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1.
J Med Chem ; 64(8): 4947-4959, 2021 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-33825469

RESUMO

Hapten-specific endogenous antibodies are naturally occurring antibodies present in human blood. Herein, we investigated a new strategy in which small-molecule haptens were utilized as naturally occurring antibody binders for peptide half-life extension. The glucagon-like peptide 1 receptor agonist exendin 4 was site-specifically functionalized with the dinitrophenyl (DNP) hapten at the C-terminus via sortase A-mediated ligation. The resulting Ex4-DNP conjugates retained GLP-1 receptor activation potency in vitro and had a similar in vivo acute glucose-lowering effect comparable to that of native Ex4. Pharmacokinetic studies and hypoglycemic duration tests demonstrated that the Ex4-DNP conjugates displayed significantly elongated half-lives and improved long-acting antidiabetic activity in the presence of endogenous anti-DNP antibodies. In chronic treatment studies, once-daily administration of optimal conjugate 7 demonstrated more beneficial effects without prominent toxicity compared with Ex4. This strategy provides a new approach and represents an alternative to the well-established peptide-Fc fusion strategy to improve the peptide half-life and the therapeutic efficacy.


Assuntos
Anticorpos/sangue , Exenatida/química , Haptenos/química , Hipoglicemiantes/síntese química , Sequência de Aminoácidos , Animais , Anticorpos/imunologia , Complexo Antígeno-Anticorpo/química , Complexo Antígeno-Anticorpo/metabolismo , Glicemia/análise , Diabetes Mellitus Experimental/tratamento farmacológico , Dinitrobenzenos/química , Dinitrobenzenos/imunologia , Desenho de Fármacos , Feminino , Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Teste de Tolerância a Glucose , Meia-Vida , Haptenos/imunologia , Hipoglicemiantes/metabolismo , Hipoglicemiantes/uso terapêutico , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Camundongos , Camundongos Endogâmicos C57BL
3.
Chem Commun (Camb) ; 55(73): 10952-10955, 2019 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-31441915

RESUMO

Triggering antibody-mediated innate immune mechanisms to kill cancer cells is an attractive therapeutic avenue. In this context, recruitment of endogenous antibodies to the cancer cell surface could be a viable alternative to the use of monoclonal antibodies. We report on antibody-recruiting polymers containing multiple antibody-binding hapten motifs and cyclooctynes that can covalently conjugate to azides introduced onto the glycocalyx of cancer cells by metabolic labeling with azido sugars.


Assuntos
Resinas Acrílicas/química , Anticorpos/imunologia , Azidas/metabolismo , Dinitrobenzenos/imunologia , Hexosaminas/metabolismo , Resinas Acrílicas/síntese química , Animais , Azidas/química , Linhagem Celular Tumoral , Química Click , Reação de Cicloadição , Ciclo-Octanos/síntese química , Ciclo-Octanos/química , Dinitrobenzenos/síntese química , Dinitrobenzenos/química , Fluorescência , Corantes Fluorescentes/química , Glicocálix/metabolismo , Hexosaminas/química , Humanos , Camundongos , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Estudo de Prova de Conceito , Esferoides Celulares/metabolismo
4.
Angew Chem Int Ed Engl ; 56(42): 13036-13040, 2017 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-28793176

RESUMO

Systemic fungal infections represent an important public health concern, and new antifungal agents are highly desirable. Herein, we describe the design, synthesis, and biological evaluation of a novel class of antifungal compounds called antibody-recruiting molecules targeting fungi (ARM-Fs). Our approach relies on the use of non-peptidic small molecules, which selectively bind fungal cells and recruit endogenous antibodies to their surfaces, resulting in immune-mediated clearance. Using the opportunistic fungal pathogen Candida albicans as a model, we identified a highly specific bifunctional molecule able to mediate the engulfment and phagocytosis of C. albicans cells by human immune cells in biologically relevant functional assays. This work represents a novel therapeutic approach to treating fungal illness with significant potential to complement and/or combine with existing treatment strategies.


Assuntos
Anticorpos/imunologia , Subpopulações de Linfócitos B/imunologia , Candida albicans/imunologia , Acetilglucosamina/química , Anticorpos/metabolismo , Subpopulações de Linfócitos B/citologia , Candida albicans/metabolismo , Candida albicans/patogenicidade , Quitina/química , Quitina/metabolismo , Dinitrobenzenos/química , Dinitrobenzenos/imunologia , Desenho de Fármacos , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Células HL-60 , Interações Hospedeiro-Patógeno , Humanos , Fagocitose
5.
Chembiochem ; 16(14): 2007-10, 2015 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-26185102

RESUMO

Haptens, such as dinitrophenyl (DNP) are small molecules that induce strong immune responses when attached to proteins or peptides and, as such, have been exploited for diverse applications. We engineered a Methanosarcina barkeri pyrrolysyl-tRNA synthetase (mbPylRS) to genetically encode a DNP-containing unnatural amino acid, N(6) -(2-(2,4-dinitrophenyl)acetyl)lysine (DnpK). Although this moiety was unstable in Escherichia coli, we found that its stability was enhanced in mammalian HEK 293T cells and was able to induce selective interactions with anti-DNP antibodies. The capability of genetically introducing DNP into proteins is expected to find broad applications in biosensing, immunology, and therapeutics.


