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1.
J Allergy Clin Immunol ; 142(2): 557-568.e6, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29074459

RESUMO

BACKGROUND: IgE-immune complexes (IgE-ICs) have been shown to enhance antibody and T-cell responses in mice by targeting CD23 (FcεRII), the low-affinity receptor for IgE on B cells. In humans, the mechanism by which CD23-expressing cells take up IgE-ICs and process them is not well understood. OBJECTIVE: To investigate this question, we compared the fate of IgE-ICs in human B cells and in CD23-expressing monocyte-derived dendritic cells (moDCs) that represent classical antigen-presenting cells and we aimed at studying IgE-dependent antigen presentation in both cell types. METHODS: B cells and monocytes were isolated from peripheral blood, and monocytes were differentiated into moDCs. Both cell types were stimulated with IgE-ICs consisting of 4-hydroxy-3-iodo-5-nitrophenylacetyl (NIP)-specific IgE JW8 and NIP-BSA to assess binding, uptake, and degradation dynamics. To assess CD23-dependent T-cell proliferation, B cells and moDCs were pulsed with IgE-NIP-tetanus toxoid complexes and cocultured with autologous T cells. RESULTS: IgE-IC binding was CD23-dependent in B cells, and moDCs and CD23 aggregation, as well as IgE-IC internalization, occurred in both cell types. Although IgE-ICs were degraded in moDCs, B cells did not degrade the complexes but recycled them in native form to the cell surface, enabling IgE-IC uptake by moDCs in cocultures. The resulting proliferation of specific T cells was dependent on cell-cell contact between B cells and moDCs, which was explained by increased upregulation of costimulatory molecules CD86 and MHC class II on moDCs induced by B cells. CONCLUSIONS: Our findings argue for a novel model in which human B cells promote specific T-cell proliferation on IgE-IC encounter. On one hand, B cells act as carriers transferring antigen to more efficient antigen-presenting cells such as DCs. On the other hand, B cells can directly promote DC maturation and thereby enhance T-cell stimulation.


Assuntos
Complexo Antígeno-Anticorpo/metabolismo , Antígenos/metabolismo , Linfócitos B/imunologia , Células Dendríticas/imunologia , Imunoglobulina E/metabolismo , Soroalbumina Bovina/metabolismo , Linfócitos T/imunologia , Apresentação do Antígeno , Complexo Antígeno-Anticorpo/imunologia , Antígenos/imunologia , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Técnicas de Cocultura , Humanos , Imunização , Imunoglobulina E/imunologia , Ativação Linfocitária , Nitro-Hidroxi-Iodofenilacetato/química , Ligação Proteica , Receptores de IgE/metabolismo , Soroalbumina Bovina/química , Soroalbumina Bovina/imunologia
2.
Scand J Immunol ; 85(1): 43-50, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27864913

RESUMO

IgM molecules circulate in serum as large polymers, mainly pentamers, which can be transported by the poly-Ig receptor (pIgR) across epithelial cells to mucosal surfaces and released as secretory IgM (SIgM). The mucosal SIgM molecules have non-covalently attached secretory component (SC), which is the extracellular part of pIgR which is cleaved from the epithelial cell membrane. Serum IgM antibodies do not contain SC and have previously been shown to make a conformational change from 'a star' to a 'staple' conformation upon reaction with antigens on a cell surface, enabling them to activate complement. However, it is not clear whether SIgM similarly can induce complement activation. To clarify this issue, we constructed recombinant chimeric (mouse/human) IgM antibodies against hapten 5-iodo-4-hydroxy-3-nitro-phenacetyl (NIP) and in addition studied polyclonal IgM formed after immunization with a meningococcal group B vaccine. The monoclonal and polyclonal IgM molecules were purified by affinity chromatography on a column containing human SC in order to isolate joining-chain (J-chain) containing IgM, followed by addition of excess amounts of soluble SC to create SIgM (IgM J+ SC+). These SIgM preparations were tested for complement activation ability and shown to be nearly as active as the parental IgM J+ molecules. Thus, SIgM may offer protection against pathogens at mucosal surface by complement-mediated cell lysis or by phagocytosis mediated by complement receptors present on effector cells on mucosa.


