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1.
Am J Transplant ; 13(12): 3103-13, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24168235

RESUMO

Antithymocyte globulin (ATG) preparations are used for treatment and prevention of graft rejection episodes, graft versus host disease and aplastic anemia. The immunomodulatory and immuosuppressive properties of ATGs are mediated by their interaction with a large variety of antigens expressed on immune and nonimmune cell populations. We have conducted a comprehensive analysis on antibody specificities contained in rabbit ATGs in clinical use, ATG-Fresenius (ATG-F) and Thymoglobulin (THG). We have used retroviral expression cloning to identify novel ATG antigens and demonstrate that together with ATG antigens described earlier, these molecules account for the majority of ATG antibodies directed to human cells. Moreover, we have employed cell lines engineered to express antigens at high levels to quantify the antibodies directed to each ATG antigen. We have used cell lines expressing the T cell receptor complex, CD2 and CD28 to remove antibodies to these antigens from ATG preparations and demonstrate that this treatment abrogated the ability of ATGs to induce activation and forkhead box P3 expression in T cells. Comprehensive information and differences on the antigens targeted by ATG-F and THG as well as novel approaches to assess their functional properties are the basis for a better understanding of their immunomodulatory capacities and might eventually translate into improved ATG-based regimen.


Assuntos
Soro Antilinfocitário/química , Linfócitos T/imunologia , Animais , Anticorpos/química , Especificidade de Anticorpos , Antígenos CD2/metabolismo , Antígenos CD28/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Biblioteca Gênica , Rejeição de Enxerto/prevenção & controle , Humanos , Terapia de Imunossupressão , Imunossupressores/química , Leucócitos Mononucleares/citologia , Coelhos , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/efeitos dos fármacos
2.
Biochim Biophys Acta ; 1800(4): 430-8, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20036718

RESUMO

BACKGROUND: Characterization of gene products originating from undefined open reading frames and delineation of biological functions has become the task after the human genome has been decoded. METHODS: We cloned the human C20orf 116 and defined its transcript in liver, kidney and various brain regions by Northern analysis. Antibodies against recombinant protein used for immunofluorescence and immunoblots confirmed its expression in these tissues. With the focus on kidney, its tubular expression and presence in glomerula were shown. RESULTS: A 28 aa long signal peptide predicted by in silico analysis is reflected by visualization of size variants of approximately 3kDa difference suggesting a signal peptidase cleavage of the proform. Cell compartment separation confirmed the presence of Dashurin in peroxisomes/mitochondria, microsomes, cytosol and nucleus. This is in line with green fluorescent protein (GFP)-Dashurin fusion protein shuttling between cytosol and nucleus. Luciferase reporter studies revealed a 2-3 fold increase of promoter activities upon over-expression. Bioinformatic analysis identified a PCI-domain at the C-terminus providing protein-protein interaction capabilities. CONCLUSION: Our present findings suggest the involvement of Dashurin in gene transcription or mRNA translation. GENERAL SIGNIFICANCE: Dashurin shares the PCI-domain with three multisubunit protein complexes (26S proteasome, COP9 signalosome and eIF3 translation initiation factor).


Assuntos
Cromossomos Humanos Par 20/genética , Genoma Humano , Rim/fisiologia , Fases de Leitura Aberta , Complexo de Endopeptidases do Proteassoma/genética , Proteínas/genética , Proteínas Adaptadoras de Transdução de Sinal , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , Mapeamento Cromossômico , Clonagem Molecular/métodos , Proteínas de Fluorescência Verde/genética , Humanos , Leucemia Mieloide Aguda/genética , Neoplasias Hepáticas/genética , Luciferases/genética , Biossíntese de Proteínas , RNA Mensageiro/genética , Proteínas Recombinantes de Fusão/genética , Transcrição Gênica , Transfecção , Translocação Genética
3.
Am J Transplant ; 11(1): 138-45, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21199353

RESUMO

The polyclonal rabbit antithymocyte globulins (ATGs), Thymoglobulin and ATG-Fresenius S, are widely used for prevention and therapy of allograft rejection and graft versus host disease. Dendritic cells (DC) govern immune responses and thus the interaction of ATGs with these cells could potentially contribute to the clinical effects of ATG therapy. Currently there is little information on the DC-antigens targeted by ATGs. In this study we have used a new methodology to identify DC surface antigens recognized by ATGs. By screening an eukaryotic expression library generated from DC with ATGs we could identify several novel ATG antigens including CD81, CD82, CD98, CD99 and CD147. Furthermore, we engineered cells to express previously described ATG antigens and probed them with Thymoglobulin and ATG-Fresenius S. Our results demonstrated strong binding to some but not all of these molecules. We show that previously described antigens and antigens identified in this study account for around 80% of the DC reactivity of ATGs. Analysis of molecules induced by ATG-DC interaction are more in support for an activation of these cells by ATGs than for a specific induction of a tolerogenic DC phenotype.