Assuntos
Aminoacil-tRNA Sintetases/genética , Dinitrobenzenos/química , Haptenos/química , Haptenos/genética , Lisina/análogos & derivados , Methanosarcina barkeri/enzimologia , Dinitrobenzenos/imunologia , Código Genético , Engenharia Genética , Células HEK293 , Haptenos/imunologia , Humanos , Lisina/química , Lisina/genética , Lisina/imunologia , Methanosarcina barkeri/genética
6.
J Clin Invest ; 122(5): 1700-11, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22523067

RESUMO

Allergic contact dermatitis is the most frequent occupational disease in industrialized countries. It is caused by CD8(+) T cell-mediated contact hypersensitivity (CHS) reactions triggered at the site of contact by a variety of chemicals, also known as weak haptens, present in fragrances, dyes, metals, preservatives, and drugs. Despite the myriad of potentially allergenic substances that can penetrate the skin, sensitization is relatively rare and immune tolerance to the substance is often induced by as yet poorly understood mechanisms. Here we show, using the innocuous chemical 2,4-dinitrothiocyanobenzene (DNTB), that cutaneous immune tolerance in mice critically depends on epidermal Langerhans cells (LCs), which capture DNTB and migrate to lymph nodes for direct presentation to CD8(+) T cells. Depletion and adoptive transfer experiments revealed that LCs conferred protection from development of CHS by a mechanism involving both anergy and deletion of allergen-specific CD8(+) T cells and activation of a population of T cells identified as ICOS(+)CD4(+)Foxp3(+) Tregs. Our findings highlight the critical role of LCs in tolerance induction in mice to the prototype innocuous hapten DNTB and suggest that strategies targeting LCs might be valuable for prevention of cutaneous allergy.


Assuntos
Linfócitos T CD8-Positivos/fisiologia , Dermatite Alérgica de Contato/imunologia , Fatores de Transcrição Forkhead/metabolismo , Células de Langerhans/fisiologia , Linfócitos T Reguladores/fisiologia , Alérgenos/imunologia , Animais , Apresentação de Antígeno , Antígenos CD/metabolismo , Linfócitos T CD8-Positivos/imunologia , Comunicação Celular , Movimento Celular , Células Cultivadas , Dermatite Alérgica de Contato/patologia , Dinitrobenzenos/imunologia , Epiderme/imunologia , Epiderme/patologia , Tolerância Imunológica , Proteína Coestimuladora de Linfócitos T Induzíveis/metabolismo , Células de Langerhans/imunologia , Linfonodos/patologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo
7.
Int Immunopharmacol ; 12(4): 675-81, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22330086

RESUMO

Degranulation inhibitors in plants are widely used for prevention and treatment of immediate-type allergy. We previously isolated a new ellagic acid glucoside, okicamelliaside (OCS), from Camellia japonica leaves for use as a potent degranulation inhibitor. Crude extracts from leaves also suppressed allergic conjunctivitis in rats. In this study, we evaluated the in vivo effect of OCS using a pure sample and performed in vitro experiments to elucidate the mechanism underlying the extraordinary high potency of OCS and its aglycon. The IC(50) values for degranulation of rat basophilic leukemia cells (RBL-2H3) were 14 nM for OCS and 3 µM for aglycon, indicating that the two compounds were approximately 2 to 3 orders of magnitude more potent than the anti-allergic drugs ketotifen fumarate, DSCG, and tranilast (0.17, 3, and >0.3 mM, respectively). Antigen-induced calcium ion (Ca(2+)) elevation was significantly inhibited by OCS and aglycon at all concentrations tested (p<0.05). Upstream of the Ca(2+) elevation in the principle signaling pathway, phosphorylation of Syk (Tyr525/526) and PLCγ-1 (Tyr783 and Ser1248) were inhibited by OCS and aglycon. In DNA microarray-screening test, OCS inhibited expression of proinflammatory cytokines [interleukin (IL)-4 and IL-13], cytokine-producing signaling factors, and prostaglandin-endoperoxidase 2, indicating that OCS broadly inhibits allergic inflammation. During passive cutaneous anaphylaxis in mice, OCS significantly inhibited vascular hyperpermeability by two administration routes: a single intraperitoneal injection at 10 mg/kg and per os at 5 mg/kg for 7 days (p<0.05). These results suggest the potential for OCS to alleviate symptoms of immediate-type allergy.