Assuntos
Ativação do Complemento , Proteínas do Sistema Complemento/imunologia , Imunoglobulina M/imunologia , Vacinas Meningocócicas/imunologia , Animais , Citotoxicidade Celular Dependente de Anticorpos , Cápsulas Bacterianas/imunologia , Humanos , Imunidade Humoral , Camundongos , Membrana Mucosa/imunologia , Nitro-Hidroxi-Iodofenilacetato/imunologia , Conformação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Componente Secretório/imunologia
3.
EMBO J ; 35(21): 2371-2381, 2016 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-27634959

RESUMO

How the B-cell antigen receptor (BCR) is activated upon interaction with its cognate antigen or with anti-BCR antibodies is not fully understood. We have recently shown that B-cell activation is accompanied by the opening of the pre-organized BCR oligomers, an observation that strengthens the role of receptor reorganization in signalling. We have now analysed the BCR oligomer opening and signalling upon treatment with different monovalent stimuli. Our results indicate that monovalent antigens are able to disturb and open the BCR oligomer, but that this requires the presence and activity of the Src family kinase (SFK) Lyn. We have also shown that monovalent Fab fragments of anti-BCR antibodies can open the BCR oligomers as long as they directly interact with the antigen-binding site. We found that monovalent antigen binding opens both the IgM-BCR and IgD-BCR, but calcium signalling is only seen in cells expressing IgM-BCR; this provides a molecular basis for IgM- and IgD-BCR functional segregation.


Assuntos
Nitro-Hidroxi-Iodofenilacetato/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Soroalbumina Bovina/metabolismo , Quinases da Família src/metabolismo , Animais , Linfócitos B/metabolismo , Sinalização do Cálcio , Linhagem Celular , Imunoglobulina D/metabolismo , Imunoglobulina M/metabolismo , Camundongos Transgênicos , Peptídeos/metabolismo , Baço/citologia
4.
J Immunol ; 194(11): 5497-508, 2015 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-25904551

RESUMO

Engineering of the constant Fc part of monoclonal human IgG1 (hIgG1) Abs is an approach to improve effector functions and clinical efficacy of next-generation IgG1-based therapeutics. A main focus in such development is tailoring of in vivo half-life and transport properties by engineering the pH-dependent interaction between IgG and the neonatal Fc receptor (FcRn), as FcRn is the main homeostatic regulator of hIgG1 half-life. However, whether such engineering affects binding to other Fc-binding molecules, such as the classical FcγRs and complement factor C1q, has not been studied in detail. These effector molecules bind to IgG1 in the lower hinge-CH2 region, structurally distant from the binding site for FcRn at the CH2-CH3 elbow region. However, alterations of the structural composition of the Fc may have long-distance effects. Indeed, in this study we show that Fc engineering of hIgG1 for altered binding to FcRn also influences binding to both the classical FcγRs and complement factor C1q, which ultimately results in alterations of cellular mechanisms such as Ab-dependent cell-mediated cytotoxicity, Ab-dependent cellular phagocytosis, and Ab-dependent complement-mediated cell lysis. Thus, engineering of the FcRn-IgG1 interaction may greatly influence effector functions, which has implications for the therapeutic efficacy and use of Fc-engineered hIgG1 variants.


Assuntos
Anticorpos Monoclonais/genética , Complemento C1q/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Imunoglobulina G/genética , Receptores Fc/imunologia , Receptores de IgG/imunologia , Anticorpos Monoclonais/imunologia , Afinidade de Anticorpos/genética , Afinidade de Anticorpos/imunologia , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Linhagem Celular , Células HEK293 , Éxons Codificadores da Região de Dobradiça/genética , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Imunoglobulina G/imunologia , Nitro-Hidroxi-Iodofenilacetato/imunologia , Fagocitose/imunologia , Engenharia de Proteínas , Receptores Fc/genética , Receptores de IgG/genética , Ressonância de Plasmônio de Superfície
5.
J Exp Med ; 207(4): 711-9, 2010 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-20231378

RESUMO

Signals processed through the B cell antigen receptor (BCR) control both the proliferation and differentiation of B lymphocytes. How these different signaling modes are established at the BCR is poorly understood. We show that a conserved arginine in the tail sequence of the Igalpha subunit of the BCR is methylated by the protein arginine methyltransferase 1. This modification negatively regulates the calcium and PI-3 kinase pathways of the BCR while promoting signals leading to B cell differentiation. Thus, Igalpha arginine methylation can play an important role in specifying the outcome of BCR signaling.


Assuntos
Arginina/metabolismo , Linfócitos B/citologia , Linfócitos B/metabolismo , Diferenciação Celular , Receptores de Antígenos de Linfócitos B/metabolismo , Sequência de Aminoácidos , Animais , Arginina/genética , Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/metabolismo , Subpopulações de Linfócitos B/transplante , Linfócitos B/imunologia , Linfócitos B/transplante , Células da Medula Óssea/citologia , Células da Medula Óssea/imunologia , Antígenos CD79/genética , Antígenos CD79/metabolismo , Sinalização do Cálcio/imunologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Linhagem Celular , Humanos , Interleucina-7/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ativação Linfocitária/fisiologia , Metilação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Dados de Sequência Molecular , Nitro-Hidroxi-Iodofenilacetato/imunologia , Fosforilação/imunologia , Células Precursoras de Linfócitos B/citologia , Células Precursoras de Linfócitos B/efeitos dos fármacos , Células Precursoras de Linfócitos B/metabolismo , Ligação Proteica/imunologia , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/genética , Receptores de Antígenos de Linfócitos B/genética , Homologia de Sequência de Aminoácidos , Baço/citologia , Baço/imunologia , Quinase Syk , Transfecção
6.
Scand J Immunol ; 70(6): 553-64, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19906198

