RESUMO
A small CD3+ T-cell population, that lacks both CD4 and CD8 molecules, defined as double negative (DN), is expanded in the peripheral blood of patients with systemic lupus erythematosus, produces IL-17 and accumulates in the kidney during lupus nephritis. Since IL-17 production is enhanced in salivary gland infiltrates of patients with primary Sjögren's syndrome (pSS), we aimed to investigate whether DN T cells may be involved in the pathogenesis of salivary gland damage. Fifteen patients with SS and 15 normal controls (NC) were enrolled. Peripheral blood mononuclear cells were stimulated with anti-CD3 antibody and cultured in presence or absence of dexamethasone (Dex). Phenotypic characterization was performed by flow cytometry in freshly isolated cells and after culture. Minor salivary glands (MSG) from pSS were processed for immunofluorescence staining. Total circulating DN T cells were increased in pSS compared to NC (4.7±0.4% vs 2.6±0.4%). NC and pSS freshly isolated DN T cells produce consistent amounts of IL-17 (67.7±5.6 in NC vs 69.2±3.3 in pSS). Notably, DN T cells were found in the pSS-MSG infiltrate. Dex was able to down-regulate IL-17 in vitro production in NC (29±2.6% vs 15.2±1.9% vs 13±1.6%) and pSS (49±4.8% vs 16±3.8% vs 10.2±0.8%) conventional Th17 cells and in DN T cells of NC (80±2.8% vs 3.8±2.1% vs 4.2±1.8%), but not of pSS (81±1.5% vs 85.4±0.8% vs 86.2±1.7%). DN T cells are expanded in pSS PB, produce IL-17 and infiltrate pSS MSG. In pSS, conventional Th17 cells are inhibited by Dex, but DN T cells appear to be resistant to this effect. Taken together, these data suggest a key role of this T-cell subset in the perpetuation of chronic sialoadenitis and eventually in pSS prognosis.
Assuntos
Dexametasona/uso terapêutico , Glucocorticoides/uso terapêutico , Interleucina-17/imunologia , Glândulas Salivares/imunologia , Síndrome de Sjogren/tratamento farmacológico , Síndrome de Sjogren/imunologia , Linfócitos T/imunologia , Complexo CD3 , Antígenos CD8 , Células Cultivadas , Resistência a Medicamentos , Feminino , Humanos , Interleucina-17/biossíntese , Masculino , Pessoa de Meia-Idade , Síndrome de Sjogren/sangueRESUMO
We recently identified a new acute myeloid leukemia (AML) subtype characterized by mutations at exon-12 of the nucleophosmin (NPM) gene and aberrant cytoplasmic expression of NPM protein (NPMc+). NPMc+ AML accounts for about 35% of adult AML and it is associated with normal karyotype, wide morphological spectrum, CD34-negativity, high frequency of FLT3-ITD mutations and good response to induction therapy. In an attempt to identify a human cell line to serve as a model for the in vitro study of NPMc+ AML, we screened 79 myeloid cell lines for mutations at exon-12 of NPM. One of these cell lines, OCI/AML3, showed a TCTG duplication at exon-12 of NPM. This mutation corresponds to the type A, the NPM mutation most frequently observed in primary NPMc+ AML. OCI/AML3 cells also displayed typical phenotypic features of NPMc+ AML, that is, expression of macrophage markers and lack of CD34, and the immunocytochemical hallmark of this leukemia subtype, that is, the aberrant cytoplasmic expression of NPM. The OCI/AML3 cell line easily engrafts in NOD/SCID mice and maintains in the animals the typical features of NPMc+ AML, such as the NPM cytoplasmic expression. For all these reasons, the OCI/AML3 cell line represents a remarkable tool for biomolecular studies of NPMc+ AML.