Assuntos
Antígenos CD/imunologia , Soro Antilinfocitário/imunologia , Células Dendríticas/imunologia , Animais , Soro Antilinfocitário/uso terapêutico , Humanos , Camundongos , Coelhos
4.
J Exp Med ; 163(3): 654-64, 1986 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-2419470

RESUMO

We observed that certain E-receptor antibodies (CD2 antibodies) can induce proliferation of resting human T cells in the presence of PMA, while other CD2 antibodies fail to have such an effect. The same CD2 antibodies that were mitogenic in the presence of PMA (9.6, X11, VIT13), but not the nonreactive ones, were also able to induce T cell proliferation via the so-called alternative pathway of T cell activation, i.e., when added pairwise in certain combinations to T cells in the absence of PMA. While the simultaneous addition of two comitogenic CD2 antibodies (9.6 or X11 plus VIT13) or the addition of a single nonmitogenic CD3 antibody (VIT3) led to a clearcut elevation of intracellular Ca++ levels, no such effect could be observed after the addition of one CD2 antibody alone. Even in the presence of PMA, one comitogenic CD2 antibody alone was unable to trigger a significant Ca++ response, although this combination induced a proliferative response. These data indicate that, distinguishable by their influence on free cytoplasmic Ca++, there are two different mechanisms of T cell activation via CD2. While simultaneous triggering with two antibodies leads to cell proliferation preceded by an increase of Ca++ levels, stimulation with one antibody plus PMA results in proliferation without a measurable early Ca++ response. We conclude that T cells treated by certain CD2 antibodies alone already recognize an activation signal probably unrelated to Ca++ homeostasis, a signal that can further be developed by PMA to result in a completely developed proliferative response.


Assuntos
Proteínas de Transporte/imunologia , Ativação Linfocitária , Forbóis/farmacologia , Receptores de Antígenos de Linfócitos T/imunologia , Receptores Imunológicos/imunologia , Linfócitos T/imunologia , Acetato de Tetradecanoilforbol/farmacologia , Anticorpos Monoclonais/imunologia , Complexo Antígeno-Anticorpo , Antígenos CD2 , Cálcio/metabolismo , Citoplasma/metabolismo , Epitopos , Humanos , Receptores Imunológicos/fisiologia , Formação de Roseta , Linfócitos T/fisiologia
5.
J Exp Med ; 184(5): 1769-79, 1996 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-8920865

RESUMO

Resting T cells spontaneously adhere in a selective manner to potent accessory cells, such as dendritic cells (DC) and lymphoblastoid B blasts (LCL). Here we demonstrate that leukosialin (CD43) and major histocompatibility complex class I molecules (MHC-I) might play a critical role in this process. T cell conjugate formation with monocyte-derived DC (md-DC) and LCL could be strongly inhibited by either preincubating T cells with Fab fragments of CD43 monoclonal antibody (mAb) 6F5 or by preincubating md-DC or LCL with MHC-I mAb W6/32. Intact CD43 mAb 6F5, in contrast to monovalent Fab fragments, enhanced T cell adhesiveness by transactivating CD2 binding to CD58 molecules. Interestingly, induction of this proadhesive signal via CD43 with intact 6F5 mAb was found to revert mAb W6/32-mediated inhibition of T cell conjugate formation. These observations indicated that CD43 cross-linkage mimics and monovalent mAb 6F5 inhibits interaction of T cell CD43 with a stimulatory ligand on opposing cells, presumably MHC-I. For the demonstration of direct physical interaction between CD43 on T cells and MHC-I-coated beads it was necessary, however, to ligate CD2 on T cells with a stimulatory pair of CD2 mAbs (VIT13 plus TS2/18). This suggests that CD2 ligation crosswise upregulates CD43 binding avidity for MHC-I and that both adhesion molecule pairs (CD43/MHC-I and CD2/CD58) act in concert to induce and mediate T cell conjugate formation with certain cell types.


Assuntos
Células Apresentadoras de Antígenos/imunologia , Antígenos CD , Adesão Celular , Antígenos de Histocompatibilidade Classe I/imunologia , Complexo Principal de Histocompatibilidade/imunologia , Sialoglicoproteínas/imunologia , Linfócitos T/imunologia , Linfócitos B/imunologia , Antígenos CD2/metabolismo , Linhagem Celular , Células Dendríticas/imunologia , Células-Tronco Hematopoéticas/imunologia , Humanos , Leucossialina , Sialoglicoproteínas/genética , Ativação Transcricional
6.
J Exp Med ; 159(6): 1784-9, 1984 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-6202820

RESUMO

As opposed to normal human skin where HLA-DR expression is restricted to the Langerhans cell (LC) population, HLA-DR, but not HLA-DS antigens can be readily detected on keratinocytes (KC) in certain disease states, i.e., cutaneous T cell lymphoma (CTCL), graft-vs-host disease (GVHD), and lichen planus (LP). To clarify the cellular origin of KC-bound HLA-DR antigens, we used a monoclonal antibody directed against determinants solely expressed on the cytoplasmic HLA-DR gamma chain (VIC-Y1) and observed that, by immunofluorescence, KC displaying HLA-DR alpha/beta complexes on their surface uniformly displayed cytoplasmic VIC-Y1 reactivity. In view of the crucial role of the gamma chain for HLA-DR biosynthesis, we conclude that HLA-DR antigens on KC are actively synthesized by these cells.


