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1.
Nat Biotechnol ; 15(7): 632-6, 1997 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-9219264

RESUMO

Monospecific antibody fragments produced in bacteria lack the Fc portion of antibodies, and are therefore unable to recruit natural effector functions. We describe the use of a bispecific antibody fragment (diabody) to recruit the whole spectrum of antibody effector functions by retargeting serum immunoglobulin (Ig). One arm of the diabody was directed against the target antigen, and the other against the serum Ig. The bispecific diabodies were able to recruit complement, induce mononuclear phagocyte respiratory burst and phagocytosis, and promote synergistic cytotoxicity towards colon carcinoma cells in conjunction with CD8+ T-cells. Further, by virtue of binding to serum Ig their half-life (beta-phase) was increased fivefold compared to a control diabody of the same molecular weight. Such bispecific diabodies may provide an attractive alternative to monoclonal antibodies for serotherapy.


Assuntos
Anticorpos Biespecíficos/farmacologia , Imunoglobulinas/sangue , Animais , Anticorpos Biespecíficos/sangue , Anticorpos Biespecíficos/genética , Antígenos , Sequência de Bases , Biotecnologia , Proteínas do Sistema Complemento/metabolismo , Citotoxicidade Imunológica , Primers do DNA/genética , Meia-Vida , Humanos , Imunização Passiva , Muramidase/imunologia , Fagocitose , Receptores de IgG/metabolismo , Células Tumorais Cultivadas
2.
Mol Immunol ; 30(5): 469-78, 1993 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-8385266

RESUMO

This study investigates the role played by Fc gamma RI and Fc gamma RII in triggering the respiratory burst induced in human monocyte-like U937 cells by monoclonal chimaeric anti-NIP antibodies expressing the human IgG subclass and mouse IgG2b heavy chains. Respiratory burst activity was measured as superoxide generation. Four separate lines of evidence indicate a predominant role for Fc gamma RI in triggering superoxide generation induced by erythrocytes sensitized with up to the maximum of 100,000 IgG molecules per cell. Firstly, erythrocytes sensitized with mouse IgG2b anti-NIP antibodies which are not recognized by human Fc gamma RI, did not induce a response but when residue Glu-235 was replaced by Leu to give the lower hinge sequence of mouse IgG2a which is recognized by Fc gamma RI, the mutant bound to Fc gamma RI and induced a response equal to 80% of that given by chimaeric human IgG3. Chimaeric human IgG3 antibodies with amino acid substitutions in the lower hinge showed reduced activity and the greatest reductions (< 32% of wild type antibody activity) were associated with changes at Leu-235 which is critical for recognition by Fc gamma RI. Secondly, chimaeric human IgG4 antibodies which are not recognized by Fc gamma RII, were able to induce superoxide generation. The rank order of abilities of chimaeric human IgG subclass antibodies to induce responses was IgG3 > IgG1 > IgG4 > > IgG2. Thirdly, responses induced by chimaeric human IgG were inhibited by concns of monomeric human IgG3 in the nM range. Finally, chimaeric human IgG3 induced responses were inhibited by anti-Fc gamma RI, but not anti-Fc gamma RII monoclonal antibodies. Consistent with a major role for Fc gamma RI in triggering the responses of U937 cells, erythrocytes sensitized with chimaeric human IgG3 did not induce superoxide generation by neutrophils which express Fc gamma RII and Fc gamma RIII, or eosinophils which express Fc gamma RII, but neither of which expresses Fc gamma RI.