Assuntos
Éxons/genética , Regulação Leucêmica da Expressão Gênica , Leucemia Promielocítica Aguda/genética , Mutação/genética , Proteínas Nucleares/genética , Animais , Antígenos CD34/metabolismo , Biomarcadores/metabolismo , Citoplasma/metabolismo , Análise Mutacional de DNA , Humanos , Cariotipagem , Leucemia Promielocítica Aguda/metabolismo , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Pessoa de Meia-Idade , Proteínas Nucleares/metabolismo , NucleofosminaAssuntos
Cromossomos Humanos Par 3/genética , Cromossomos Humanos Par 5/genética , Regulação Leucêmica da Expressão Gênica , Leucemia Mieloide/tratamento farmacológico , Leucemia Mieloide/genética , Proteínas Nucleares/genética , Proteínas de Fusão Oncogênica/genética , Doença Aguda , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Humanos , Proteínas Nucleares/biossíntese , Nucleofosmina , Proteínas de Fusão Oncogênica/análise , Proteínas de Fusão Oncogênica/biossíntese , Frações Subcelulares/química , Translocação Genética/genéticaRESUMO
In acute myeloid leukaemia (AML), nucleophosmin-1 (NPM1) mutations create a nuclear export signal (NES) motif and disrupt tryptophans at NPM1 C-terminus, leading to nucleophosmin accumulation in leukaemic cell cytoplasm. We investigated how nucleophosmin NES motifs (two physiological and one created by the mutation) regulate traffic and interaction of mutated NPM1, NPM1wt and p14(ARF). Nucleophosmin export into cytoplasm was maximum when the protein contained all three NES motifs, as naturally occurs in NPM1-mutated AML. The two physiological NES motifs mediated NPM1 homo/heterodimerization, influencing subcellular distribution of NPM1wt, mutated NPM1 and p14(ARF) in a 'dose-dependent tug of war' fashion. In transfected cells, excess doses of mutant NPM1 relocated completely NPM1wt (and p14(ARF)) from the nucleoli to the cytoplasm. This distribution pattern was also observed in a proportion of NPM1-mutated AML patients. In transfected cells, excess of NPM1wt (and p14(ARF)) relocated NPM1 mutant from the cytoplasm to the nucleoli. Notably, this distribution pattern was not observed in AML patients where the mutant was consistently cytoplasmic restricted. These findings reinforce the concept that NPM1 mutants are naturally selected for most efficient cytoplasmic export, pointing to this event as critical for leukaemogenesis. Moreover, they provide a rationale basis for designing small molecules acting at the interface between mutated NPM1 and other interacting proteins.
Assuntos
Transporte Ativo do Núcleo Celular/fisiologia , Leucemia Mieloide/genética , Proteínas de Neoplasias/genética , Sinais de Exportação Nuclear/genética , Proteínas Nucleares/genética , Mapeamento de Interação de Proteínas , Proteína Supressora de Tumor p14ARF/química , Transporte Ativo do Núcleo Celular/genética , Doença Aguda , Animais , Nucléolo Celular/metabolismo , Transformação Celular Neoplásica/genética , Citoplasma/metabolismo , Dimerização , Sistemas de Liberação de Medicamentos , Humanos , Leucemia Mieloide/metabolismo , Camundongos , Células NIH 3T3/metabolismo , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Sinais de Exportação Nuclear/fisiologia , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Nucleofosmina , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Relação Estrutura-Atividade , Transfecção , Proteína Supressora de Tumor p14ARF/metabolismoRESUMO
On the basis of the experience derived from the study of 2,000 biopsies, the authors critically review the problem of the newly generated monoclonal antibodies (mAbs) working in paraffin sections. The reliability and drawbacks of the most commonly used reagents are examined, with special reference to their employment in the diagnosis of malignant lymphomas. The authors underline that only a few mAbs are really specific, since most reagents give rise to a certain number of cross-reactivities. In the light of this, a wide panel of antibodies should always be applied in the diagnosis of lymphoid tumours in order to avoid possible misinterpretations, which can actually lead to wrong therapeutical decisions.