Assuntos
Epiderme/imunologia , Antígenos de Histocompatibilidade Classe II/análise , Queratinas/metabolismo , Dermatopatias/imunologia , Imunofluorescência , Doença Enxerto-Hospedeiro/complicações , Antígenos HLA-DR , Humanos , Líquen Plano/imunologia , Linfoma/complicações , Dermatopatias/etiologia , Linfócitos T
7.
J Exp Med ; 171(5): 1431-42, 1990 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-2139695

RESUMO

We present here the molecular characterization of a new activation-induced surface structure on human T lymphocytes, termed LA45, with high homology (93% at protein level) to MHC class I molecules. Antigen modulation and sequential immunoprecipitation experiments revealed that LA45 and HLA class I proteins do not crossreact with the corresponding antibodies. Furthermore, LA45 is not associated with beta 2-m. On the other hand, we could show that the separation of HLA-A,B,C and beta 2m molecules, induced by SDS-denaturation, leads to a conformational change in the heavy chain in such a way that it becomes reactive with LA45. The 90/45 kD LA45 proteins thus appear to be non-beta 2m-associated MHC class I alpha chains that are selectively expressed by activated but not by resting human T lymphocytes.


Assuntos
Antígenos CD/análise , Antígenos de Histocompatibilidade Classe I/análise , Ativação Linfocitária , Linfócitos T/imunologia , Microglobulina beta-2/análise , Sequência de Aminoácidos , Anticorpos Monoclonais , Antígenos de Diferenciação de Linfócitos T/análise , Sequência de Bases , Western Blotting , Complexo CD3 , Células Cultivadas , Clonagem Molecular , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Substâncias Macromoleculares , Dados de Sequência Molecular , Receptores de Antígenos de Linfócitos T/análise , Homologia de Sequência do Ácido Nucleico
8.
J Exp Med ; 181(4): 1381-90, 1995 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-7535337

RESUMO

The glycosylphosphatidylinositol (GPI)-anchored membrane protein urokinase plasminogen activator-receptor (uPA-R; CD87) is one of the key molecules involved in migration of leukocytes and tumor cells. uPA bound to uPA-R provides the cell proteolytic potential used for degradation of extracellular matrix. uPA-R is also involved in induction of cell adhesion and chemotaxis. Here, we provide a molecular explanation for these uPA-R-related cellular events. By size fractionation of monocyte lysate and affinity isolation on its natural ligand uPA, we demonstrate uPA-R as a component of a receptor complex of relatively large size. Reprecipitation and immunoblotting techniques allowed us to detect the protein tyrosine kinases (PTKs) p60fyn, p53/56lyn, p58/64hck, and p59fgr as components of this "uPA-R complex". Activation of monocytes even with enzymatically inactivated uPA resulted in induction of tyrosine phosphorylation, suggesting modulation of uPA-R-associated PTKs upon ligand binding. In spite of their presence in large complexes, we did not find the GPI-linked proteins CD14, CD58, and CD59 in the uPA-R complex, which indicates the presence of different receptor domains containing GPI-linked proteins in monocytes. However, we identified the leukocyte integrins LFA-1 and CR3 as components of the uPA-R complex as indicated by coisolation of these molecules, as well as by cocapping and comodulation of uPA-R and leukocyte integrins on the monocyte surface. The assemblage of uPA-R, PTKs and membrane spanning beta 2-integrins in one receptor complex indicates functional cooperation. In regard to the involvement of these molecules in pericellular proteolysis, signal transduction, as well as adhesion and chemotactic movement, we suggest uPA-R complex as a potential cellular device for cell migration.


Assuntos
Integrinas/análise , Monócitos/química , Complexos Multienzimáticos/análise , Receptores Proteína Tirosina Quinases/análise , Receptores de Superfície Celular/análise , Transdução de Sinais , Quinases da Família src , Antígenos CD/análise , Antígenos CD18 , Adesão Celular , Movimento Celular , Humanos , Antígeno-1 Associado à Função Linfocitária/análise , Substâncias Macromoleculares , Antígeno de Macrófago 1/análise , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas Tirosina Quinases/análise , Proteínas Proto-Oncogênicas/análise , Proteínas Proto-Oncogênicas c-fyn , Proteínas Proto-Oncogênicas c-hck , Receptores de Ativador de Plasminogênio Tipo Uroquinase
9.
J Exp Med ; 187(7): 1019-28, 1998 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-9529318