Assuntos
Monócitos/imunologia , Fagócitos/metabolismo , Receptores de IgG/fisiologia , Explosão Respiratória/imunologia , Anticorpos Monoclonais/imunologia , Bucladesina/farmacologia , Linhagem Celular , Eosinófilos/imunologia , Eritrócitos/imunologia , Humanos , Imunoglobulina G/fisiologia , Isotipos de Imunoglobulinas , Interferon gama/farmacologia , Neutrófilos/imunologia , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes , Formação de Roseta , Superóxidos/metabolismo , Acetato de Tetradecanoilforbol/farmacologia
3.
Mol Immunol ; 30(3): 233-41, 1993 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-8381917

RESUMO

This study investigates the capacity of a complexed aglycosylated chimaeric human IgG3 antibody to induce the respiratory burst in human monocyte-like U937 cells. It demonstrates that the aglycosylated antibody, prepared by cell culture in tunicamycin, retains significant capacity to trigger this effector function which was assayed as superoxide generation. Erythrocytes sensitized with near maximal levels of aglycosylated IgG3 were able to trigger > 80% of the superoxide generation triggered by the glycosylated antibody from U937 cells induced to differentiate by interferon gamma and the aglycosylated IgG3 gave half maximal responses at sensitization levels only 72% higher than those required by the glycosylated form. Aglycosylated IgG3 was, however, much less effective in triggering superoxide generation by interferon gamma treated U937 cells at low sensitization levels as threshold responses required only 60 glycosylated IgG3 molecules per erythrocyte compared with 16,000 aglycosylated molecules. In addition, these studies indicate significant differences between the target cell to effector cell ratios which permit IgG sensitized erythrocytes to stimulate the respiratory burst and those which stimulate ADCC in the same effector cell type.


Assuntos
Imunoglobulina G/imunologia , Fagócitos/metabolismo , Proteínas Recombinantes de Fusão/imunologia , Explosão Respiratória , Relação Dose-Resposta Imunológica , Glicosilação , Humanos , Técnicas In Vitro , Interferon gama/farmacologia , Receptores de IgG/análise , Formação de Roseta , Superóxidos/metabolismo , Acetato de Tetradecanoilforbol/farmacologia
4.
Mol Immunol ; 29(1): 53-9, 1992 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-1530984

RESUMO

Important mammalian defensive functions such as phagocytosis are triggered in leukocytes by the interaction of the Fc region of IgG with cell surface receptors (Fc gamma R). The CH2 domain of IgG has been implicated previously as the site of interaction with human and mouse Fc gamma R. This domain was mapped for interaction with mouse Fc gamma R11 expressed by the macrophage-like cell line P388D1, using two panels of a total of 32 site-directed mutants of mouse IgG2b and chimeric human IgG3 monoclonal antibodies. Two potential binding sites have been identified: one in or within the vicinity of the lower hinge site on IgG for human Fc gamma R1, and one within the binding site on IgG for Clq. The three mutant IgGs (Gly 237----Ala, Asn 297----Ala, and Glu 318----Ala) which do not interact in complexed form also fail to bind as monomers. A 1H NMR study of the three non-binding monomeric mutants suggests that the mutations are largely site-specific, indicating that IgG interacts with mouse Fc gamma R11 at two regions within the CH2 domain. This interaction dictates phagocytosis mediated by Fc gamma R11 of the P388D1 cell line.


Assuntos
Antígenos de Diferenciação/metabolismo , Regiões Constantes de Imunoglobulina/metabolismo , Cadeias gama de Imunoglobulina/metabolismo , Receptores Fc/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , Análise Mutacional de DNA , Imunoglobulina G/metabolismo , Técnicas In Vitro , Espectroscopia de Ressonância Magnética , Camundongos , Modelos Moleculares , Fagocitose , Receptores de IgG , Proteínas Recombinantes/metabolismo , Formação de Roseta , Relação Estrutura-Atividade
5.
J Immunol Methods ; 122(1): 97-103, 1989 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-2547875

RESUMO

A simple method for isolating highly purified eosinophils from human blood is described. Buffy coats from normal individuals (eosinophil counts less than 0.4 x 10(9)/litre) were centrifuged through a two layer Percoll density gradient, to produce a granulocyte fraction containing neutrophils and eosinophils. Neutrophils were extracted from this fraction using a monoclonal antibody (CLB FcR gran 1) against CD 16 (Fc gamma R III) in a direct or indirect selection procedure using immunomagnetic beads (Dynabeads). This negative immunoselection produced eosinophils of greater purity and with a superior capacity to mount a respiratory burst than eosinophils isolated by a method employing metrizamide.