Assuntos
Anticorpos Monoclonais , Anticorpos Antineoplásicos , Biomarcadores Tumorais/análise , Linfoma/diagnóstico , Anticorpos Monoclonais/imunologia , Anticorpos Antineoplásicos/imunologia , Especificidade de Anticorpos , Antígenos de Diferenciação/imunologia , Antígenos de Neoplasias/imunologia , Reações Cruzadas , Diagnóstico Diferencial , Humanos , Imuno-Histoquímica/métodos , Imunofenotipagem/métodos , Linfoma/classificação , Linfoma/imunologia , Linfoma/patologia , Proteínas de Neoplasias/imunologia , Células-Tronco Neoplásicas/imunologia , Células-Tronco Neoplásicas/patologia , Parafina , Prognóstico , Proteínas Proto-Oncogênicas/imunologia , Proteínas Proto-Oncogênicas c-bcl-2RESUMO
The expression of the gut intra-epithelial T-cell associated molecule HML-1, a trimeric protein of 150, 125, 105 kD, was studied in 75 T-cell lymphomas of different subtypes: 20 T-lymphoblastic lymphomas/leukaemias; 50 nodal peripheral T-cell lymphomas; and five intestinal T-cell lymphomas. Our results confirm: (i) the usefulness of the HML-1 monoclonal antibody as an immunohistochemical marker for intestinal T-cell lymphomas: and (ii) the lack of reactivity of HML-1 with nodal peripheral T-cell lymphomas. Moreover, expression of the HML-1 molecule was found for the first time in a case of T-lymphoblastic lymphoma/leukaemia. The patient presented with a mediastinal mass which consisted of HML-1 + neoplastic cells displaying a phenotypic profile consistent with early thymocytes. Genes coding for the alpha, beta, gamma and delta chains of the T-cell receptor were in a germline configuration. The neoplastic cells could have been derived from the small subset of HML-1 + thymocytes detectable in the cortex of normal human thymus.
Assuntos
Antígenos de Neoplasias/análise , Biomarcadores Tumorais/análise , Integrinas , Neoplasias Intestinais/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/imunologia , Adolescente , Anticorpos Monoclonais , Antígenos de Diferenciação de Linfócitos T/análise , Southern Blotting , Antígenos CD8 , Humanos , Linfoma de Células T/imunologia , Masculino , Neoplasias do Mediastino/imunologia , Receptores de Interleucina-2/análise , Linfócitos T/imunologiaRESUMO
The authors critically review the problem of Hodgkin's disease (HD) in the light of new morphological, immunohistochemical, kinetic, genotypic, and virological findings. These support the lymphoid origin of neoplastic Hodgkin's and Reed-Sternberg cells, because of regular expression of the CD30 lymphoid activation antigen and frequent detection of B- or T-cell phenotypic and/or genotypic markers. It is possible to hypothesize the release of cytokines by tumoral elements as well as the presence of specific cytokine receptors on their surface. This might explain some clinical and pathological features, such as fever, loss of weight, eosinophilia and attraction of reactive elements that make up the composite cellular milieu of typical HD. Integration of monoclonal EBV in the genoma of neoplastic elements has repeatedly been shown, and this might play an essential role in the pathogenesis of the disease. On the basis of present concepts, the borderlines between HD and some categories of non-Hodgkin's lymphomas--especially the anaplastic large cell forms--have become somewhat blurred. Additional research work in the field of HD is desirable and might pave the way for new and more effective therapeutic approaches, designed on the basis of the natural history of the neoplasm.