RESUMO

Polymorphonuclear granulocytes (PMNs) are thought to fulfill their role in host defense primarily via phagocytosis and release of cytotoxic compounds and to be inefficient in antigen presentation and stimulation of specific T cells. Dendritic cells (DCs), in contrast, are potent antigen-presenting cells with the unique capacity to initiate primary immune responses. We demonstrate here that highly purified lactoferrin-positive immediate precursors of end-stage neutrophilic PMN (PMNp) can be reverted in their functional maturation program and driven to acquire characteristic DC features. Upon culture with the cytokine combination granulocyte/macrophage colony-stimulating factor plus interleukin 4 plus tumor necrosis factor alpha, they develop DC morphology and acquire molecular features characteristic for DCs. These molecular changes include neo-expression of the DC-associated surface molecules cluster of differentiation (CD)1a, CD1b, CD1c, human leukocyte antigen (HLA)-DR, HLA-DQ, CD80, CD86, CD40, CD54, and CD5, and downregulation of CD15 and CD65s. Additional stimulation with CD40 ligand induces also expression of CD83 and upregulates CD80, CD86, and HLA-DR. The neutrophil-derived DCs are potent T cell stimulators in allogeneic, as well as autologous, mixed lymphocyte reactions (MLRs), whereas freshly isolated neutrophils are completely unable to do so. In addition, neutrophil-derived DCs are at least 10,000 times more efficient in presenting soluble antigen to autologous T cells when compared to freshly isolated monocytes. Also, in functional terms, these neutrophil-derived DCs thus closely resemble "classical" DC populations.


Assuntos
Diferenciação Celular/fisiologia , Células Dendríticas/citologia , Neutrófilos/metabolismo , Apresentação de Antígeno/imunologia , Antígenos CD/imunologia , Ligante de CD40 , Contagem de Células , Células Cultivadas , Citometria de Fluxo , Regulação da Expressão Gênica no Desenvolvimento/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Histocitoquímica , Humanos , Interleucina-4/farmacologia , Lactoferrina/metabolismo , Glicoproteínas de Membrana/farmacologia , Neutrófilos/citologia , Fenótipo , Linfócitos T/metabolismo , Fator de Necrose Tumoral alfa/farmacologia
10.
J Exp Med ; 191(6): 937-48, 2000 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-10727456

RESUMO

The specificity of immunoglobulins and alpha/beta T cell receptors (TCRs) provides a framework for the molecular basis of antigen recognition. Yet, evolution has preserved a separate lineage of gamma/delta antigen receptors that share characteristics of both immunoglobulins and alpha/beta TCRs but whose antigens remain poorly understood. We now show that T cells of the major tissue gamma/delta T cell subset recognize nonpolymorphic CD1c molecules. These T cells proliferated in response to CD1+ presenter cells, lysed CD1c+ targets, and released T helper type 1 (Th1) cytokines. The CD1c-reactive gamma/delta T cells were cytotoxic and used both perforin- and Fas-mediated cytotoxicity. Moreover, they produced granulysin, an important antimicrobial protein. Recognition of CD1c was TCR mediated, as recognition was transferred by transfection of the gamma/delta TCR. Importantly, all CD1c-reactive gamma/delta T cells express V delta 1 TCRs, the TCR expressed by most tissue gamma/delta T cells. Recognition by this tissue pool of gamma/delta T cells provides the human immune system with the capacity to respond rapidly to nonpolymorphic molecules on professional antigen presenting cells (APCs) in the absence of foreign antigens that may activate or eliminate the APCs. The presence of bactericidal granulysin suggests these cells may directly mediate host defense even before foreign antigen-specific T cells have differentiated.


Assuntos
Antígenos CD1/metabolismo , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Sequência de Aminoácidos , Anti-Infecciosos/metabolismo , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Antígenos CD1/biossíntese , Antígenos de Diferenciação de Linfócitos T/biossíntese , Sequência de Bases , Diferenciação Celular/imunologia , Linhagem Celular , Testes Imunológicos de Citotoxicidade , Citotoxicidade Imunológica , Rearranjo Gênico da Cadeia delta dos Receptores de Antígenos dos Linfócitos T , Rearranjo Gênico da Cadeia gama dos Receptores de Antígenos dos Linfócitos T , Humanos , Imunidade Inata , Ativação Linfocitária , Glicoproteínas de Membrana/fisiologia , Dados de Sequência Molecular , Perforina , Proteínas Citotóxicas Formadoras de Poros , Receptores de Antígenos de Linfócitos T gama-delta/genética , Receptores de Antígenos de Linfócitos T gama-delta/fisiologia , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/microbiologia , Células Th1/imunologia , Células Th1/metabolismo , Receptor fas/fisiologia
11.
Br J Cancer ; 99(1): 151-9, 2008 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-18594539