Assuntos
Separação Celular/métodos , Eosinófilos , Técnicas Imunológicas , Antígenos de Diferenciação/imunologia , Centrifugação com Gradiente de Concentração , Eosinófilos/imunologia , Humanos , Medições Luminescentes , Magnetismo , Metrizamida , Neutrófilos/imunologia , Povidona , Receptores Fc/imunologia , Receptores de IgG , Dióxido de Silício
6.
Leuk Res ; 22(4): 379-82, 1998 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-9669843

RESUMO

The clonal cells of patients with B-chronic lymphocytic leukaemia (B-CLL)--which essentially reside in a resting configuration--are characterised by a relative refractoriness to the normal signals for B cell growth and differentiation. Previously it has been shown that, using an in vitro culture system where CD40 is hyper-crosslinked by monoclonal antibody (mAb) held on CD32-transfected mouse L cells, the clonal block in B-CLL cells can be released with a resultant high rate of DNA synthesis ensuing. In the present study, we report that such release can be achieved purely with soluble reagents whereby co-operative epitopes on CD40 are targeted by the combined use of mAb and soluble recombinant CD40L. Substantial levels of DNA synthesis were induced under such conditions in 7/18 patients using CD40-targeted reagents alone and in 16/18 patients in the additional presence of interleukin 4. Possible extrapolation of these findings to novel therapeutic modalities could be envisaged.


Assuntos
Antígenos CD40/imunologia , Epitopos/imunologia , Leucemia Linfocítica Crônica de Células B/imunologia , Leucemia Linfocítica Crônica de Células B/patologia , Anticorpos Monoclonais/farmacologia , Linfócitos B/citologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Antígenos CD40/metabolismo , Ligante de CD40 , Ciclo Celular , Células Clonais/citologia , Células Clonais/efeitos dos fármacos , Células Clonais/imunologia , DNA/biossíntese , DNA/efeitos dos fármacos , Sinergismo Farmacológico , Feminino , Humanos , Interleucina-4/farmacologia , Ligantes , Masculino , Glicoproteínas de Membrana/farmacologia , Proteínas Recombinantes/farmacologia , Solubilidade
7.
Leuk Res ; 14(11-12): 1007-17, 1990.
Artigo em Inglês | MEDLINE | ID: mdl-2280600

RESUMO

Fractionation of mononuclear cells from human fetal liver provides a cell population at early stages of myeloid differentiation which, when cultured, generates neutrophils and macrophages for up to a month. These studies describe the further purification of an undifferentiated myeloid blast cell population by rosette sedimentation of unwanted cells, after coating these cells with monoclonal antibodies which identify macrophages and erythroblasts. In culture, the purified blast cells generated only neutrophils and macrophages. When treated with 10 nM PMA, 62% of the purified cells were induced to differentiate towards macrophages within 48 h. PMA-induced cells acquired morphological features of macrophages and synthesized alpha-naphthyl acetate esterase. The differentiation of the remaining blast cells towards neutrophils, seen in untreated cultures, was completely inhibited by PMA, as revealed by the absence of increases in the numbers of cells expressing lactoferrin and an antigen which appears at the promyelocyte stage of differentiation. Thus, PMA effects intracellular changes which both promote monopoiesis and inhibit granulopoiesis, suggesting a reciprocal interaction between intracellular processes which regulate the capacity for the two pathways of maturation. The purified blast cell population provides a good model system for studies of molecular events which regulate the expression of macrophage characteristics.