Assuntos
Doença de Hodgkin , Antígenos CD/análise , Antígenos de Diferenciação/análise , Antígenos de Neoplasias/análise , Citocinas/metabolismo , Genes de Imunoglobulinas , Herpesvirus Humano 4/isolamento & purificação , Herpesvirus Humano 4/patogenicidade , Doença de Hodgkin/genética , Doença de Hodgkin/imunologia , Doença de Hodgkin/microbiologia , Doença de Hodgkin/patologia , Humanos , Imunofenotipagem , Linfoma/classificação , Linfoma/patologia , Proteínas de Neoplasias/metabolismo , Células de Reed-Sternberg/microbiologia , Células de Reed-Sternberg/patologiaRESUMO
A new anti-macrophage monoclonal antibody (PG-M1) was produced by immunizing BALB/c mice with fresh spleen cells from a patient with Gaucher's disease. PG-M1 reacts strongly with a fixative-resistant epitope of an intracytoplasmic molecule, selectively expressed by virtually all macrophages of the human body. Although attempts to immunoprecipitate the molecule recognized by PG-M1 have failed so far, the reactivity of the antibody with COS-1 and WOP cells transfected with a human complementary DNA clone encoding for the CD68 antigen suggests that PG-M1 is a new member of the CD68 cluster. However, unlike other CD68 antibodies (KP1, EBM11, etc.), which react with both macrophages and myeloid cells, PG-M1 detects a fixative-resistant epitope on the macrophage-restricted form of the CD68 antigen. In 957 routinely fixed, paraffin-embedded samples, PG-M1 showed a more restricted reactivity with elements of the monocyte/macrophage lineage than the previously described monoclonal antibodies MAC-387 (anti-calgranulins), KP1 (CD68) and Ki-M1P. Among hematological malignancies, PG-M1 only labels acute leukemias of M4 and M5 type and rare examples of malignant histiocytosis/true histiocytic sarcoma. In contrast, acute leukemias of the M1, M2, M3, M6, M7, and L1-L3 types, non-Hodgkin's lymphomas, and Hodgkin and Reed-Sternberg cells of Hodgkin's disease are consistently PG-M1-negative. In the daily diagnostic practice, PG-M1 seems to be particularly valuable for the diagnosis of myelomonocytic or monocytic leukemia and neoplasms of true histiocytic origin in routine paraffin sections.
Assuntos
Anticorpos Monoclonais/imunologia , Antígenos CD/imunologia , Antígenos de Diferenciação Mielomonocítica/imunologia , Epitopos , Fixadores/farmacologia , Macrófagos/imunologia , Formação de Anticorpos , Antígenos CD/análise , Antígenos de Diferenciação Mielomonocítica/análise , Linhagem Celular , DNA/genética , Resistência a Medicamentos , Doenças Hematológicas/imunologia , Sistema Hematopoético , Humanos , Inflamação/imunologia , Doenças Linfáticas/imunologia , Neoplasias/imunologia , Testes de Precipitina , Valores de Referência , TransfecçãoRESUMO
The ability of the Ber-H2 (CD30) monoclonal antibody (mAb) to target in vivo Hodgkin (H) and Reed-Sternberg (R-S) cells was investigated in six patients with advanced Hodgkin's disease (HD). The patients were injected with scaled-up quantities of 'cold' Ber-H2 mixed-up to a small dose of 131I-labelled Ber-H2, and in vivo binding of the antibody to H and R-S cells was assessed by immunohistological analysis of tumour biopsies and immunoscintigraphy. Only 50% of tumour sites were imaged at scintigraphy by the 131I-labelled Ber-H2. In contrast, immunohistological studies on tissue biopsies, taken 24-72 h following the mAb injection, showed that H and R-S cells in all tumour sites, including those that were not imaged by immunoscintigraphy, were specifically and strongly labelled in vivo by the injected Ber-H2, at a dose as low as 30-50 mg of antibody. In vivo binding of a single dose of Ber-H2 mAb to H and R-S cells did not result in any anti-tumour effect. The excellent in vivo targeting of H and R-S cells with the Ber-H2 mAb may have been the result of multiple favourable factors, including: (a) the restricted expression of the CD30 antigen in normal human tissues; (b) the low level of soluble CD30 in the serum of our patients; and (c) the high affinity of the Ber-H2 mAb for the CD30 molecule. The immunohistological results presented in this study provide a strong argument for using the Ber-H2 mAb as a carrier for delivering cytotoxic agents (isotopes or toxins) to neoplastic cells of HD refractory to conventional therapy.