Assuntos
Antialérgicos/farmacologia , Degranulação Celular/efeitos dos fármacos , Ácido Elágico/análogos & derivados , Glucosídeos/farmacologia , Leucemia Basofílica Aguda/imunologia , Anafilaxia Cutânea Passiva/efeitos dos fármacos , Animais , Antígenos/imunologia , Cálcio/imunologia , Linhagem Celular Tumoral , Ciclo-Oxigenase 2/genética , Citocinas/genética , Dinitrobenzenos/imunologia , Regulação para Baixo , Ácido Elágico/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Imunoglobulina E/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Masculino , Camundongos , Camundongos Endogâmicos ICR , Análise de Sequência com Séries de Oligonucleotídeos , Anafilaxia Cutânea Passiva/imunologia , Proteínas Tirosina Quinases/imunologia , Ratos , Quinase Syk
8.
Eur J Immunol ; 41(4): 1004-13, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21360700

RESUMO

Rapid IgE desensitization provides temporary tolerization for patients who have presented severe hypersensitivity reactions to food and drugs, protecting them from anaphylaxis, but the underlying mechanisms are still incompletely understood. Thus, here we develop an effective and reproducible in vitro model of rapid IgE desensitization for mouse BM-derived mast cells (BMMCs) under physiologic calcium conditions, and we characterize its antigen specificity and primary events. BMMCs were challenged with DNP-human serum albumin conjugated (DNP-HSA) and/or OVA antigens, delivered either as a single dose (activation) or as increasing sequential doses (desensitization). Compared to activated cells, desensitized BMMCs had impaired degranulation, calcium flux, secretion of arachidonic acid products, early and late TNF-α production, IL-6 production, and phosphorylation of STAT6 and p38 mitogen-activated protein kinase (p38 MAPK). OVA-desensitized cells responded to DNP and DNP-desensitized cells responded to OVA, proving specificity. Internalization of specific antigen, IgE and high-affinity receptor for IgE (FcεRI) were impaired in desensitized BMMCs. Our results demonstrate that rapid IgE desensitization is antigen specific and inhibits early and late mast cell activation responses and internalization of the antigen/IgE/FcεRI complexes.


Assuntos
Antígenos/imunologia , Imunoglobulina E/imunologia , Mastócitos/imunologia , Receptores de IgE/imunologia , Animais , Células Cultivadas , Dinitrobenzenos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Fatores de Tempo
9.
J Immunol ; 183(11): 7576-84, 2009 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-19890059

RESUMO

Contact allergy to environmental xenobiotics is a common and important problem, but it is unclear why some chemicals are potent sensitizers and others weak/nonsensitizers. We explored this by investigating why similar chemicals, 2,4-dinitrochlorobenzene (DNCB) and 2,4-dinitrothiocyanobenzene (DNTB), differ in their ability to induce contact hypersensitivity (CHS). DNCB induced CHS in humans, whereas at similar doses DNTB did not. However, following DNCB sensitization, DNTB elicited CHS in vivo and stimulated DNCB-responsive T cells in vitro, suggesting that differences in response to these compounds lie in the sensitization phase. In contrast to DNCB, DNTB failed to induce emigration of epidermal Langerhans cells in naive individuals. Examination for protein dinitrophenylation in skin revealed that DNCB penetrated into the epidermis, whereas DNTB remained bound to a thiol-rich band within the stratum corneum. DNTB reacted rapidly with reduced glutathione in vitro and was associated with a decrease in the free thiol layer in the stratum corneum, but not in the nucleated epidermis. By contrast, DNCB required GST facilitation to react with gluthathione and, following penetration through the stratum corneum, depleted thiols in the viable epidermis. Chemical depletion of the thiol-rich band or removing it by tape stripping allowed increased penetration of DNTB into the epidermis. Our results suggest that the dissimilar sensitizing potencies of DNCB and DNTB in humans are determined by a previously undescribed outer epidermal biochemical redox barrier, a chemical component of the innate immune defense mechanisms that defend against sensitization by highly reactive environmental chemicals.


Assuntos
Dermatite de Contato/imunologia , Dinitrobenzenos/imunologia , Dinitroclorobenzeno/imunologia , Pele/química , Pele/imunologia , Xenobióticos/imunologia , Adulto , Dinitroclorobenzeno/farmacologia , Exposição Ambiental , Feminino , Humanos , Imunidade Inata , Irritantes/imunologia , Irritantes/farmacologia , Queratinócitos/efeitos dos fármacos , Masculino , Oxirredução/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Testes Cutâneos , Xenobióticos/farmacologia
10.
Talanta ; 79(4): 1142-8, 2009 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-19615523