Assuntos
Hematopoese/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Monócitos/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Acetato de Tetradecanoilforbol/farmacologia , Diferenciação Celular/efeitos dos fármacos , Feto , Células-Tronco Hematopoéticas/citologia , Humanos , Fígado/citologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Monócitos/citologia , Neutrófilos/citologia
8.
Adv Exp Med Biol ; 406: 139-44, 1996.
Artigo em Inglês | MEDLINE | ID: mdl-8910680

RESUMO

B lymphocytes are subject to selection within germinal centers following somatic hyper-mutation on immunoglobulin variable region genes based on their ability to bind antigen with high affinity. Non-selected cells die by apoptosis. Tumors with features of germinal center B cells include follicular center cell lymphoma and Burkitt's lymphoma. We have used the latter extensively as a neoplastic model of germinal center cells and have compared directly the behaviour of cell lines derived from biopsy material with that of the normal counterparts. Here we describe some of our findings in the two systems with regard to signals regulating survival and apoptosis.


Assuntos
Apoptose/imunologia , Linfócitos B/imunologia , Transdução de Sinais/imunologia , Animais , Linfoma de Burkitt/patologia , Sobrevivência Celular/imunologia , Centro Germinativo/imunologia , Humanos , Células Tumorais Cultivadas
9.
Cell Death Differ ; 20(5): 698-708, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23392124

RESUMO

Cells dying by apoptosis are normally cleared by phagocytes through mechanisms that can suppress inflammation and immunity. Molecules of the innate immune system, the pattern recognition receptors (PRRs), are able to interact not only with conserved structures on microbes (pathogen-associated molecular patterns, PAMPs) but also with ligands displayed by apoptotic cells. We reasoned that PRRs might therefore interact with structures on apoptotic cells - apoptotic cell-associated molecular patterns (ACAMPs) - that are analogous to PAMPs. Here we show that certain monoclonal antibodies raised against the prototypic PAMP, lipopolysaccharide (LPS), can crossreact with apoptotic cells. We demonstrate that one such antibody interacts with a constitutively expressed intracellular protein, laminin-binding protein, which translocates to the cell surface during apoptosis and can interact with cells expressing the prototypic PRR, mCD14 as well as with CD14-negative cells. Anti-LPS cross reactive epitopes on apoptotic cells colocalised with annexin V- and C1q-binding sites on vesicular regions of apoptotic cell surfaces and were released associated with apoptotic cell-derived microvesicles (MVs). These results confirm that apoptotic cells and microbes can interact with the immune system through common elements and suggest that anti-PAMP antibodies could be used strategically to characterise novel ACAMPs associated not only with apoptotic cells but also with derived MVs.


Assuntos
Anticorpos Monoclonais/imunologia , Apoptose/imunologia , Lipopolissacarídeos/imunologia , Receptores de Reconhecimento de Padrão/imunologia , Proteínas de Fase Aguda/imunologia , Animais , Anexina A5/metabolismo , Células COS , Proteínas de Transporte/imunologia , Linhagem Celular , Chlorocebus aethiops , Complemento C1q/metabolismo , Epitopos/imunologia , Células HEK293 , Humanos , Imunidade Inata , Laminina/metabolismo , Receptores de Lipopolissacarídeos , Glicoproteínas de Membrana/imunologia , Estrutura Molecular , Septinas/imunologia
11.
Immunol Today ; 12(12): 429-31, 1991 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-1686171

RESUMO

It is becoming increasingly clear that regulation of MHC antigen expression by viruses and oncogenes, leading to either immune evasion or autoimmunity, is widespread and important in disease. At a recent meeting*, which brought together workers interested in tumour immunology, viral infection and the MHC, a number of mechanisms for the regulation of MHC antigen expression were revealed and the importance of balanced expression of MHC gene products to effective immunity was underlined.