RESUMO

To enable detailed analyses of cell interactions in tumour development, new epithelial and mesenchymal cell lines were established from human hepatocellular carcinoma by spontaneous outgrowth in culture. We obtained several hepatocarcinoma (HCC)-, B-lymphoblastoid (BLC)-, and myofibroblastoid (MF)-lines from seven cases. In-depth characterisation included cell kinetics, genotype, tumourigenicity, expression of cell-type specific markers, and proteome patterns. Many functions of the cells of origin were found to be preserved. We studied the impact of the mesenchymal lines on hepatocarcinogenesis by in vitro assays. BLC- and MF-supernatants strongly increased the DNA replication of premalignant hepatocytes. The stimulation by MF-lines was mainly attributed to HGF secretion. In HCC-cells, MF-supernatant had only minor effects on cell growth but enhanced migration. MF-lines also stimulated neoangiogenesis through vEGF release. BLC-supernatant dramatically induced death of HCC-cells, which could be largely abrogated by preincubating the supernatant with TNFbeta-antiserum. Thus, the new cell lines reveal stage-specific stimulatory and inhibitory interactions between mesenchymal and epithelial tumour cells. In conclusion, the new cell lines provide unique tools to analyse essential components of the complex interplay between the microenvironment and the developing liver cancer, and to identify factors affecting proliferation, migration and death of tumour cells, neoangiogenesis, and outgrowth of additional malignancy.


Assuntos
Carcinoma Hepatocelular/fisiopatologia , Comunicação Celular , Neoplasias Hepáticas/fisiopatologia , Animais , Linhagem Celular Tumoral , Células Epiteliais , Humanos , Camundongos , Ratos
12.
J Clin Invest ; 104(7): 957-65, 1999 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-10510336

RESUMO

Human rhinoviruses (HRVs) are the predominant cause of the common cold. Although this disease is per se rather harmless, HRV infection is considered to set the stage for more dangerous pathogens in vivo. Here we demonstrate that HRV-14, a member of the major group HRV family, can efficiently inhibit antigen-induced T-cell proliferation and T-cell responses to allogeneic monocytes. HRV-14 triggered a significant downregulation of MHC class II molecules on monocytes. Moreover, supernatants from monocytes cultured in the presence of HRV-14 strongly reduced the allogeneic T-cell stimulatory property of untreated monocytes and monocyte-derived dendritic cells (md-DCs), whereas Epstein Barr virus-transformed B-lymphoblastoid cells were not sensitive. Analysis of the supernatant revealed that HRV-14 induced the production of significant amounts of the immunosuppressive cytokine IL-10. The important T-cell stimulatory cytokine IL-12 or the proinflammatory cytokines IL-1beta or TNF-alpha were not detected or were only minimally detected. Finally, monocytes pretreated with HRV-14 were greatly inhibited in their production of IL-12 upon stimulation with IFN-gamma/LPS. These observations suggest that altered cytokine production in mononuclear phagocytes upon interaction with HRV downmodulates appropriate immune responses during the viral infection.


Assuntos
Interleucina-10/genética , Monócitos/imunologia , Monócitos/virologia , Rhinovirus/imunologia , Linfócitos T/imunologia , Antígenos CD/análise , Células Cultivadas , Enterotoxinas/imunologia , Regulação da Expressão Gênica/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Células HeLa , Humanos , Interleucina-1/análise , Interleucina-10/biossíntese , Interleucina-4/farmacologia , Cinética , Ativação Linfocitária/efeitos dos fármacos , Muromonab-CD3/farmacologia , Proteínas Recombinantes/farmacologia , Staphylococcus aureus , Linfócitos T/efeitos dos fármacos , Toxoide Tetânico/farmacologia , Fator de Necrose Tumoral alfa/análise
13.
J Natl Cancer Inst ; 73(1): 7-11, 1984 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-6204096

RESUMO

The 3-fucosyl-N-acetyllactosamine structure, a sugar sequence contained in the human milk oligosaccharide lacto-N-fucopentaose III, is recognized by most of the granulocyte-specific monoclonal antibodies (MoAb) reported in the literature, including the six MoAb from our laboratory. Blast cells from patients with acute myeloblastic leukemia (AML) displayed a heterogeneous reaction pattern when they were exposed to MoAb against this moiety, and the proportion of reactive cells in individual cell samples was highly variable. The intensity of the reaction was strongly enhanced by neuraminidase treatment of AML blasts, and reactive structures were exposed on previously negative AML blast cells. Surprisingly, this granulocyte-associated antigen was exposed by desialylation not only on malignant myeloid precursor cells but also on common acute lymphoblastic leukemia cells. No such effect was seen when normal peripheral blood lymphocytes, lymphocytes from patients with chronic lymphatic leukemia, or blast cells from patients with B-cell acute lymphoblastic leukemia, acute erythroid leukemia, and acute megakaryoblastic leukemia were treated with neuraminidase.