RESUMO

A surface plasmon resonance (SPR) immunosensor for detection of 2,4-dinitrotoluene (2,4-DNT), which is a signature compound of 2,4,6-trinitrotoluene-related explosives, was developed by using a novel oligo (ethylene glycol) (OEG)-based sensor surface. A rabbit polyclonal antibody against 2,4-DNT (anti-DNPh-KLH-400 antibody) was prepared, and the avidity for 2,4-DNT and recognition capability were investigated by indirect competitive ELISA. The sensor surface was fabricated by immobilizing a 2,4-DNT analog onto an OEG-based self-assembled monolayer formed on a gold surface via an OEG linker. The fabricated surface was characterized by Fourier-transform infrared-refractive absorption spectrometry (FTIR-RAS). The immunosensing of 2,4-DNT is based on the indirect competitive principle, in which the immunoreaction between the anti-DNPh-KLH-400 antibody and 2,4-DNT on the sensor surface was inhibited in the presence of free 2,4-DNT in solution. The limit of detection for the immunosensor, calculated as three times the standard deviation of a blank value, was 20 pg mL(-1), and the linear dynamic range was found to be between 1 and 100 ng mL(-1). Additionally, the fabricated OEG-based surface effectively prevented non-specific adsorption of proteins, and the specific response to anti-DNPh-KLH-400 antibody was maintained for more than 30 measurement cycles.


Assuntos
Dinitrobenzenos/análise , Substâncias Explosivas/análise , Imunoensaio/métodos , Polietilenoglicóis/química , Ressonância de Plasmônio de Superfície/métodos , Absorção , Animais , Anticorpos/imunologia , Ligação Competitiva , Bovinos , Dinitrobenzenos/química , Dinitrobenzenos/imunologia , Substâncias Explosivas/química , Substâncias Explosivas/imunologia , Feminino , Haptenos/imunologia , Peso Molecular , Coelhos , Espectroscopia de Infravermelho com Transformada de Fourier , Propriedades de Superfície
11.
J Am Chem Soc ; 131(26): 9361-7, 2009 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-19534466

RESUMO

This paper describes a method for the purification of monoclonal antibodies (rat anti-2,4-dinitrophenyl IgG: IgG(DNP); and mouse antidigoxin IgG: IgG(Dgn)) from ascites fluid. This procedure (for IgG(DNP)) has three steps: (i) precipitation of proteins heavier than immunoglobulins with ammonium sulfate; (ii) formation of cyclic complexes of IgG(DNP) by causing it to bind to synthetic multivalent haptens containing multiple DNP groups; (iii) selective precipitation of these dimers, trimers, and higher oligomers of the target antibody, followed by regeneration of the free antibody. This procedure separates the targeted antibody from a mixture of antibodies, as well as from other proteins and globulins in a biological fluid. This method is applicable to antibodies with a wide range of monovalent binding constants (0.1 microM to 0.1 nM). The multivalent ligands we used (derivatives of DNP and digoxin) isolated IgG(DNP) and IgG(Dgn) from ascites fluid in yields of >80% and with >95% purity. This technique has two advantages over conventional chromatographic methods for purifying antibodies: (i) it is selective for antibodies with two active Fab binding sites (both sites are required to form the cyclic complexes) over antibodies with one or zero active Fab binding sites; (ii) it does not require chromatographic separation. It has the disadvantage that the structure of the hapten must be compatible with the synthesis of bi- and/or trivalent analogues.


Assuntos
Anticorpos Monoclonais/isolamento & purificação , Digoxina/imunologia , Dinitrobenzenos/imunologia , Haptenos/química , Imunoglobulina G/isolamento & purificação , Sulfato de Amônio , Animais , Anticorpos Monoclonais/imunologia , Ascite/imunologia , Sítios de Ligação de Anticorpos , Precipitação Química , Cromatografia em Gel , Digoxina/química , Dimerização , Dinitrobenzenos/química , Haptenos/imunologia , Imunoglobulina G/imunologia , Camundongos , Modelos Moleculares , Ratos
12.
Toxicol Appl Pharmacol ; 238(2): 120-32, 2009 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-19427879

RESUMO

Dendritic cell (DC) maturation in response to contact sensitizers is a crucial step in the induction of sensitization reactions; however the underlying mechanism of activation remains unknown. To test whether the extent of protein haptenation is a determinant in DC maturation, we tested the effect of five dinitrophenyl (DNP) analogues of different reactivity, on maturation markers in the cell line, THP-1. The potencies of the test compounds in upregulating CD54 levels, inducing IL-8 release and triggering p38 MAPK phosphorylation did not correlate with their ability to deplete intracellular glutathione (GSH) levels or cause cell toxicity. However, the compounds' potency at inducing p38 phosphorylation was significantly associated with the amount of intracellular protein adducts formed (p<0.05). Inhibition experiments show that, at least for DNFB, p38 MAP kinase signalling controls compound-specific changes in CD54 expression and IL-8 release. 2D-PAGE analysis revealed that all the DNP analogues appeared to bind similar proteins. The analogues failed to activate NFkB, however, they activated Nrf2, which was used as a marker of oxidative stress. Neither GSH depletion, by use of buthionine sulfoximine, nor treatment with the strongly lysine-reactive hapten penicillin elicited maturation. We conclude that protein haptenation, probably through reactive cysteine residues may be a trigger for maturation events in this in vitro model and that p38 activation may be a discriminatory marker for the classification of potency of chemical sensitizers.