Assuntos
Regulação da Expressão Gênica , Antígenos de Histocompatibilidade/biossíntese , Complexo Principal de Histocompatibilidade , Oncogenes , Viroses/imunologia , Animais , Antígenos de Neoplasias/biossíntese , Antígenos de Neoplasias/genética , Moléculas de Adesão Celular/biossíntese , Moléculas de Adesão Celular/genética , Transformação Celular Neoplásica/genética , Transformação Celular Viral , Citocinas/fisiologia , Regulação Neoplásica da Expressão Gênica , Regulação Viral da Expressão Gênica , Antígenos HLA/biossíntese , Antígenos HLA/genética , Antígenos de Histocompatibilidade/genética , Humanos , Vírus Oncogênicos/genética , Vírus Oncogênicos/fisiologia , Fenômenos Fisiológicos Virais , Vírus/genética
12.
Blood ; 89(3): 919-28, 1997 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-9028323

RESUMO

The ability to maintain germinal center (GC) B cells in culture should facilitate studies on the molecular and cellular events which accompany affinity maturation and the generation of memory in T-dependent responses. We have investigated the ability of cytokines to maintain human tonsillar GC B cells (IgD-/CD39-/CD38+/CD77+) in the "CD40 culture system". In the absence of added cytokines, CD40 monoclonal antibody held on CD32-transfected L cells effectively sustained DNA synthesis in GC B cells for a maximum 3 to 4 days. Of the following cytokines (interleukin-1 beta [IL-1 beta], IL-2, IL-3, IL-4, IL-6, IL-7, IL-10, and stem cell factor), only IL-2 and IL-4 provided a significant enhancement to DNA synthesis in the CD40 culture system; this was modest and short-term. Following a study on the cooperative activity between pairs of cytokines, triple combinations were identified that could maintain high levels of GC B-cell stimulation for at least 10 days. IL-10 was a common component of these synergistic cytokine cocktails, which were IL-10 + IL-4 + IL-7; IL-10 + IL-3 + IL-7; IL-10 + IL-1 beta + IL-2; IL-10 + IL-1 beta + IL-3, and IL-10 + IL-3 + IL-6. Culture of GC B cells with these cytokine combinations resulted in a net increase in viable cell numbers of 50% to 100% whereas total cell numbers increased up to fourfold. Cells recovered from these cultures retained a GC B-cell phenotype with a significant proportion being CD38+/CD44-, features characteristic of centroblasts. Studies with metabolically inactive CD32-L cells supported a role for stromal cell-derived soluble factors in maintaining GC B cells in vitro.


Assuntos
Linfócitos B/citologia , Linfócitos B/imunologia , Centro Germinativo/citologia , Centro Germinativo/imunologia , Animais , Anticorpos Monoclonais/farmacologia , Linfócitos B/efeitos dos fármacos , Antígenos CD40/imunologia , Antígenos CD40/fisiologia , Células Cultivadas , Citocinas/farmacologia , Replicação do DNA/efeitos dos fármacos , Replicação do DNA/imunologia , Combinação de Medicamentos , Sinergismo Farmacológico , Centro Germinativo/efeitos dos fármacos , Humanos , Interleucina-10/farmacologia , Células L/metabolismo , Camundongos , Receptores de IgG/genética , Receptores de IgG/fisiologia , Transfecção
13.
Eur J Immunol ; 23(5): 1165-8, 1993 May.
Artigo em Inglês | MEDLINE | ID: mdl-7682959