Assuntos
Antígenos de Neoplasias/análise , Epitopos/análise , Leucemia Linfoide/imunologia , Antígenos CD15/análise , Oligossacarídeos/análise , Ácidos Siálicos , Animais , Anticorpos Monoclonais , Plaquetas/imunologia , Linhagem Celular , Humanos , Hibridomas/imunologia , Imunoglobulina M/análise , Leucócitos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Neuraminidase , Plasmocitoma/imunologia , Valores de Referência
14.
Leukemia ; 4(4): 278-81, 1990 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-1694941

RESUMO

Within normal hemopoiesis, the intranuclear DNA polymerase TdT seems to be exclusively expressed by T and B lymphoid precursor cells. Double staining experiments showed that TdT can also be expressed in blast cells of certain acute myeloid leukemias. Recent reports described a very strong association between TdT expression and rearrangements of IgH and TcR genes in such AML specimens, suggesting a predominant lymphoid commitment of these TdT positive AML blasts. When submitting 24 serologically and morphologically well-characterized TdT positive AML specimens for additional genotypic analysis to determine the IgH and TcR gene configuration, we observed that only four had clonally rearranged IgH and/or TcR genes, whereas 20 had germ line configuration. This frequency is clearly lower than previously reported and not necessarily different from rearrangement frequencies reported for TdT negative AML (4-40%). It would seem to us, therefore, that the expression of TdT in otherwise well-defined AML blasts is not necessarily associated with a higher frequency of immunoglobulin and/or T cell receptor gene rearrangement.


Assuntos
Antígenos de Diferenciação de Linfócitos T/imunologia , DNA Nucleotidilexotransferase/metabolismo , Rearranjo Gênico do Linfócito T/genética , Cadeias Pesadas de Imunoglobulinas/genética , Leucemia Mieloide Aguda , Receptores de Antígenos de Linfócitos T/genética , Antígenos CD7 , Humanos , Leucemia Monocítica Aguda/enzimologia , Leucemia Monocítica Aguda/genética , Leucemia Monocítica Aguda/imunologia , Leucemia Mieloide Aguda/enzimologia , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/imunologia , Leucemia Mielomonocítica Aguda/enzimologia , Leucemia Mielomonocítica Aguda/genética , Leucemia Mielomonocítica Aguda/imunologia , Leucemia Promielocítica Aguda/enzimologia , Leucemia Promielocítica Aguda/genética , Leucemia Promielocítica Aguda/imunologia , Fenótipo
15.
J Leukoc Biol ; 53(5): 541-9, 1993 May.
Artigo em Inglês | MEDLINE | ID: mdl-8099115

RESUMO

Several carbohydrate structures on human granulocytes have been discussed as potential ligands for C-type lectins (selectins) on endothelial cells. Among them are the lacto-series type II chain antigens sialyl-Lewis(x) (SLe(x), Lewis(x) (Le(x)), and VIM2. We demonstrated in this study that monoclonal antibodies (mAbs) to Le(x) and to SLe(x), but not other anticarbohydrate mAbs (VIM2, CDw17, CD24), can stimulate granulocytes to form homotypic aggregates. This effect was particularly noticeable with three distinct anti-Le(x) mAbs (3C6, 4D1, 6C7). Much less impressive effects were also seen with nine other anti-Le(x) mAbs and with the anti-SLe(x) mAb CSLEX1. Aggregation was shown to be an active process. It is temperature and energy dependent, requires divalent cations, and is selective in terms of mAb specificity. Anti-Le(x)-induced homoaggregate formation could be inhibited with CD11b mAb JML-H11 and CD54 (ICAM1) mAb LB-2 and thus seems to be associated with activation of the beta 2-integrin cytoadhesion pathway.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Antígenos CD/fisiologia , Granulócitos/citologia , Granulócitos/imunologia , Leucócitos/citologia , Antígenos CD15/imunologia , Anticorpos Monoclonais/análise , Especificidade de Anticorpos , Antígenos CD/análise , Antígenos CD/metabolismo , Antígenos CD11 , Antígenos CD18 , Adesão Celular/efeitos dos fármacos , Adesão Celular/imunologia , Moléculas de Adesão Celular/análise , Moléculas de Adesão Celular/metabolismo , Moléculas de Adesão Celular/fisiologia , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Glicolipídeos/análise , Glicolipídeos/imunologia , Glicoproteínas/análise , Glicoproteínas/imunologia , Granulócitos/fisiologia , Humanos , Immunoblotting , Molécula 1 de Adesão Intercelular , Leucócitos/imunologia , Leucócitos/fisiologia , Antígeno de Macrófago 1/análise , Antígeno de Macrófago 1/metabolismo , Antígeno de Macrófago 1/fisiologia , Temperatura
16.
Mol Immunol ; 31(12): 885-93, 1994 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-8065372