Assuntos
Apresentação de Antígeno/efeitos dos fármacos , Células Dendríticas/imunologia , Dermatite Alérgica de Contato/imunologia , Dinitrobenzenos/farmacologia , Haptenos/efeitos dos fármacos , Apresentação de Antígeno/imunologia , Linhagem Celular Tumoral , Células Dendríticas/citologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Dermatite Alérgica de Contato/metabolismo , Dinitrobenzenos/imunologia , Glutationa/metabolismo , Haptenos/imunologia , Haptenos/metabolismo , Humanos , Leucemia Monocítica Aguda , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Xenobióticos/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
13.
Clin Exp Allergy ; 39(3): 417-25, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19032356

RESUMO

BACKGROUND: Occupational exposure to chemicals is an important cause of asthma. Recent studies indicate that IgE antibodies enhance sensitization to chemicals in the skin. OBJECTIVE: We investigated whether IgE might similarly promote the development of airway inflammation following inhalation of a contact sensitizer. METHODS: A model of chemical-induced asthma is described in which introduction of the low-molecular-weight compound, trinitrobenzene sulphonic acid (TNBS), via the respiratory tract was used for both sensitization and challenge. The role of IgE antibodies in the immune response to inhaled TNBS in this model was assessed by comparing the responses of wild-type (WT) and IgE-deficient (IgE(-/-)) mice on the BALB/c background. Reconstitution of circulating IgE levels by intravenous injection of IgE antibodies into IgE(-/-) mice before sensitization was performed to confirm the role of IgE in any differences observed between the responses of WT and IgE(-/-) mice. RESULTS: Intranasal challenge of TNBS-sensitized (but not sham-sensitized control mice) induced intense pulmonary inflammation. Macrophages, eosinophils and lymphocytes, including T, B, natural killer and natural killer T cells, were recruited to the airway and the animals displayed bronchial hyperresponsiveness (BHR) to methacholine. Serum levels of murine mast cell protease-1 (mMCP-1) were elevated suggesting mast cell activation. In contrast, the development of airway inflammation, recruitment of lymphocytes, induction of BHR and production of mMCP-1 were all significantly attenuated in IgE-deficient mice. Reconstitution of IgE(-/-) mice with IgE (of unrelated antigen specificity) before sensitization partially restored these features of asthma. CONCLUSION: Our data indicate that IgE antibodies non-specifically enhance the development of airway inflammation induced by exposure to chemical antigens.


Assuntos
Asma/etiologia , Haptenos/imunologia , Imunoglobulina E/imunologia , Inflamação/etiologia , Resistência das Vias Respiratórias/efeitos dos fármacos , Resistência das Vias Respiratórias/fisiologia , Animais , Asma/induzido quimicamente , Asma/patologia , Asma/fisiopatologia , Testes de Provocação Brônquica , Líquido da Lavagem Broncoalveolar/citologia , Contagem de Células , Quimiocina CCL2/sangue , Dinitrobenzenos/imunologia , Modelos Animais de Doenças , Eosinófilos/citologia , Imunização/métodos , Imunoglobulina E/sangue , Imunoglobulina E/genética , Imunoglobulina E/farmacologia , Inflamação/induzido quimicamente , Inflamação/patologia , Inflamação/fisiopatologia , Células Matadoras Naturais/citologia , Pulmão/efeitos dos fármacos , Pulmão/patologia , Pulmão/fisiopatologia , Metaplasia/patologia , Cloreto de Metacolina , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Células T Matadoras Naturais/citologia , Doenças Profissionais/imunologia , Linfócitos T/citologia , Ácido Trinitrobenzenossulfônico/imunologia
14.
J Immunol Methods ; 339(2): 195-204, 2008 Dec 31.
Artigo em Inglês | MEDLINE | ID: mdl-18854191

RESUMO

Antibodies against hydrophobic antigens are common in several autoimmune diseases. However, detection of such antibodies by standard immune-assays, such as ELISA, is problematic, in part because of the problems with coating hydrophobic molecules onto polystyrene multi-well plates. We describe a novel method of stably associating hydrophobic antigens to ELISA plates. By mixing the antigen with a hydrophobic molecule containing a hydrophilic anchor, we generate mixed lipid aggregates that can attach to ELISA plates, and are resistant to detergent wash. Using the ganglioside GM-1 and phosphatidylethanolamine conjugated to the hapten DNP (dinitrophenyl) as model antigens, we show that hydrophobic antigens incorporated into mixed lipid aggregates expose their antigenic determinants in a correct configuration. The detection limit of both GM-1 and DNP-PE was considerably improved compared to when these antigens were coated on ELISA plates using organic solvents. Furthermore GM-1 incorporated into mixed lipid aggregates can be detected by specific antibodies in patient serum. The method of incorporating hydrophobic antigens into mixed lipid aggregates for stable association to ELISA plates can presumably be applied to a vast array of hydrophobic antigens, and may well be developed into a large scale screening system for serum reactivity towards different hydrophobic antigens.


