiinflammatory cytokine. However its use in the clinic is hampered by side effects. We here describe the identification of a novel synthetic peptide, termed Ph8, derived from α-helix C of IL-4, which interacts with IL-4 receptor α (IL-4Rα). Employing various cultured genetically engineered cell lines and primary lymphocytes, surface plasmon resonance, qPCR, ELISA and immunoblotting techniques we found that Ph8 bound IL-4Rα and mimicked the anti-inflammatory effects of IL-4 by inhibiting TNF-α production by macrophages in vitro. It induced phosphorylation of STAT6 65kD but inhibited phosphorylation of STAT6 110 kD induced by IL-4 in a B-cell line that expressed the type I receptor. It also inhibited the IL-4-stimulated expression of a STAT6-inducible reporter gene in cells that expressed the type II receptor. Ph8 inhibited the proliferation of Th1/2 cells and downregulated the production of IFN-γ in stimulated Th1 cells. Moreover, Ph8 did not induce any shift in Th1/Th2 profile. This is a favorable effect and it is indicating that Ph8 could block general T cell activation and inflammatory responses without further inducing the side effects generally associated with IL-4 signaling. These data collectively show that Ph8 is only a partial agonist of IL-4 mimicking its desirable properties. In agreement, Ph8 treatment of rats with collagen-induced arthritis, a Th1- and antibody- mediated disease of joint, delayed the manifestation of chronic inflammation and reduced acute inflammation in carrageenan-induced edema. Our findings indicate that Ph8 is a promising potential drug candidate for the treatment of inflammatory diseases.


Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Subunidade alfa de Receptor de Interleucina-4/metabolismo , Interleucina-4/farmacologia , Fragmentos de Peptídeos/farmacologia , Animais , Artrite Experimental/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Edema/tratamento farmacológico , Células HEK293 , Humanos , Interferon gama/metabolismo , Interleucina-4/análogos & derivados , Interleucina-4/química , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fragmentos de Peptídeos/química , Fosforilação/efeitos dos fármacos , Ligação Proteica , Ratos , Ratos Wistar , Fator de Transcrição STAT6/metabolismo , Células Th1/efeitos dos fármacos , Células Th1/metabolismo , Fator de Necrose Tumoral alfa/biossíntese
4.
Nat Chem Biol ; 8(12): 990-8, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23103943

RESUMO

Cytokines dimerize their receptors, with the binding of the 'second chain' triggering signaling. In the interleukin (IL)-4 and IL-13 system, different cell types express varying numbers of alternative second receptor chains (γc or IL-13Rα1), forming functionally distinct type I or type II complexes. We manipulated the affinity and specificity of second chain recruitment by human IL-4. A type I receptor-selective IL-4 'superkine' with 3,700-fold higher affinity for γc was three- to ten-fold more potent than wild-type IL-4. Conversely, a variant with high affinity for IL-13Rα1 more potently activated cells expressing the type II receptor and induced differentiation of dendritic cells from monocytes, implicating the type II receptor in this process. Superkines showed signaling advantages on cells with lower second chain numbers. Comparative transcriptional analysis reveals that the superkines induce largely redundant gene expression profiles. Variable second chain numbers can be exploited to redirect cytokines toward distinct cell subsets and elicit new actions, potentially improving the selectivity of cytokine therapy.


Assuntos
Citocinas/fisiologia , Interleucina-4/análogos & derivados , Interleucina-4/farmacologia , Linfócitos T CD4-Positivos/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Células Dendríticas/efeitos dos fármacos , Citometria de Fluxo , Perfilação da Expressão Gênica , Humanos , Fatores Imunológicos/farmacologia , Interleucina-4/química , Mutação/fisiologia , Fenótipo , Fosforilação , Engenharia de Proteínas , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Interleucina-4/efeitos dos fármacos , Fator de Transcrição STAT6/metabolismo , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade
6.
Lancet ; 370(9596): 1422-31, 2007 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-17950857