RESUMO

The C1q binding epicentre on IgG molecules involves residues Asp(270), Lys(322), Pro(329) and Pro(331) in the C(H)2 domain. IgG1 and IgG3 are usually the most efficient of the four human IgG subclasses in activating complement and they both share all these residues. To reveal possible differences in the structural requirement for complement activation, we created a number of NIP (5-iodo-4-hydroxy-3-nitro-phenacetyl) specific IgG1 and IgG3 antibodies with parallel mutations in or near the putative C1q binding site. The mutants were tested simultaneously for antibody induced, antibody-dependent complement-mediated lysis (ADCML) at high and low antigen concentration on the target cells using sera of human, rabbit and guinea pig as complement source. In addition, we tested the antibodies against target cells decorated with the NP hapten, which has 10-fold lower affinity for the antibodies compared to the NIP hapten. We also used ELISA methods to measure complement activation. We observed a clear difference between IgG1 and IgG3 localized to residues Asp(270), Leu(334), Leu(335). For all these residues, and especially for Asp(270), IgG1 was heavily reduced in complement activation, while IgG3 was only moderated reduced, by alanine substitution. This difference was independent of the long hinge region of IgG3, demonstrated by hinge region truncation of this isotype such that it resembles that of IgG1. This report indicates the presence of structural differences between human IgG1 and IgG3 in the C1q binding site, and points to a specialization of the two isotypes with respect to complement activation.


Assuntos
Sítios de Ligação de Anticorpos/imunologia , Ativação do Complemento/imunologia , Complemento C1q/imunologia , Imunoglobulina G/imunologia , Animais , Afinidade de Anticorpos/genética , Afinidade de Anticorpos/imunologia , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Sítios de Ligação de Anticorpos/genética , Complemento C1q/metabolismo , Cobaias , Humanos , Imunoglobulina G/química , Imunoglobulina G/genética , Mutação , Nitro-Hidroxi-Iodofenilacetato/imunologia , Coelhos
7.
Scand J Immunol ; 70(3): 277-87, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19703017

RESUMO

Immunoglobulin (IgG) has the ability to suppress the Ab response against the Ag to which it binds. Although the mechanism remains unclear, this phenomenon has physiological relevance and is used clinically in Rh prophylaxis. As suppression works well in mice lacking the inhibitory FcgammaRIIB, the two most likely explanations are that IgG masks epitopes and/or that IgG increases the clearance of Ag. In the present study, mice were immunized with sheep red blood cells (SRBC) to which the hapten 5-iodo-4-hydroxyl-3-nitrophenacetyl (NIP) was conjugated at high or low density and the ability of IgG anti-NIP to suppress the Ab response to NIP and SRBC was assayed. Only the NIP-specific response was suppressed when mice were immunized with SRBC-NIP(low), whereas both NIP- and SRBC-specific responses were suppressed when SRBC-NIP(high) was used. This is best explained by epitope masking; at high epitope density, IgG also blocks neighbouring epitopes from recognition by B cells. We also examined the effects of IgG-mediated suppression on T-cell responses directly in vivo. While IgG anti-SRBC administered with sheep red blood cells ovalbumin (SRBC-OVA) almost completely suppressed the anti-SRBC and anti-OVA Ab responses, the OVA-specific T-cell response was still 50% of that observed in control mice. This is probably the result of decreased Ag exposure as IgG-bound SRBC were cleared faster from the bloodstream and were found at lower concentration in the spleen than unbound SRBC. These results suggest that both Ag clearance and epitope masking occurs during IgG-mediated suppression, but that under physiological circumstances epitope masking is the predominant mechanism.