Assuntos
Autoanticorpos/sangue , Autoantígenos/química , Doenças Autoimunes/sangue , Gangliosídeo G(M1)/química , Autoanticorpos/imunologia , Autoantígenos/imunologia , Doenças Autoimunes/imunologia , Dinitrobenzenos/química , Dinitrobenzenos/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Gangliosídeo G(M1)/imunologia , Haptenos/química , Haptenos/imunologia , Humanos , Interações Hidrofóbicas e Hidrofílicas , Lipossomos/química , Lipossomos/imunologia , Fosfatidiletanolaminas/química , Fosfatidiletanolaminas/imunologia , Sensibilidade e Especificidade
15.
Chem Pharm Bull (Tokyo) ; 56(9): 1264-9, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18758098

RESUMO

Eleven 3-substituted isocoumarins and a benzylidenephthalide were synthesized through thermal cyclization reaction of delta- and gamma-ketoamides, respectively. Subsequent deprotection of the hydroxyl groups of the resulting isocoumarin and benzylidenephthalide compounds afforded thunberginols A, B, and F, respectively, which originated from the processed leaves of Hydrangea macrophylla SERINGE var. thunbergii MAKINO. The synthesized isocoumarins and thunberginols were evaluated for their anti-allergic activity, in which thunberginol B exhibited the highest inhibitory potency on the degranulation of RBL-2H3 cells induced by antigen. Structure-activity relationship studies were carried out to determine the necessary substituents on the 3-phenylisocoumarin skeleton for inhibitory activity.


Assuntos
Degranulação Celular/efeitos dos fármacos , Degranulação Celular/imunologia , Isocumarinas/síntese química , Antígenos/imunologia , Antígenos/farmacologia , Linhagem Celular , Ciclização , Dinitrobenzenos/imunologia , Hydrangea/química , Indicadores e Reagentes , Espectroscopia de Ressonância Magnética , Folhas de Planta/química , Soroalbumina Bovina/imunologia , Soroalbumina Bovina/farmacologia , Espectrometria de Massas por Ionização por Electrospray , Espectrofotometria Infravermelho , Relação Estrutura-Atividade
16.
Vet Immunol Immunopathol ; 126(1-2): 64-73, 2008 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-18692907

RESUMO

Members of the Camelidae family possess a functional class of antibodies devoid of light chains (known as heavy chain antibodies, HCAbs). Three IgG isotypes have been identified (IgG(1), IgG(2) and IgG(3)); IgG(2) and IgG(3) are HCAbs whereas the IgG(1) has the conventional structure. Different subtypes of IgG(1) (IgG(1a) and IgG(1b)) and IgG(2) (IgG(2a), IgG(2b) and IgG(2c)) have been classified according to variations in the amino acids sequence of the hinge region. The single variable domain of HCAbs has been referred as VHH. Until now, the relative amount of each subclass has been inferred, but the lack of highly specific antibodies against HCAbs has been a limitation for their quantification. In a previous work, we produced specific polyclonal antibodies against IgG(2a), IgG(2b), IgG(2c) and IgG(3) by immunizing rabbits with synthetic and recombinant peptides corresponding to their hinge region. In this work we produced specific antisera against llama IgM and IgG(1). The anti-IgG(1) serum was obtained by immunizing rabbits with a recombinant fusion protein formed by GST fused to the CH(1) domain of the IgG(1). The anti-IgM serum was obtained by immunizing rabbits with IgM heavy chain. All these antisera were useful for the development of ELISAs for the measurement of IgM, total IgG and IgG subclasses. Sera from llamas (n=20) analyzed by ELISA gave the following values of immunoglobulins: IgG(1)=6.168+/-1.628 mg/ml; IgG(2)=0.684+/-0.310 mg/ml; IgG(3)=1.232+/-0.410 mg/ml; total IgG=8.933+/-1.815 mg/ml and IgM=1.027+/-0.308 mg/ml. These results indicate that HCAbs represent almost 25% of total IgG and the IgG(3) subtype is the predominant HCAb. We also analyzed the primary humoral immune response after immunization llamas with different antigens (BSA, BSA-DNP and dextran). Although it has been described that a few VHH clones are very efficient in the interaction with haptens, in this case the response against DNP was characterized by a delayed appearance of HCAbs in comparison with that of IgG(1). No anti-dextran response was observed in any of the isotypes analyzed.