RESUMO

Loading of peptides onto DR molecules was studied by characterizing precursors of the mature peptide-DR complexes expressed at the surface of B cells. Since invariant chain (Ii) prevents binding of peptides by DR molecules, it was speculated that analysis of complexes between DR heterodimers and proteolytic fragments of Ii offers the possibility to examine how DR molecules and peptides assemble. Using a procedure combining a two-step affinity chromatography and gel filtration, we isolated from leupeptin-treated B cells complexes between DR molecules and N-terminal Ii fragments previously called "leupeptin-induced polypeptides" (LIP; Blum and Cresswell, 1988, Proc. natn. Acad. Sci. U.S.A. 85, 3975-3979). It was observed that the most prominent LIP fragment has a relative molecular mass (M(r)) of 16 kDa. In addition, we show that this polypeptide species does not bear N-linked glycans, indicating that this fragment does not extend beyond residue 129 of Ii. Similarly to DR alpha beta heterodimers associated with the full length 33 and 35 kDa Ii forms, DR alpha beta heterodimers associated with LIP fragments are unstable in sodium dodecyl sulfate (SDS) at ambient temperature, whereas mature DR alpha beta heterodimers are resistant to dissociation with SDS. These results are indirect evidence that LIP-DR complexes are devoid of bound peptides. This possibility was supported by showing that LIP-DR complexes fail to bind a radioiodinated tetanus toxin peptide (125I-p2), while DR molecules, which are spontaneously released from complexes with LIP fragments, bind the labeled peptide. These results demonstrate that association with LIP fragments is sufficient to prevent binding of peptides by DR molecules. This notion was further documented by showing that binding of 125I-p2 on DR heterodimers is inhibited by preparations of LIP fragment. By contrast, a soluble recombinant fragment corresponding to the extracytoplasmic region of Ii did not block 125I-p2 binding. The results presented in this study indicate that the cytoplasmic and/or transmembrane region of Ii is required to prevent peptide binding by DR molecules, while the extracytoplasmic portion of Ii, though capable of associating with DR molecules, lacks the capacity to block peptide binding.


Assuntos
Antígenos de Diferenciação de Linfócitos B , Antígenos HLA-DR/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Leupeptinas/farmacologia , Sequência de Aminoácidos , Linfócitos B/imunologia , Linhagem Celular Transformada , Dados de Sequência Molecular , Tamanho da Partícula , Fragmentos de Peptídeos/metabolismo , Ligação Proteica/efeitos dos fármacos
17.
Exp Hematol ; 13(11): 1211-6, 1985 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-4065266

RESUMO

In the present study we evaluated the reactivity of monoclonal cytotoxic antibodies directed against myeloid differentiation antigens with hemopoietic precursor cells. VIM-D5 and VIM-2 inhibit the proliferation of clusters and colony formation after seven days of incubation. Day-14 CFU-GM are not affected by these antibodies. After complement-mediated cytolysis with VIM-2, the number of BFU-e was significantly reduced; however, this effect was largely abrogated by addition of leukocyte-conditioned medium to the cultures as an exogenous source of burst-promoting activity. Furthermore, the maturation of myeloid progenitor cells has been examined by delayed treatment with VIM-D5 and complement during the in vitro culture period. In these experiments a different maturation behavior of day-7 and day-14 CFU-GM was demonstrated. To study whether a cryptic carbohydrate structure is present on more immature CFU-GM, the effect of neuraminidase treatment of myeloid progenitor cells on reactivity with VIM-D5 was tested.


Assuntos
Antígenos de Superfície/análise , Hematopoese , Células-Tronco Hematopoéticas/imunologia , Anticorpos Monoclonais , Diferenciação Celular , Células Cultivadas , Eritropoese , Granulócitos/citologia , Granulócitos/imunologia , Humanos , Monócitos/citologia , Monócitos/imunologia , Neuraminidase
18.
Exp Hematol ; 28(5): 575-83, 2000 May.
Artigo em Inglês | MEDLINE | ID: mdl-10812248

RESUMO

OBJECTIVE: Because of its potent immunosuppressive properties in vitro as well as in vivo, we studied the effect of 1,25-dihydroxyvitamin D(3) (calcitriol) on differentiation, maturation, and function of dendritic cells (DC). MATERIALS AND METHODS: Monocyte-derived DCs were generated with GM-CSF plus IL-4, and maturation was induced by a 2-day exposure to TNFalpha. DCs were derived from CD34(+) progenitors using SCF plus GM-CSF plus TNFalpha. For differentiation studies, cells were exposed to calcitriol at concentrations of 10(-)(9)- 10(-7) M at days 0, 6, and 8, respectively. The obtained cell populations were evaluated by morphology, phenotype, and function. RESULTS: When added at day 0, calcitriol blocked DC differentiation from monocytes and inhibited the generation of CD1a(+) cells from progenitor cells while increasing CD14(+) cells. Exposure of immature DCs to calcitriol at day 6 resulted in a loss of the DC-characteristic surface molecule CD1a, downregulation of the costimulatory molecules CD40 and CD80, and MHC class II expression, whereas the monocyte/macrophage marker CD14 was clearly reinduced. In addition, calcitriol hindered TNFalpha-induced DC maturation, which is usually accompanied with induction of CD83 expression and upregulation of costimulatory molecules. In contrast, the mature CD83(+) DCs remained CD1a(+)CD14(-) when exposed to calcitriol. The capacity of cytokine-treated cells to stimulate allogeneic and autologous T cells and to take up soluble antigen was inhibited by calcitriol. CONCLUSION: The potent suppression of DC differentiation, the reversal of DC phenotype, and function in immature DCs, as well as the inhibition of DC maturation by calcitriol, may explain some of its immunosuppressive properties.