Assuntos
Formação de Anticorpos , Antígenos/imunologia , Epitopos/imunologia , Imunoglobulina G/imunologia , Nitro-Hidroxi-Iodofenilacetato/imunologia , Baço/imunologia , Transferência Adotiva , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Epitopos/metabolismo , Eritrócitos/imunologia , Imunoglobulina G/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Nitro-Hidroxi-Iodofenilacetato/metabolismo , Ovinos , Baço/metabolismo
8.
Immunity ; 30(1): 44-55, 2009 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-19135393

RESUMO

B cells are activated in vivo after the B cell receptors (BCRs) bind to antigens captured on the surfaces of antigen-presenting cells. Antigen binding results in BCR microclustering and signaling; however, the molecular nature of the signaling-active BCR clusters is not well understood. Using single-molecule imaging techniques, we provide evidence that within microclusters, the binding of monovalent membrane antigens results in the assembly of immobile signaling-active BCR oligomers. The oligomerization depends on interactions between the membrane-proximal Cmicro4 domains of the membrane immunoglobulin that are both necessary and sufficient for assembly. Antigen-bound BCRs that lacked the Cmicro4 domain failed to cluster and signal, and conversely, Cmicro4 domains alone clustered spontaneously and activated B cells. These results support a unique mechanism for the initiation of BCR signaling in which antigen binding induces a conformational change in the Fc portion of the BCR, revealing an interface that promotes BCR clustering.


Assuntos
Linfócitos B/imunologia , Membrana Celular/fisiologia , Regiões Constantes de Imunoglobulina/fisiologia , Molécula 1 de Adesão Intercelular/metabolismo , Nitro-Hidroxi-Iodofenilacetato/metabolismo , Receptores de Antígenos de Linfócitos B/fisiologia , Animais , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais , Quinase Syk
9.
J Immunol ; 174(9): 5358-66, 2005 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-15843533

RESUMO

It is believed that Ag in immune complexes (ICs) on follicular dendritic cells (FDCs) selects high affinity B cells and promotes affinity maturation. However, selection has been documented in the absence of readily detectable ICs on FDCs, suggesting that FDC-ICs may not be important. These results prompted experiments to test the hypothesis that IC-bearing murine FDCs can promote high affinity IgG responses by selecting B cells after stimulating naive IgM(+) cells to mature and class switch. Coculturing naive lambda(+) B cells, FDCs, (4-hydroxy-3-nitrophenyl)acetyl-chicken gamma-globulin (CGG) + anti-CGG ICs, and CGG-primed T cells resulted in FDC-lymphocyte clusters and production of anti-4-hydroxy-5-iodo-3-nitrophenyl acetyl. Class switching was indicated by a shift from IgM to IgG, and affinity maturation was indicated by a change from mostly low affinity IgM and IgG in the first week to virtually all high affinity IgG anti-4-hydroxy-5-iodo-3-nitrophenyl acetyl in the second week. Class switching and affinity maturation were easily detectable in the presence of FDCs bearing appropriate ICs, but not in the absence of FDCs. Free Ag plus FDCs resulted in low affinity IgG, but affinity maturation was only apparent when FDCs bore ICs. Class switching is activation-induced cytidine deaminase (AID) dependent, and blocking FDC-CD21 ligand-B cell CD21 interactions inhibited FDC-IC-mediated enhancement of AID production and the IgG response. In short, these data support the concept that ICs on FDCs can promote AID production, class switching, and maturation of naive IgM(+) B cells, and further suggest that the IC-bearing FDCs help select high affinity B cells that produce high affinity IgG.


Assuntos
Complexo Antígeno-Anticorpo/fisiologia , Sítios de Ligação de Anticorpos , Células Dendríticas Foliculares/imunologia , Células Dendríticas Foliculares/metabolismo , Switching de Imunoglobulina/imunologia , Imunoglobulina G/biossíntese , Imunoglobulina G/metabolismo , Imunoglobulina M/biossíntese , Animais , Complexo Antígeno-Anticorpo/metabolismo , Linfócitos B/enzimologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Comunicação Celular/imunologia , Células Cultivadas , Técnicas de Cocultura , Citidina Desaminase/biossíntese , Ligantes , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Nitro-Hidroxi-Iodofenilacetato/imunologia , Receptores de Complemento 3d/metabolismo , Receptores de Complemento 3d/fisiologia , Fatores de Tempo
10.
J Immunol ; 172(5): 2925-34, 2004 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-14978095

RESUMO

Mature, naive B cells coexpress IgD and IgM with identical binding sites. In this study, the binding properties of such IgM and IgD are compared to determine how size and shape may influence their ability to bind Ag and thus function as receptors. To dissect their intrinsic binding properties, recombinant IgM and IgD were produced in soluble form as monomers of the basic H(2)L(2) Ab architecture, each with two Ag binding sites. Since these sites are connected with a hinge region in IgD and structural Ig domains in IgM, the two molecules differ significantly in this region. The results show that IgD exhibited the larger angle and longer distance between its binding sites, as well as having the greater flexibility. Relative functional affinity was assessed on two antigenic surfaces with high or low epitope density, respectively. At high epitope density, IgM had a higher functional affinity for the Ag compared with IgD. The order was reversed at low epitope density due to a decrease in the functional affinity of IgM. Studies of binding kinetics showed similar association rates for both molecules. The dissociation rate, however, was slower for IgM at high epitope density and for IgD at low epitope density. Taken together, the results show that IgM and IgD with identical Ag binding regions have different Ag binding properties.