Assuntos
Formação de Anticorpos/fisiologia , Antígenos/imunologia , Camelídeos Americanos/sangue , Ensaio de Imunoadsorção Enzimática/veterinária , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Animais , Camelídeos Americanos/imunologia , Dextranos/imunologia , Dinitrobenzenos/imunologia , Feminino , Regulação da Expressão Gênica , Imunoglobulina G/classificação , Imunoglobulina M/classificação , Masculino , Soroalbumina Bovina/imunologia
17.
J Immunol ; 180(12): 7869-77, 2008 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-18523250

RESUMO

The Tec family tyrosine kinase, IL-2-inducible T cell kinase (Itk), is expressed in T cells and mast cells. Mice lacking Itk exhibit impaired Th2 cytokine secretion; however, they have increased circulating serum IgE, but exhibit few immunological symptoms of allergic airway responses. We have examined the role of Itk in mast cell function and FcepsilonRI signaling. We report in this study that Itk null mice have reduced allergen/IgE-induced histamine release, as well as early airway hyperresponsiveness in vivo. This is due to the increased levels of IgE in the serum of these mice, because the transfer of Itk null bone marrow-derived cultured mast cells into mast cell-deficient W/W(v) animals is able to fully rescue histamine release in the W/W(v) mice. Further analysis of Itk null bone marrow-derived cultured mast cells in vitro revealed that whereas they have normal degranulation responses, they secrete elevated levels of cytokines, including IL-13 and TNF-alpha, particularly in response to unliganded IgE. Analysis of biochemical events downstream of the FcepsilonRI revealed little difference in overall tyrosine phosphorylation of specific substrates or calcium responses; however, these cells express elevated levels of NFAT, which was largely nuclear. Our results suggest that the reduced mast cell response in vivo in Itk null mice is due to elevated levels of IgE in these mice. Our results also suggest that Itk differentially modulates mast cell degranulation and cytokine production in part by regulating expression and activation of NFAT proteins in these cells.


Assuntos
Mastócitos/enzimologia , Mastócitos/imunologia , Proteínas Tirosina Quinases/fisiologia , Alérgenos/administração & dosagem , Animais , Transplante de Medula Óssea/imunologia , Hiper-Reatividade Brônquica/enzimologia , Hiper-Reatividade Brônquica/genética , Hiper-Reatividade Brônquica/imunologia , Hiper-Reatividade Brônquica/prevenção & controle , Degranulação Celular/genética , Degranulação Celular/imunologia , Células Cultivadas , Citocinas/metabolismo , Dinitrobenzenos/imunologia , Modelos Animais de Doenças , Liberação de Histamina/genética , Liberação de Histamina/imunologia , Imunoglobulina E/administração & dosagem , Imunoglobulina E/biossíntese , Imunoglobulina E/metabolismo , Mastócitos/metabolismo , Camundongos , Camundongos Congênicos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Tirosina Quinases/deficiência , Proteínas Tirosina Quinases/genética , Receptores de IgE/metabolismo , Receptores de IgE/fisiologia , Transdução de Sinais/genética , Transdução de Sinais/imunologia
18.
J Immunol ; 180(12): 8040-7, 2008 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-18523267

RESUMO

Mast cell stimulation by Ag is followed by the opening of Ca(2+)-activated K(+) channels, which participate in the orchestration of mast cell degranulation. The present study has been performed to explore the involvement of the Ca(2+)-activated K(+) channel K(Ca)3.1 in mast cell function. To this end mast cells have been isolated and cultured from the bone marrow (bone marrow-derived mast cells (BMMCs)) of K(Ca)3.1 knockout mice (K(Ca)3.1(-/-)) and their wild-type littermates (K(Ca)3.1(+/+)). Mast cell number as well as in vitro BMMC growth and CD117, CD34, and FcepsilonRI expression were similar in both genotypes, but regulatory cell volume decrease was impaired in K(Ca)3.1(-/-) BMMCs. Treatment of the cells with Ag, endothelin-1, or the Ca(2+) ionophore ionomycin was followed by stimulation of Ca(2+)-activated K(+) channels and cell membrane hyperpolarization in K(Ca)3.1(+/+), but not in K(Ca)3.1(-/-) BMMCs. Upon Ag stimulation, Ca(2+) entry but not Ca(2+) release from intracellular stores was markedly impaired in K(Ca)3.1(-/-) BMMCs. Similarly, Ca(2+) entry upon endothelin-1 stimulation was significantly reduced in K(Ca)3.1(-/-) cells. Ag-induced release of beta-hexosaminidase, an indicator of mast cell degranulation, was significantly smaller in K(Ca)3.1(-/-) BMMCs compared with K(Ca)3.1(+/+) BMMCs. Moreover, histamine release upon stimulation of BMMCs with endothelin-1 was reduced in K(Ca)3.1(-/-) cells. The in vivo Ag-induced decline in body temperature revealed that IgE-dependent anaphylaxis was again significantly (by approximately 50%) blunted in K(Ca)3.1(-/-) mice. In conclusion, K(Ca)3.1 is required for Ca(2+)-activated K(+) channel activity and Ca(2+)-dependent processes such as endothelin-1- or Ag-induced degranulation of mast cells, and may thus play a critical role in anaphylactic reactions.