RESUMO

Cross-linking of surface Ig (sIg) on resting B cells can generate intracellular signals; however, for T-dependent antigens to promote growth and differentiation additional surface receptors must be engaged. Ligation of CD40 can stimulate B cell proliferation in the presence of interleukin-4. A recently identified counterstructure for CD40 is found on T helper cells and is believed to represent the cognate ligand for B cell activation. This study investigates the role of CD40 as an accessory molecule in sIg-dependent B cell activation. Simultaneous ligation of sIg and CD40 by monoclonal antibodies (mAb) in the presence of mouse L cells which express human Fc gamma receptor type II (Fc gamma RII-L cells) results in potent stimulation of small resting B cells. When CD40 is co-ligated, picomolar concentrations of mouse IgG1 anti-mu, and anti-delta mAb can stimulate B cell proliferation. This requires interaction of the anti-Ig mAb with the Fc gamma RII-L cells: a mouse IgG2a anti-mu mAb which is not recognized by Fc gamma RII, was > or = 1000-fold less effective. These findings suggest a mechanism for B cell activation whereby engagement of T cells via CD40 and its cognate ligand provides potent enhancement of signals delivered through sIg. Based on these observations, models for the activation of B cells by T-dependent antigens are presented.


Assuntos
Antígenos CD/fisiologia , Antígenos de Diferenciação de Linfócitos B/fisiologia , Linfócitos B/imunologia , Ativação Linfocitária , Receptores de Antígenos/fisiologia , Anticorpos Monoclonais/imunologia , Antígenos CD40 , Humanos , Interleucina-4/farmacologia , Receptores de Antígenos de Linfócitos B/imunologia , Receptores de IgG/fisiologia , Linfócitos T/imunologia
14.
Immunol Rev ; 163: 59-76, 1998 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-9700502

RESUMO

The Fc region of human IgG expresses interaction sites for many effector ligands. In this review the topographical distributions of ten of these sites are discussed in relation to functional requirement. It is apparent that interaction sites localised to the inter-CH2-CH3 domain region of the Fc allow for functional divalency, whereas sites localised to the hinge proximal region of the CH2 domain are functionally monovalent, with expression of the latter sites being particularly dependent on glycosylation. All x-ray crystal structures for Fc and Fc-ligand complexes report that the protein structure of the hinge proximal region of the CH2 domain is "disordered", suggesting "internal mobility". We propose a model in which such "internal mobility" results in the generation of a dynamic equilibrium between multiple conformers, certain of which express interaction sites specific to individual ligands. The emerging understanding of the influence of oligosaccharide/protein interactions on protein conformation and biological function of IgG antibodies suggests a potential to generate novel glycoforms of antibody molecules having unique profiles of effector functions.


Assuntos
Fragmentos Fc das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/fisiologia , Imunoglobulina G/química , Imunoglobulina G/fisiologia , Conformação Proteica , Animais , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Configuração de Carboidratos , Proteínas de Transporte/metabolismo , Glicosilação , Humanos , Fragmentos Fc das Imunoglobulinas/metabolismo , Imunoglobulina G/metabolismo , Ligantes , Lectinas de Ligação a Manose , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Receptores de IgG/metabolismo , Fator Reumatoide/metabolismo
15.
J Rheumatol ; 12(3): 432-6, 1985 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-3930719

RESUMO

IgG rheumatoid factors (RF) may play an important role in the pathogenesis of rheumatoid arthritis (RA). Our study investigates the relationship between class specific RF levels measured by radioimmunoassay and disease activity in patients with RA undergoing chrysotherapy. Nineteen patients were treated with 20 mg disodium aurothiomalate weekly for 6 months. Rheumatoid disease activity was assessed before and after 6 months' treatment and the level of IgG, IgA and IgM RF measured. There were significant falls in disease activity (p less than 0.005), IgA RF (p less than 0.005) IgG RF (p less than 0.005) and SCAT (p less than 0.025), but not IgM RF, over the 6 month treatment period. No correlation was found between absolute levels of IgA, IgG or IgM RF and disease activity before or after 6 months' therapy but there was a highly significant linear correlation between reduction in IgG RF levels and fall in disease activity (r = 0.642, p less than 0.005) with treatment.