Assuntos
Sítios de Ligação de Anticorpos , Imunoglobulina D/genética , Imunoglobulina D/metabolismo , Imunoglobulina M/metabolismo , Nitro-Hidroxi-Iodofenilacetato/imunologia , Receptores de Antígenos de Linfócitos B/fisiologia , Proteínas Recombinantes de Fusão/metabolismo , Sequência de Aminoácidos , Animais , Especificidade de Anticorpos/genética , Sítios de Ligação de Anticorpos/genética , Linhagem Celular Tumoral , Ensaio de Imunoadsorção Enzimática , Epitopos/metabolismo , Vetores Genéticos , Imunoglobulina D/biossíntese , Imunoglobulina D/ultraestrutura , Imunoglobulina M/biossíntese , Imunoglobulina M/genética , Imunoglobulina M/ultraestrutura , Cinética , Camundongos , Microscopia Imunoeletrônica , Dados de Sequência Molecular , Nitro-Hidroxi-Iodofenilacetato/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/ultraestrutura , Tiocianatos/química , Transfecção
11.
J Immunol ; 171(11): 5707-17, 2003 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-14634078

RESUMO

CD40 is an important costimulatory molecule for B cells as well as dendritic cells, monocytes, and other APCs. The ligand for CD40, CD154, is expressed on activated T cells, NK cells, mast cells, basophils, and even activated B cells. Although both CD40(-/-) and CD154(-/-) mice have impaired ability to isotype switch, form germinal centers, make memory B cells, and produce Ab, it is not entirely clear whether these defects are intrinsic to B cells, to other APCs, or to T cells. Using bone marrow chimeric mice, we investigated whether CD40 or CD154 must be expressed on B cells for optimal B cell responses in vivo. We demonstrate that CD40 expression on B cells is required for the generation of germinal centers, isotype switching, and sustained Ab production, even when other APCs express CD40. In contrast, the expression of CD154 on B cells is not required for the generation of germinal centers, isotype switching, or sustained Ab production. In fact, B cell responses are completely normal when CD154 expression is limited exclusively to Ag-specific T cells. These results suggest that the interaction of CD154 expressed by activated CD4 T cells with CD40 expressed by B cells is the primary pathway necessary to achieve B cell activation and differentiation and that CD154 expression on B cells does not noticeably facilitate B cell activation and differentiation.


Assuntos
Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/metabolismo , Antígenos CD40/biossíntese , Ligante de CD40/fisiologia , Animais , Subpopulações de Linfócitos B/citologia , Subpopulações de Linfócitos B/virologia , Transplante de Medula Óssea/imunologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Antígenos CD40/genética , Antígenos CD40/fisiologia , Ligante de CD40/biossíntese , Ligante de CD40/genética , Diferenciação Celular/imunologia , Epitopos de Linfócito T/imunologia , Vírus da Influenza A/imunologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Nitro-Hidroxi-Iodofenilacetato/administração & dosagem , Nitro-Hidroxi-Iodofenilacetato/imunologia , Ovalbumina/administração & dosagem , Ovalbumina/imunologia , Quimera por Radiação/imunologia
12.
J Endocrinol ; 176(2): 267-74, 2003 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-12553875

RESUMO

The soy phytoestrogen, genistein, induces thymic atrophy when administered to ovariectomized mice by injection or in the diet. Injected genistein also causes decreased humoral immunity, but the effects of genistein on cell-mediated immunity have not been addressed. Here we examined effects of injected and dietary genistein on cell-mediated immune responses. Female C57BL/6 mice (25- to 27-days-old) were ovariectomized, then placed on phytoestrogen-free feed 5 days later. Seven days after ovariectomy, they were given daily subcutaneous injections of either dimethylsulfoxide (DMSO) or genistein (8, 20, 80 mg/kg) for 28 days; some mice were given 80 mg/kg genistein plus the anti-estrogen ICI 182,780 (5 mg/kg/week). Cell-mediated immune response was tested by analyzing the delayed-type hypersensitivity (DTH) response to a hapten, 4-hydroxy-3-nitrophenyl acetyl succinimide (NP-O-SU), at the end of treatment. Reversibility of the effects of genistein was tested by measuring the DTH response in mice that were given genistein (20 or 80 mg/kg) for 28 days, then allowed to recover for 28 days. To determine if dietary genistein could affect cell-mediated immunity, mice ovariectomized as above were fed genistein at 0, 1000 or 1500 parts per million (ppm) for 28 days. There was a 46-67% decrease in the DTH response in the footpads of mice injected with 8-80 mg/kg genistein compared with controls (P<0.05 vs control for all treatment groups); these effects were reversible. On histopathological examination of the feet, there was decreased cell infiltration in genistein-treated animals compared with controls, and the numbers of CD4(+) and CD8(+) T cells in popliteal lymph nodes were reduced. The effects of genistein are mediated through both estrogen receptor (ER) and non-ER pathways, as the anti-estrogen ICI 182,780 only partially blocked the effects of genistein on the DTH response. Dietary genistein (1000 or 1500 ppm) decreased cell-mediated immunity while producing serum genistein concentrations in the physiological range for humans under certain nutritional conditions. Further work is needed to determine if dietary genistein and phytoestrogen exposure can produce effects on cell-mediated immunity in humans or other animals under various nutritional conditions.


