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1.
J Allergy Clin Immunol ; 121(2): 441-447.e5, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17949802

RESUMO

BACKGROUND: A human Fcgamma-Fcepsilon fusion protein (GE2) designed to inhibit FcepsilonRI signaling by coaggregating FcepsilonRI with the inhibitory receptor FcgammaRIIB has been shown to inhibit mast cell activation and block cutaneous anaphylaxis. A critical issue remained as to whether the mechanism of GE2 inhibition is competition for IgE binding or inhibitory signaling through FcgammaRIIB. OBJECTIVE: Our aim was to define the in vitro and in vivo mechanism of action of a mouse homolog of GE2 (mGE) and to assess the potential of human GE2 (hGE2) for therapeutic administration. METHODS: The in vitro activity of mGE on mediator release and signaling pathways was characterized in IgE-sensitized bone marrow-derived mast cells (BMMCs). The in vivo activity of mGE was examined in mouse passive cutaneous and passive systemic anaphylaxis models, and the therapeutic activity of hGE2 was evaluated in Ascaris suum-sensitized cynomolgus monkeys. RESULTS: mGE inhibited release of histamine and cytokines by BMMCs from wild-type mice but not by BMMCs from FcgammaRIIB-deficient mice. In mice mGE blocked IgE-dependent anaphylaxis mediated by mast cells with sustained efficacy. In BMMCs mGE decreased spleen tyrosine kinase and extracellular signal-regulated kinases 1/2 phosphorylation and induced FcgammaRIIB phosphorylation and the subsequent recruitment of SH2 domain-containing inositol polyphosphate 5' phosphatase (SHIP) 1 and SH2 domain-containing protein tyrosine phosphatase (SHP) 1/2 phosphatases. When administered therapeutically, hGE2 protected sensitized monkeys from local anaphylaxis for 3 weeks. CONCLUSION: mGE-mediated inhibition of mast cell activation is associated with inhibitory signaling through FcgammaRIIB that results from activation of SHIP-1 and SHP-1/2 phosphatases.


Assuntos
Mastócitos/efeitos dos fármacos , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Receptores de IgG/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Domínios de Homologia de src , Anafilaxia/prevenção & controle , Animais , Ascaris suum , Células da Medula Óssea/efeitos dos fármacos , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Imunização , Imunoglobulina E/metabolismo , Inositol Polifosfato 5-Fosfatases , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Macaca fascicularis , Camundongos , Camundongos Endogâmicos , Camundongos Knockout , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases , Monoéster Fosfórico Hidrolases/química , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Proteínas Tirosina Fosfatases/química , Proteínas Tirosina Quinases/metabolismo , Receptores de IgE/metabolismo , Proteínas Recombinantes de Fusão/genética , Quinase Syk , Receptor 4 Toll-Like/metabolismo
2.
J Allergy Clin Immunol ; 120(2): 462-8, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17543377

RESUMO

BACKGROUND: GE2, a human bifunctional Fcgamma-Fcvarepsilon fusion protein cross-links FcgammaRIIb and FcvarepsilonRI on human mast cells and basophils and results in inhibition of FcvarepsilonRI-mediated functions. OBJECTIVE: Three modified Fcgamma-Fcvarepsilon (GE) proteins were compared with GE2 for their effect on inhibition of FcvarepsilonRI-mediated cellular responses. METHODS: GE2 was modified to potentially improve its therapeutic efficacy by increasing binding to FcgammaRIIb (GE S mutant) and decreasing binding to FcgammaRIII (GE H mutant) or reversing the Fcgamma and Fcvarepsilon domains and removing nonhuman linker sequences (E2G). These proteins were tested for their ability to bind a basophil-like cell line, block FcvarepsilonRI-mediated degranulation in human basophils, and inhibit passive cutaneous anaphylaxis in human FcvarepsilonRIalpha-transgenic mice. RESULTS: All 4 GE proteins bound cells that express FcvarepsilonRI and FcgammaRIIb, although the original GE2 retained the strongest ability to bind to these cells. E2G was as effective as GE2 in its ability to inhibit anti-Fel d 1 IgE-mediated histamine release from human basophils and block passive cutaneous anaphylaxis reactions. The GE S and GE H mutants were less effective. CONCLUSION: Optimization of GE2 as an inhibitor of FcvarepsilonRI-mediated functions showed that effectiveness was maintained when potentially immunogenic linker sequences were removed and Ig domain positions were reversed, but specific residue changes within the IgG C(H)2 domain aimed at enhancing GE2's inhibitory function by increasing FcgammaRII binding or additionally decreasing FcgammaRIII binding were not beneficial. CLINICAL IMPLICATIONS: GE2 and E2G molecules are effective inhibitors of FcvarepsilonRI-mediated degranulation and are of interest as potential therapeutics for IgE-mediated allergic reactions.