Assuntos
Artrite Reumatoide/imunologia , Fator Reumatoide/classificação , Adulto , Artrite Reumatoide/tratamento farmacológico , Feminino , Tiomalato Sódico de Ouro/uso terapêutico , Humanos , Imunoglobulina A/metabolismo , Imunoglobulina G/metabolismo , Imunoglobulina M/metabolismo , Masculino , Pessoa de Meia-Idade , Fator Reumatoide/metabolismo , Fatores de Tempo
16.
J Rheumatol ; 12(3): 427-31, 1985 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-4045841

RESUMO

A simple radioimmunoassay suitable for the routine estimation of IgG rheumatoid factors (IgG RF) has been developed. This involves determination of the amount of IgG RF binding to highly purified human IgG Fc on microtiter plates using a radiolabelled F(ab')2 preparation of an antihuman IgG Fd antiserum. A correction for nonspecific binding is made by subtracting sample reactivity with bovine serum albumin. Significantly elevated IgG RF levels were found only in patients with symmetrical peripheral erosive polyarthritis and in some patients with "mixed connective tissue disease."


Assuntos
Artrite Reumatoide/diagnóstico , Imunoglobulina G/análise , Radioimunoensaio/métodos , Fator Reumatoide/análise , Adulto , Artrite Reumatoide/imunologia , Humanos , Fragmentos Fc das Imunoglobulinas , Pessoa de Meia-Idade , Doença Mista do Tecido Conjuntivo/diagnóstico , Doença Mista do Tecido Conjuntivo/imunologia
17.
Ann Rheum Dis ; 45(8): 684-90, 1986 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-3488715

RESUMO

Four assays of rheumatoid factor (RF) have been measured on serum from 213 individuals from 13 families containing at least two sufferers from classical or definite rheumatoid arthritis (RA). Families were not uniformly RF positive or negative, and there was no evidence that non-RA RF positivity was inherited. Four individuals developed definite RA over a two year period, showing that the family members were at increased risk of RA. IgG RF and latex RF assays predicted the RA in the four cases. An association of RF positivity in RA with DR4 was observed, but this may be related to disease severity.


Assuntos
Artrite Reumatoide/diagnóstico , Fator Reumatoide/análise , Artrite Reumatoide/genética , Artrite Reumatoide/imunologia , Reações Falso-Positivas , Antígeno HLA-DR4 , Antígenos de Histocompatibilidade Classe II/análise , Humanos , Prognóstico
18.
J Pathol ; 159(2): 143-9, 1989 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-2530324

RESUMO

The resting human microglia have previously been shown to be cells of dendritic morphology expressing class II MHC antigens and macrophage specific antigens by immunocytochemical techniques. To examine the relationship between the microglia and the family of dendritic antigen presenting cells (APC), normal white matter from eight normal adults with no neurological disease at autopsy was examined by immunocytochemical techniques to localize antibodies to leukocyte common antigen (LCA), HLA-DR, CD1 (T6), CD4 (T4), and glial fibrillary acidic protein. In addition, enzyme histochemical staining for ATPase, non-specific esterase (NSE), and acid phosphatase (ACP) was performed. The normal microglia are ATPase +ve, NSE -ve, ACP -ve, HLA-DR +ve, LCA +ve, CD1 (T6) +ve and weakly CD4 (T4) +ve. This specialized phenotype closely resembles that of Langerhans cells and suggests that microglia are not simply quiescent phagocytes, but may have a primary role as microenvironmentally specialized APC. The finding of weak anti-CD4 (T4) immunoreactivity supports suggestions for a central role for this cell in infection of the central nervous system by human immunodeficiency virus type 1.