Assuntos
Calcitriol/farmacologia , Diferenciação Celular/efeitos dos fármacos , Células Dendríticas/citologia , Monócitos/citologia , Antígenos CD/análise , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Células Dendríticas/efeitos dos fármacos , Endocitose , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Antígenos HLA-D/imunologia , Humanos , Imunofenotipagem , Interleucina-4/farmacologia , Monócitos/efeitos dos fármacos , Valores de Referência
19.
Hum Gene Ther ; 8(14): 1651-8, 1997 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-9322867

RESUMO

A retroviral-vector encoding the low affinity nerve growth factor receptor (LNGFR) was used to transduce dendritic cells (DCs) generated from CD34+ cord blood (CB) progenitor cells under serum-free conditions. Transduction efficiency was monitored by flow cytometry (FACS) using a specific monoclonal antibody. Prior to retroviral infections, CD34+ CB cells were stimulated for 60 h in a serum-free medium containing a DC differentiation inducing cytokine cocktail: stem cell factor (SCF), granulocyte/macrophage-colony stimulating factor (GM-CSF), tumor necrosis factor alpha (TNFalpha), and transforming growth factor beta 1 (TGF-beta1). Addition of flt3-ligand (FL) to the aforementioned growth factors significantly enhanced cell expansion (41.7+/-11.5 fold vs. 22.5+/-4.7 fold without FL) and generation of CD1a+ DCs (mean 45.7+/-9.8% vs. 28+/-6.5% without FL, n = 4,p = 0.01). Furthermore, FL significantly increased the proportion of CD1a+LNGFR+ cells (mean 10%+/-4.4% vs. 6%+/-2.4 without FL n = 4, p = 0.03). When serum-free viral supernatants were used to infect DCs progenitors under entirely serum-free conditions and with the most potent cytokine combination, approximately one-third of the CD1a+ DCs generated co-expressed the LNGFR gene. Moreover, the transduced gene was also identified in more mature CD1a+CD80+ and CD1a+CD86+ DCs after 12-14 days of culture. In addition, transduced CD1a+ DCs maintained their functional properties, stimulating allogeneic T cells with similar efficiency as nontransduced CD1a+ DCs. Thus, the serum-free system described allows efficient generation and transduction of CD1a+ DCs derived from CD34+ progenitor cells and may be very useful for future therapeutic applications of DCs.


Assuntos
Antígenos CD34/análise , Células Dendríticas , Técnicas de Transferência de Genes , Células-Tronco Hematopoéticas , Retroviridae/genética , Antígenos CD1/análise , Células Cultivadas , Meios de Cultura Livres de Soro , Citocinas/farmacologia , Células Dendríticas/química , Sangue Fetal/citologia , Expressão Gênica , Humanos , Ativação Linfocitária , Proteínas de Membrana/farmacologia , Receptores de Fator de Crescimento Neural/genética , Linfócitos T/imunologia
20.
J Invest Dermatol ; 93(5): 600-9, 1989 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-2529318

RESUMO

Freshly isolated murine epidermal Langerhans cells (LC) are weak stimulators of resting T cells. Upon culture their phenotype changes, their stimulatory activity increases significantly, and they come to resemble lymphoid dendritic cells. Resident murine LC, therefore, might represent a reservoir of immature dendritic cells. We have now used enzyme cytochemistry, a panel of some 80 monoclonal antibodies, and immunofluorescence microscopy or two-color flow cytometry, as well as transmission electron microscopy, to analyse the phenotype and morphology of human LC before and after 2-4 d of bulk epidermal cell culture. In addition, LC were enriched from bulk epidermal cell cultures, and their stimulatory capacity was tested in the allogeneic mixed leukocyte reaction and the oxidative mitogenesis assay. Cultured human LC resembled human lymphoid dendritic cells in morphology, phenotype, and function. Specifically, LC became non-adherent upon culture and developed sheet-like processes (so-called "veils"), decreased their surface ATP/ADP'ase activity, and lost nonspecific esterase activity. As in the mouse, surface expression of MHC class I and II antigens increased significantly, and FcII receptors were significantly reduced. Markers that are expressed by dendritic cells (like CD40) appeared on LC following culture. Cultured human LC were potent T-cell stimulators. Our findings support the view that resident human LC, like murine LC, represent immature precursors of lymphoid dendritic cells in skin-draining lymph nodes.


Assuntos
Células Dendríticas/fisiologia , Células de Langerhans/fisiologia , Anticorpos Monoclonais , Antígenos de Diferenciação , Antígenos de Superfície/análise , Moléculas de Adesão Celular/análise , Células Cultivadas , Células Dendríticas/citologia , Células Dendríticas/enzimologia , Citometria de Fluxo , Imunofluorescência , Antígenos de Histocompatibilidade , Humanos , Técnicas In Vitro , Células de Langerhans/citologia , Células de Langerhans/enzimologia , Antígenos Comuns de Leucócito , Ativação Linfocitária , Complexo Principal de Histocompatibilidade , Microscopia Eletrônica , Receptores Fc/análise
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