RESUMO

BACKGROUND: Increases in T helper (Th) 2 cytokine concentrations have been seen in atopic asthma, with interleukin 4 and interleukin 13 thought to have a role in the physiological response to allergen challenge. Our aim was to assess the therapeutic effect of pitrakinra, an interleukin-4 variant that targets allergic Th2 inflammation by potently inhibiting the binding of interleukin 4 and interleukin 13 to interleukin-4Ralpha receptor complexes. METHODS: In two independent randomised, double-blind, placebo-controlled, parallel group phase 2a clinical trials, patients with atopic asthma were treated with pitrakinra or placebo via two routes. In study 1, patients were randomly assigned to receive either 25 mg pitrakinra (n=12) or placebo (n=12) by subcutaneous injection once daily. In study 2, patients were randomly assigned to receive either 60 mg pitrakinra (n=16) or placebo (n=16) by nebulisation twice daily. Inhaled allergen challenge was done before and after 4 weeks of treatment. The primary endpoint for study 1 was maximum percentage decrease in forced expiratory volume in 1 s (FEV1) over 4-10 h after allergen challenge, whereas that in study 2 was average percentage decrease in FEV(1) over 4-10 h after allergen challenge. All patients except those with baseline data only were included in our analyses. These trials are registered with ClinicalTrials.gov, numbers NCT00535028 and NCT00535031. FINDINGS: No patients dropped out or were lost to follow-up in study 1; in study 2, two patients in the placebo group and one in the pitrakinra group dropped out or were lost to follow-up. These individuals had baseline data only, and were excluded from the analyses. In study 1, there was a 17.1% maximum percentage decrease in FEV1 in the pitrakinra group; by contrast, the maximum decrease was 23.1% in the placebo group (difference 6%, 95% CI -4.37 to 16.32; p=0.243). In study 2, there was a 4.4% average percentage decrease in FEV1 in the pitrakinra group; by contrast, the average percentage decrease was 15.9% in the placebo group (3.7 [95% CI 2.08-6.25] times lower in the pitrakinra group; p=0.0001). There were fewer asthma-related adverse events (p=0.069) and fewer adverse events requiring beta-agonist rescue (p=0.031) after subcutaneous administration of pitrakinra than with placebo. There were too few asthma-related adverse events in study 2 to assess the effect of inhalation of pitrakinra on adverse events. INTERPRETATION: Local treatment, targeted at inhibition of interleukins 4 and 13 in the lung, could substantially diminish the symptoms of asthma.


Assuntos
Antiasmáticos/uso terapêutico , Asma/tratamento farmacológico , Testes de Provocação Brônquica/métodos , Interleucina-4/análogos & derivados , Adulto , Antiasmáticos/efeitos adversos , Método Duplo-Cego , Feminino , Volume Expiratório Forçado/efeitos dos fármacos , Humanos , Interleucina-4/metabolismo , Masculino , Receptores de Interleucina-4/antagonistas & inibidores
7.
Cell Immunol ; 191(2): 161-7, 1999 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-9973539

RESUMO

IL-4 is a pleiotropic cytokine which exerts its actions on various lineages of hematopoietic and nonhematopoietic cells. This cytokine is one of the central regulators of immunity in health and disease states. An alternative splice variant, in which the second of four exons is omitted, has been recently described and designated as IL-4delta2. The variant has been previously described as a potential naturally occurring antagonist of human IL-4 (hIL-4)-stimulated T cell proliferation. In this study, we investigated the effects of recombinant human (rh) IL-4delta2 on monocytes and B cells. In monocytes, rhIL-4delta2 blocked inhibitory action of hIL-4 on LPS-induced cyclooxygenase-2 expression and subsequent prostaglandin E2 secretion. In B cells, rhIL-4delta2 was an antagonist of the hIL-4-induced synthesis of IgE and expression of CD23. Our results broaden the spectrum of hIL-4-antagonistic activities of rhIL-4delta2, thus creating the background for the potential use of rhIL-4delta2 as a therapeutic anti-hIL-4 agent.