Assuntos
Genisteína/efeitos adversos , Imunidade Celular/efeitos dos fármacos , Reguladores de Crescimento de Plantas/farmacologia , Animais , Dieta , Feminino , Genisteína/sangue , Genisteína/farmacologia , Injeções , Camundongos , Camundongos Endogâmicos C57BL , Nitro-Hidroxi-Iodofenilacetato , Ovariectomia , Soja
13.
Biomol Eng ; 18(3): 109-16, 2001 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-11566602

RESUMO

A major objective in vaccine development is the design of reagents that give a strong, specific T cell response. Targeting of antigens to antigen presenting cells (APC) results in enhanced antigen presentation and T cell activation. In this paper, we describe a novel targeting reagent denoted 'Troy-bodies', namely recombinant antibodies with APC-specificity and with T cell epitopes integrated in their C regions. We have made such antibodies with V regions specific for either IgD or MHC class II, and five different T cell epitopes have been tested. All epitopes could be introduced into loops of C domains without disrupting immunoglobulin (Ig) folding. Four have been tested in T cell activation studies, and all could be released and presented by APC. Furthermore, whether IgD- or MHC-specific, the molecules tested enhanced T cell stimulation compared to non-specific control antibodies in vitro as well as in vivo. Using this technology, specific reagents can be designed that target selected antigenic peptides to an APC of choice. Troy-bodies may therefore be useful for manipulation of immune responses, and in particular for vaccination purposes.


Assuntos
Anticorpos/imunologia , Células Apresentadoras de Antígenos/imunologia , Epitopos/imunologia , Engenharia de Proteínas , Linfócitos T/imunologia , Animais , Anticorpos/genética , Especificidade de Anticorpos , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Imunoglobulina D/imunologia , Região Variável de Imunoglobulina/química , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos BALB C , Modelos Estruturais , Mutagênese Insercional , Nitro-Hidroxi-Iodofenilacetato , Dobramento de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/imunologia
14.
Biochem Biophys Res Commun ; 286(2): 243-9, 2001 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-11500028

RESUMO

A recombinant IgG3 antibody with Phe-243 replaced by Ala (FA243) was expressed in a CHO-K1 parental cell line. The resulting IgG-Fc-linked carbohydrate was significantly alpha2,3-sialylated (53% of glycans), as indicated by normal- and reverse-phase HPLC analyses. Following transfection of a rat alpha2,6-sialyltransferase gene into this parental cell line, IgG-Fc-linked glycans were sialylated (60% of glycans) such that the ratio of alpha2,6- to alpha2,3-linked sialic acid was 0.9:1.0. By comparison, the wild-type IgG3 (F243) is minimally sialylated (2-3% alpha2,3-linked), thus suggesting that sialylation is controlled primarily by the protein structure local to the carbohydrate and that the two sialyltransferases compete to sialylate the nascent oligosaccharide. The additional alpha2,6-sialylation affected the function of the recombinant antibody. FA243 IgG3 having both alpha2,6 and alpha2,3-sialylation restored recognition to wild-type IgG3 levels for human FcgammaRI, FcgammaRII, and target cell lysis by complement. We discuss how sialylation linkage could modulate IgG function.