Assuntos
Imunoglobulina E/fisiologia , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/deficiência , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/genética , Mastócitos/imunologia , Mastócitos/metabolismo , Anafilaxia/genética , Anafilaxia/imunologia , Anafilaxia/metabolismo , Animais , Antígenos/imunologia , Transporte Biológico Ativo/imunologia , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Cálcio/antagonistas & inibidores , Cálcio/fisiologia , Degranulação Celular/genética , Degranulação Celular/imunologia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Proliferação de Células , Tamanho Celular , Células Cultivadas , Dinitrobenzenos/imunologia , Endotelina-1/antagonistas & inibidores , Endotelina-1/fisiologia , Feminino , Regulação da Expressão Gênica/imunologia , Imunoglobulina E/biossíntese , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/biossíntese , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/fisiologia , Masculino , Mastócitos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
19.
Eur J Immunol ; 38(3): 841-54, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18236401

RESUMO

Mast cells, perhaps best known by their ability to trigger allergic reactions after stimulation through the FcepsilonRI, express the unusual phosphatidylinositol 3-kinase (PI3K)-dependent, Rac-binding protein SWAP-70. Here, we show that the IgE-mediated passive cutaneous and the systemic anaphylactic responses are strongly reduced in SWAP-70(-/-) mice. Cultured SWAP-70(-/-) immature bone marrow mast cells (BMMC) are also impaired in FcepsilonRI-mediated degranulation, which can be restored by expression of exogenous wild-type SWAP-70, but less so if a phosphatidylinositol trisphosphate (PIP(3)) binding mutant is expressed. SWAP-70 itself supports inositol-3-phosphate and PIP(3) production, the latter indicating a potential feedback from SWAP-70 towards PI3K. FcepsilonRI-stimulated transcription and release of cytokines is controlled by SWAP-70. Key FcepsilonRI signal transduction events like activation of LAT by phosphorylation, activation of Akt/PKB and of p38 MAP kinase are reduced in SWAP-70(-/-) BMMC, but ERK is strongly hyperactivated. Some requirements for SWAP-70 were apparent only under limited-strength signaling conditions. We suggest that SWAP-70 defines a new element of efficient mast cell activation upon FcepsilonRI signaling, important for the control of mast cell-dependent anaphylaxis.


Assuntos
Anafilaxia/imunologia , Proteínas de Ligação a DNA/fisiologia , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Mastócitos/fisiologia , Proteínas Nucleares/fisiologia , Receptores de IgE/imunologia , Transdução de Sinais/imunologia , Anafilaxia/induzido quimicamente , Anafilaxia/metabolismo , Animais , Degranulação Celular/fisiologia , Células Cultivadas , Citocinas/genética , Proteínas de Ligação a DNA/genética , Dinitrobenzenos/imunologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Expressão Gênica , Fatores de Troca do Nucleotídeo Guanina/genética , Imunoglobulina E/imunologia , Inositol 1,4,5-Trifosfato/metabolismo , Interleucina-4/sangue , Interleucina-4/genética , Mastócitos/citologia , Mastócitos/imunologia , Camundongos , Camundongos Endogâmicos , Camundongos Knockout , Antígenos de Histocompatibilidade Menor , Modelos Biológicos , Proteínas Nucleares/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transfecção , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
20.
ACS Chem Biol ; 2(10): 674-84, 2007 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-18041817

RESUMO

Antigen-mediated cross-linking of IgE bound to its receptor, FcRI, stimulates degranulation, phospholipid metabolism, and cytokine production in mast cells and basophils to initiate inflammatory and allergic responses. Previous studies suggested that spatial organization of the clustered receptors affects the assembly of the transmembrane signaling complexes. To investigate systematically the structural constraints in signal initiation, we utilized rigid double-stranded DNA scaffolds to synthesize ligands with tunable lengths. We characterized a series of symmetric trivalent DNA ligands with rigid spacing between 2,4-dinitrophenyl (DNP) haptenic groups in the range of 5-15 nm. These ligands all bind to anti-DNP IgE on RBL mast cells with similar avidity, and they all cross-link IgE-FcRI complexes effectively. We observe length-dependent stimulation of tyrosine phosphorylation of FcRI beta and gamma subunits and the adaptor protein LAT: the shortest ligand is approximately 5-10-fold more potent than the longest. Stimulated Ca2+ mobilization and degranulation also exhibits kinetics and magnitudes that differ as a function of ligand length. In contrast, tyrosine phosphorylation of phospholipase Cgamma1 and consequent Ca2+ release from intracellular stores do not show this dependence on ligand length. Our results with these rigid, DNA-based ligands provide direct support for receptor transphosphorylation as a key step in amplified signaling leading to degranulation, and they further reveal branching of pathways in signaling events.


Assuntos
DNA/química , Mastócitos/fisiologia , Receptores de IgE/fisiologia , Transdução de Sinais/fisiologia , Animais , Cálcio/metabolismo , Degranulação Celular , Citocalasina D/farmacologia , DNA/metabolismo , Dinitrobenzenos/imunologia , Dinitrobenzenos/metabolismo , Imunoglobulina E/metabolismo , Ligantes , Fosfatidilinositol 3-Quinases/fisiologia , Fosfolipase C gama/fisiologia , Fosforilação , Receptores de IgE/química , Tirosina/metabolismo
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