Assuntos
Células Dendríticas , Neuroglia/fisiologia , Lobo Temporal/citologia , Idoso , Idoso de 80 Anos ou mais , Antígenos de Diferenciação , Antígenos HLA-DR/análise , Antígenos de Histocompatibilidade , Humanos , Antígenos Comuns de Leucócito , Masculino , Glicoproteínas de Membrana/análise , Pessoa de Meia-Idade , Neuroglia/imunologia , Proteína Tirosina Fosfatase não Receptora Tipo 1
19.
Immunology ; 101(2): 210-7, 2000 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-11012774

RESUMO

Affinity maturation of antibody responses depends on somatic hypermutation of the immunoglobulin V genes. Hypermutation is initiated specifically in proliferating B cells in lymphoid germinal centres but the signals driving this process remain unknown. This study identifies signals that promote V gene mutation in human germinal centre (GC) B cells in vitro. Single GC B cells were cultured by limiting dilution to allow detection of mutations arising during proliferation in vitro. Cells were first cultured in the presence of CD32L cell transfectants and CD40 antibody (the 'CD40 system') supplemented with combinations of cytokines capable of supporting similar levels of CD40-dependent GC B-cell growth [interleukin (IL)-10 + IL-1beta + IL-2 and IL-10 + IL-7 + IL-4]. Components of the 'EL4 system' were then added to drive differentiation, providing sufficient immunoglobulin mRNA for analysis. Analysis of VH3 genes from cultured cells by reverse transcription-polymerase chain reaction (RT-PCR)-based single-strand conformation polymorphism indicated that the combination IL-10 + IL-1beta + IL-2 promoted active V gene mutation whereas IL-10 + IL-7 + IL-4 was ineffective. This was confirmed by sequencing which also revealed that the de novo generated mutations were located in framework and complementarity-determining regions and shared characteristics with those arising in vivo. Somatic mutation in the target GC B-cell population may therefore be actively cytokine driven and not simply a consequence of continued proliferation. The experimental approach we describe should facilitate further studies of the mechanisms underlying V gene hypermutation.


Assuntos
Linfócitos B/imunologia , Genes de Imunoglobulinas , Centro Germinativo/imunologia , Região Variável de Imunoglobulina/genética , Mutação/imunologia , Técnicas de Cultura de Células , Humanos , Interleucinas/imunologia , Polimorfismo Conformacional de Fita Simples , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/imunologia
20.
Cell Immunol ; 175(2): 141-9, 1997 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-9023419

RESUMO

In order to define an in vitro culture system allowing growth of single human germinal center B cells (GC-B), we have studied the proliferation and differentiation of human tonsillar GC-B, and subsets thereof, when cultured together with murine EL-4 thymoma cells in the "EL-4 system." The cells were analyzed and compared to resting tonsillar B cells with respect to phenotypic changes, proliferation, Ig secretion, intracellular Ig levels, and growth abilities under limiting dilution conditions. It was found that GC-B differentiated terminally to Ig-secreting cells with the phenotypic features of plasma cells in a similar manner to tonsillar resting B cells. The GC-B proliferated for 4-5 days, followed by a loss of GC-B phenotype and an increase in intracellular immunoglobulin levels. Over a 10-day culture period a larger proportion of the Ig produced by GC-B was IgG and IgA, as compared to resting B cells, indicating that these cells switched isotype more easily or had already switched in the germinal center prior to the culture period. Analysis of frequencies of Ig-producing cells revealed that 1/3.8 of GC-B and less than 1/10 of the centroblast B cell subpopulation (CB-B) differentiated toward Ig-producing cells when cultured in the EL-4 system whereas 1/1.25 and 1/1.5 of peripheral blood B cells (PBL-B) and resting tonsillar B cells did so, respectively. Taken together, these findings show that tonsillar GC-B differentiate in a similar manner to resting B cells when cultured in the EL-4 system, and we conclude that these conditions allow manipulation of GC-B in single cell cultures in vitro.


Assuntos
Linfócitos B/citologia , Diferenciação Celular , Centro Germinativo/citologia , Animais , Sobrevivência Celular , Células Cultivadas , Técnicas de Cocultura , Humanos , Imunofenotipagem , Camundongos , Tonsila Palatina/citologia , Timidina/metabolismo , Células Tumorais Cultivadas
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