Assuntos
Fragmentos Fc das Imunoglobulinas/química , Imunoglobulina G/química , Sialiltransferases/genética , Animais , Células CHO , Carboidratos/análise , Cromatografia Líquida de Alta Pressão , Ativação do Complemento , Cricetinae , Glicosilação , Humanos , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Células K562 , Mutação , Ácido N-Acetilneuramínico/análise , Nitro-Hidroxi-Iodofenilacetato/imunologia , Oligossacarídeos/análise , Ratos , Superóxidos/metabolismo , Transfecção , Células U937
15.
Curr Protoc Immunol ; Chapter 6: Unit 6.20, 2001 May.
Artigo em Inglês | MEDLINE | ID: mdl-18432811

RESUMO

This unit describes a general method for immunoenzymetric assay of mouse and human cytokines. The technique is based on use of two anti-cytokine monoclonal antibodies (MAbs), each specific for a spatially distinct determinant on the cytokine. One of these is a coating antibody, and the other is a derivatized detecting antibody. A support protocol describes chemical labeling of anti-cytokine monoclonal IgG antibodies with the NIP hapten group to produce the detecting antibody. Another support protocol describes a method for producing horseradish peroxidase (HRPO)-conjugated J4, a rat IgG1 anti-NIP monoclonal antibody that confers the anti-NIP specificity to the HRPO detection system used in the immunoenzymetric assay. The method described in this unit has allowed successful measurement of different cytokines in a wide variety of clinical samples including conditioned medium, serum and plasma, ascites, amniotic fluid, and bronchoalveolar lavage fluid.


Assuntos
Anticorpos Monoclonais/imunologia , Citocinas/análise , Citocinas/imunologia , Técnicas Imunoenzimáticas/métodos , Nitro-Hidroxi-Iodofenilacetato/imunologia , Animais , Humanos , Camundongos
16.
Cancer Gene Ther ; 7(4): 526-9, 2000 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-10811469

RESUMO

Adoptive therapy with autologous T cells expressing chimeric T-cell receptors (chTCRs) is of potential interest for the treatment of malignancy. To limit possible T-cell-mediated damage to normal tissues that weakly express the targeted tumor antigen (Ag), we have tested a strategy for the suppression of target cell recognition by engineered T cells. Jurkat T cells were transduced with an anti-hapten chTCR tinder the control of a tetracycline-suppressible promoter and were shown to respond to Ag-positive (hapten-coated) but not to Ag-negative target cells. The engineered T cells were then reacted with hapten-coated target cells at different effector to target cell ratios before and after exposure to tetracycline. When the engineered T cells were treated with tetracycline, expression of the chTCR was greatly decreased and recognition of the hapten-coated target cells was completely suppressed. Tetracycline-mediated suppression of target cell recognition by engineered T cells may be a useful strategy to limit the toxicity of the approach to cancer gene therapy.


Assuntos
Ativação Linfocitária , Nitro-Hidroxi-Iodofenilacetato/farmacologia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia , Transfecção , Citometria de Fluxo , Haptenos/farmacologia , Células HeLa , Humanos , Interleucina-2/biossíntese , Células Jurkat , Ativação Linfocitária/efeitos dos fármacos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/imunologia , Tetraciclina/farmacologia
17.
Gene Ther ; 6(4): 694-702, 1999 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-10476231

RESUMO

Using genetic engineering techniques, two strategies for changing the receptor-binding specificity of the influenza A virus haemagglutinin (HA) protein whilst preserving its membrane fusion function, have been explored. The aim was to investigate whether the HA protein could be developed as an attachment/entry protein for targeting enveloped virus gene therapy vectors to specific cell populations. In the first strategy, a single chain antibody Fv region (scFv) specific for the hapten NIP was inserted between HA1 codons 139 and 145, to create a scFv-HA chimaeric protein. This protein was shown to possess anti-NIP binding activity, but membrane fusion activity could not be demonstrated. The possibility that linking the scFv domain directly to HA may have inhibited the HA fusion function led to the development of the second strategy. This involved separating the receptor-binding and membrane fusion functions of HA on to two different molecules. The feasibility of this strategy was tested by looking for fusion between NIP-conjugated red blood cells which lacked sialic acid (the HA protein's natural receptor) and Chinese hamster ovary cells that expressed both the above anti-NIP scFv-HA chimaeric protein (as a non-fusigenic, receptor-binding molecule) and wild-type HA protein (as a fusigenic, non-binding molecule) on their surface. Cell-to-cell fusion was observed in this system, indicating that the receptor-binding function of HA can be transferred to an adjacent molecule, and also changed in its specificity, without compromising its membrane fusion activity. This finding strongly suggests that the development of a two-molecule attachment and entry system for retargeting enveloped virus gene therapy vectors, based on HA, is a viable proposition.


Assuntos
Engenharia Genética/métodos , Vetores Genéticos/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A , Proteínas do Envelope Viral/genética , Animais , Células CHO , Cricetinae , Eritrócitos , Humanos , Fusão de Membrana/genética , Nitro-Hidroxi-Iodofenilacetato/genética , Ligação Proteica/genética
18.
J Exp Med ; 189(10): 1679-84, 1999 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-10330447

RESUMO

The striking and unique structural feature of the T cell receptor (TCR) beta chain is the bulky solvent-exposed FG loop on the Cbeta domain, the size of almost half an immunoglobulin domain. The location and size of this loop suggested immediately that it could be a crucial structural link between the invariant CD3 subunits and antigen-recognizing alpha/beta chains during TCR signaling. However, functional analysis does not support the above notion, since transgene coding for TCR beta chain lacking the complete FG loop supports normal alpha/beta T cell development and function.