Assuntos
Mutação , Receptores de IgE/antagonistas & inibidores , Receptores de IgE/genética , Receptores de IgG/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacologia , Animais , Basófilos/metabolismo , Basófilos/fisiologia , Degranulação Celular/efeitos dos fármacos , Linhagem Celular , Humanos , Camundongos , Camundongos Transgênicos , Anafilaxia Cutânea Passiva/efeitos dos fármacos , Anafilaxia Cutânea Passiva/fisiologia , Isoformas de Proteínas/farmacologia , Receptores de IgE/metabolismo , Receptores de IgE/fisiologia , Receptores de IgG/metabolismo , Proteínas Recombinantes de Fusão/química
3.
Curr Opin Allergy Clin Immunol ; 4(6): 563-8, 2004 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-15640700

RESUMO

PURPOSE OF REVIEW: This review summarizes current knowledge regarding the control of human mast cell and basophil signaling and recent developments using a new therapeutic platform consisting of a human bifunctional gamma and epsilon heavy chain (Fc gamma-Fc epsilon) protein to inhibit allergic reactivity. RECENT FINDINGS: Crosslinking of Fc gamma RIIb to Fc epsilon RI on human mast cells and basophils by a genetically engineered Fc gamma-Fc epsilon protein (GE2) leads to the inhibition of mediator release upon Fc epsilon RI challenge. GE2 protein was shown to inhibit cord blood-derived mast cell and peripheral blood basophil mediator release in vitro in a dose-dependent fashion, including inhibition of human IgE reactivity to cat. IgE-driven mediator release from lung tissue was also inhibited by GE2. The mechanism of inhibition in mast cells included alterations in IgE-mediated Ca mobilization, spleen tyrosine kinase phosphorylation and the formation of downstream of kinase-growth factor receptor-bound protein 2-SH2 domain-containing inositol 5-phosphatase (dok-grb2-SHIP) complexes. Proallergic effects of Langerhan's like dendritic cells and B-cell IgE switching were also inhibited by GE2. In vivo, GE2 was shown to block passive cutaneous anaphylaxis driven by human IgE in mice expressing the human Fc epsilon RI and inhibit skin test reactivity to dust mite antigen in a dose-dependent manner in rhesus monkeys. SUMMARY: The balance between positive and negative signaling controls mast cell and basophil reactivity, which is critical in the expression of human allergic diseases. This approach using a human Fc gamma-Fc epsilon fusion protein to co-aggregate Fc epsilon RI with the Fc gamma RII holds promise as a new therapeutic platform for the immunomodulation of allergic diseases and potentially other mast cell/basophil-dependent disease states.


Assuntos
Engenharia Genética , Hipersensibilidade Imediata/genética , Hipersensibilidade Imediata/imunologia , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Animais , Basófilos/efeitos dos fármacos , Basófilos/imunologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Humanos , Mastócitos/efeitos dos fármacos , Mastócitos/imunologia , Agregação de Receptores/efeitos dos fármacos , Agregação de Receptores/imunologia , Receptores de IgE/imunologia , Receptores de IgG/imunologia , Transdução de Sinais/efeitos dos fármacos
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