Assuntos
Linfócitos B/efeitos dos fármacos , Interleucina-4/análogos & derivados , Interleucina-4/antagonistas & inibidores , Interleucina-4/genética , Monócitos/efeitos dos fármacos , Processamento Alternativo , Western Blotting , Células Cultivadas , Ciclo-Oxigenase 2 , Dinoprostona/metabolismo , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Imunoglobulina E/biossíntese , Interleucina-4/metabolismo , Isoenzimas/biossíntese , Proteínas de Membrana , Prostaglandina-Endoperóxido Sintases/biossíntese , Ligação Proteica , Radioimunoensaio , Receptores de IgE/biossíntese , Proteínas Recombinantes/farmacologia
8.
Blood ; 87(10): 4286-95, 1996 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-8639787

RESUMO

Interleukin-4 (IL-4) and IL-13 exert similar, nonadditive effects on endothelial cells, inducing vascular cell adhesion molecule-1 (VCAM-1) expression and subsequent transmigration of eosinophils. The receptor for IL-4 and IL-13 was described as a shared heteromultimeric complex in which the common gamma-chain (gamma c) subunit was essential for activity. Endothelial cell bound both cytokines with high affinity; by flow cytofluorometry and reverse transcription-polymerase chain reaction (RT-PCR), they expressed IL-4 receptor alpha (IL-4R alpha) but did not express the gamma c of the IL-2R. Radioligand cross-linking experiments followed by immunoprecipitation with the monoclonal antibody (MoAb) S697 to the IL-4R alpha showed IL-4-specific binding at 130 kD, the IL-4R alpha, and to a minor extent to a double band coimmunoprecipitated at 65 to 75 kD. [125 I]IL-13 bound predominantly to the 65- to 75- kD band and with a trace amount of binding at 130 kD. However, no ligand-cross-linked receptor was precipitated by the MoAb S697, indicating a cognate novel IL-13-binding subunit. Excess unlabeled IL-4 completely displaced IL-13 binding. Similarly, nonsignaling IL-4 (Y124D)-mutant abolished IL-4- and IL-13-mediated signal transduction. Unlabeled IL-13 competed successfully for IL-4 binding at 65 to 75 kD but was unable to completely displace Il-4 from its binding to the IL-4R alpha. The MoAb TUGh4, specific for the gamma c, failed to precipitate ligand-cross-linked IL-4R and IL-13R. Therefore, the subunit structure of the functional receptors for IL-4 and IL-13 on human endothelial cells does not use or require the common gamma c of the IL-2R.


Assuntos
Antígenos CD/metabolismo , Endotélio Vascular/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Interleucina-13/metabolismo , Interleucina-4/metabolismo , Receptores de Interleucina/metabolismo , Molécula 1 de Adesão de Célula Vascular/biossíntese , Sequência de Aminoácidos , Animais , Antígenos CD/química , Antígenos CD/genética , Sequência de Bases , Ligação Competitiva , Células CHO , Linhagem Celular Transformada , Cricetinae , Cricetulus , Endotélio Vascular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Humanos , Interleucina-13/farmacologia , Subunidade alfa1 de Receptor de Interleucina-13 , Interleucina-4/análogos & derivados , Interleucina-4/genética , Interleucina-4/farmacologia , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Inibidores de Proteínas Quinases , Receptores de Interleucina/química , Receptores de Interleucina/genética , Receptores de Interleucina-13 , Receptores de Interleucina-2/biossíntese , Receptores de Interleucina-2/química , Receptores de Interleucina-2/genética , Receptores de Interleucina-4 , Transdução de Sinais , Molécula 1 de Adesão de Célula Vascular/genética
9.
Int Arch Allergy Immunol ; 107(1-3): 304-7, 1995.
Artigo em Inglês | MEDLINE | ID: mdl-7613155

RESUMO

In the present study, it is shown that a human interleukin (IL)-4 mutant protein (IL-4.Y124D) acts as a potent IL-4 and IL-13 receptor antagonist. Human (h) IL-4.Y124D efficiently inhibits both IL-4- and IL-13-induced IgE production in vitro. In addition, hIL-4.Y124D strongly inhibits ongoing human IgE synthesis in SCID-hu mice. These inhibitory effects are specific, since human IgG levels were not significantly affected. These results confirm the notion that the IL-4 and IL-13 receptor share a common component, which is required for signal transduction. In addition, they show that relatively large antagonistic polypeptides, such as hIL-4.Y124D have potential clinical utility in reducing IgE-mediated allergic diseases.