Assuntos
Receptores de Antígenos de Linfócitos T alfa-beta/genética , Linfócitos T/imunologia , Animais , Divisão Celular , Linhagem Celular , Citometria de Fluxo , Hibridomas , Imunoglobulina G/análise , Camundongos , Camundongos Endogâmicos , Camundongos Transgênicos , Mutagênese , Nitro-Hidroxi-Iodofenilacetato/imunologia , Ovalbumina/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/química , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Deleção de Sequência , Transfecção
19.
EMBO J ; 16(12): 3553-62, 1997 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-9218797

RESUMO

The B-cell antigen receptor (BCR) internalizes bound antigen such that antigen-derived peptides become associated with emigrating major histocompatibility complex (MHC) class II molecules for presentation to T cells. Experiments with B-cell transfectants reveal that BCR confers a specificity of intracellular targeting since chimeric antigen receptors which internalize antigen by virtue of a heterologous cytoplasmic domain do not necessarily give rise to presentation. In contrast, however, previous studies have shown that antigen binding to irrelevant cell surface molecules (e.g. transferrin receptor, MHC class I) can ultimately lead to presentation. The solution to this paradox appears to be that the intracellular targeting by BCR actually reflects an acceleration of antigen delivery. Depending on the nature of the BCR-antigen interaction, this accelerated targeting can be essential in determining whether or not internalization leads to significant presentation. Physiologically, the accelerated delivery of antigen by BCR could prove of particular importance early in the immune response when antigen-BCR interaction is likely to be poor.


Assuntos
Apresentação do Antígeno , Antígenos/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Animais , Antígenos/genética , Antígenos H-2/genética , Antígenos H-2/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Imunoglobulina M/genética , Imunoglobulina M/imunologia , Lipoproteínas LDL/genética , Lipoproteínas LDL/imunologia , Camundongos , Muramidase/genética , Muramidase/imunologia , Nitro-Hidroxi-Iodofenilacetato/genética , Nitro-Hidroxi-Iodofenilacetato/imunologia , Ovalbumina/genética , Ovalbumina/imunologia , Receptores de Antígenos de Linfócitos B/genética , Células Tumorais Cultivadas
20.
J Surg Res ; 70(1): 69-74, 1997 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-9228931

RESUMO

Various gastrointestinal functions such as mucosal blood flow and mucus secretion can be influenced immunologically. Rats were systemically sensitized with 4-hydroxy-3-iodo-5-nitro-phenylacetic acid (NIP), a synthetic antigen. Mucosal release of gastrin, prostaglandin F2 alpha, 6-keto-prostaglandin F1 alpha, and leukotriene C4 was measured after intragastric or in vitro antigen challenge. Gastric protection from ethanol was determined. In sensitized rats, intragastric antigen challenge increased release of gastrin from the antral mucosa ex vivo and tended to increase release of prostaglandin F2 alpha. Likewise, antral mucosa of sensitized rats released significantly more gastrin and prostaglandin F2 alpha during in vitro antigen challenge than during incubation in the absence of antigen. Release of 6-keto-prostaglandin F1 alpha and leukotriene C4 was not affected by the immunologic reaction. Topical antigen challenge in sensitized rats reduced gastric mucosal damage caused by ethanol by 50%. The immunologically induced gastroprotection was significantly attenuated by pretreatment with indomethacin. The findings show that specific antigen challenge renders the gastric mucosa more resistant against the injurious effect of ethanol indicating that the stomach is a target organ of immunological reactions. As gastrin and prostaglandins exert potent protective effects, release of these mediators may contribute to the protective response to gastric mucosal immune activation.


Assuntos
Antígenos/imunologia , Eicosanoides/metabolismo , Etanol/toxicidade , Mucosa Gástrica/imunologia , Gastrinas/metabolismo , 6-Cetoprostaglandina F1 alfa/metabolismo , Animais , Dinoprosta/metabolismo , Mucosa Gástrica/efeitos dos fármacos , Mucosa Gástrica/metabolismo , Indometacina/farmacologia , Leucotrieno C4/metabolismo , Masculino , Nitro-Hidroxi-Iodofenilacetato/imunologia , Ratos , Ratos Wistar , Gastropatias/induzido quimicamente , Gastropatias/prevenção & controle
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