Assuntos
Imunoglobulina E/biossíntese , Interleucina-4/análogos & derivados , Receptores de Interleucina/antagonistas & inibidores , Animais , Linfócitos B/imunologia , Ligação Competitiva , Transplante Ósseo , Quimera , Transplante de Tecido Fetal , Humanos , Imunoglobulina E/genética , Interleucina-13/antagonistas & inibidores , Interleucina-13/farmacologia , Subunidade alfa1 de Receptor de Interleucina-13 , Interleucina-4/genética , Interleucina-4/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Camundongos SCID , Mutagênese Sítio-Dirigida , Receptores de Interleucina-13 , Receptores de Interleucina-4 , Transdução de Sinais/efeitos dos fármacos , Timo/embriologia , Timo/transplante , Transplante Heterólogo , Transplante Heterotópico
10.
Cytokine ; 7(4): 311-8, 1995 May.
Artigo em Inglês | MEDLINE | ID: mdl-8589260

RESUMO

In human interleukin 4(IL-4), the carboxyl and amino termini of the 129 amino acid hormone are close to each other and this region is believed to be important for binding to the IL-4 receptor (IL-4r). We constructed plasmids encoding circularly permuted IL-4 mutants with the peptide Gly-Gly-Asn-Gly-Gly (GGNGG) joining the carboxyl to the amino terminus and with new amino and carboxyl termini elsewhere. Mutant IL-4(38-37) is composed of IL-4 residues 38-129 GGNGG and 1-37. Mutant IL-4(105-104) is composed of IL-4 residues 105-129, GGNGG and 1-104. IL-4(38-37) and IL-4(105-104) were purified from E. coli to near homogeneity and retained 50-100% of the binding and proliferative activity of IL-4, and in addition retained the ability to upregulate CD23 on Burkitt's lymphoma cells. Circular dichroism studies indicated that the tertiary structures of both IL-4(38-37) and IL-4(105-104) were retained, with the former molecule most similar to native IL-4. We conclude that while both native termini of IL-4 may be near its binding site, neither is required to be free for optimum activity.


Assuntos
Interleucina-4/análogos & derivados , Sequência de Aminoácidos , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Sequência de Bases , Linfoma de Burkitt/patologia , Divisão Celular/efeitos dos fármacos , Dicroísmo Circular , Escherichia coli , Humanos , Interleucina-4/química , Interleucina-4/farmacologia , Dados de Sequência Molecular , Mutagênese , Reação em Cadeia da Polimerase , Estrutura Terciária de Proteína , Receptores de IgE/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/farmacologia , Células Tumorais Cultivadas
11.
EMBO J ; 11(9): 3237-44, 1992 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-1387082

RESUMO

Interleukin-4 (IL-4) represents a prototypic lymphokine (for a recent review see Paul, 1991). It promotes differentiation of B-cells and the proliferation of T- and B-cell, and other cell types of the lymphoid system. An antagonist of human IL-4 was discovered during the studies presented here after Tyr124 of the recombinant protein had been substituted by an aspartic acid residue. This IL-4 variant, Y124D, bound with high affinity to the IL-4 receptor (KD = 310 pM), but retained no detectable proliferative activity for T-cells and inhibited IL-4-dependent T-cell proliferation competitively (K(i) = 620 pM). The loss of efficacy in variant Y124D was estimated to be greater than 100-fold on the basis of a weak partial agonist activity for the very sensitive induction of CD23 positive B-cells. The substitution of Tyr124 by either phenylalanine, histidine, asparagine, lysine or glycine resulted in partial agonist variants with unaltered receptor binding affinity and relatively small deficiencies in efficacy. These results demonstrate that high affinity binding and signal generation can be uncoupled efficiently in a ligand of a receptor belonging to the recently identified hematopoietin receptor family. In addition we show for the first time, that a powerful antagonist acting on the IL-4 receptor system can be derived from the IL-4 protein.


Assuntos
Interleucina-4/antagonistas & inibidores , Interleucina-4/genética , Antígenos de Diferenciação de Linfócitos B/biossíntese , Linfócitos B/citologia , Ligação Competitiva , Diferenciação Celular , Células Cultivadas , Escherichia coli , Variação Genética , Humanos , Interleucina-4/análogos & derivados , Interleucina-4/metabolismo , Ativação Linfocitária , Mutagênese Sítio-Dirigida , Receptores Fc/biossíntese , Receptores de IgE , Receptores de Interleucina-4 , Receptores Mitogênicos/antagonistas & inibidores , Receptores Mitogênicos/metabolismo , Transdução de Sinais , Linfócitos T/citologia , Linfócitos T/imunologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...