Assuntos
Anticorpos Monoclonais/metabolismo , Antígenos CD/imunologia , Antígenos de Neoplasias/imunologia , Doença de Hodgkin/imunologia , Células de Reed-Sternberg/imunologia , Adulto , Anticorpos Monoclonais/uso terapêutico , Feminino , Doença de Hodgkin/diagnóstico , Humanos , Técnicas Imunoenzimáticas , Antígeno Ki-1 , Masculino , Pessoa de Meia-Idade , Tomografia Computadorizada de Emissão de Fóton Único , Tomografia Computadorizada por Raios XRESUMO
The BCL-6 gene is frequently involved in translocations occurring at the 3q27 locus and is rearranged in approximately 30% of diffuse large cell lymphomas and in a small fraction of follicular lymphomas. The BCL-6 gene encodes for a Kruppel-type zinc-finger protein, the cell/tissue expression and function of which is unknown. In this study, we describe a new monoclonal antibody (PG-B6) that is specifically directed against a fixative-sensitive epitope on the amino-terminal region of the BCL-6 protein. By immunocytochemical analysis, BCL-6 localizes in the nucleus where PG-B6 staining gives a microgranular/diffuse pattern with exclusion of the nucleoli. The main reactivity of PG-B6 in tonsil and spleen is with the nuclei of germinal center B cells, whereas B cells within the mantle and marginal zones do not express BCL-6. No other lymphoid cells in the tonsil express BCL-6 except for a subset of CD3+/CD4+ intrafollicular and interfollicular T cells. A few lymphoid cells of unknown phenotype express BCL-6 in the thymus. Extra-lymphoid BCL-6 expression includes a weak nuclear positivity of epithelia. In non-Hodgkin's lymphomas, BCL-6 expression parallels that observed in normal lymphoid compartments, eg, expression in germinal center-derived tumors (follicular and diffuse large cell lymphomas), but not in mantle cell and marginal zone lymphomas. In most diffuse large cell lymphomas, the BCL-6 protein is expressed at high levels in cases with or without BCL-6 gene rearrangements. These findings indicate that BCL-6 expression is specifically regulated during B lymphocyte development and suggest that BCL-6 may play a role during B cell differentiation in the germinal center.
Assuntos
Anticorpos Monoclonais/imunologia , Linfócitos B/metabolismo , Proteínas de Ligação a DNA/imunologia , Proteínas de Ligação a DNA/metabolismo , Tecido Linfoide/metabolismo , Linfoma de Células B/metabolismo , Proteínas Proto-Oncogênicas/imunologia , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Transcrição/imunologia , Fatores de Transcrição/metabolismo , Animais , Especificidade de Anticorpos , Tecido Linfoide/citologia , Linfoma de Células B/patologia , Linfoma não Hodgkin/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Proto-Oncogênicas c-bcl-6 , Valores de ReferênciaRESUMO
In anaplastic large-cell lymphoma (ALCL), the (2;5) chromosomal translocation creates a fusion gene encoding the 80-kD NPM-ALK hybrid protein. This report describes three new monoclonal antibodies, two of which recognize, by Western blotting, the N-terminal portion of NPM present in the NPM-ALK fusion protein and also in two other NPM fusion proteins (NPM-RARalpha and NPM-MLF1). The third antibody recognizes the C-terminal portion (deleted in NPM-ALK) and reacts only with wild-type NPM. The three antibodies immunostain wild-type NPM (in paraffin-embedded normal tissue samples) in cell nuclei and in the cytoplasm of mitotic cells. Cerebral neurones, exceptionally, show diffuse cytoplasmic labeling. In contrast to normal tissues, the two antibodies against the N-terminal portion of NPM labeled the cytoplasm of neoplastic cells, in four ALK-positive ALCL, reflecting their reactivity with NPM-ALK fusion protein, whereas the antibody to the C-terminal NPM epitope labeled only cell nuclei. Immunocytochemical labeling with these antibodies can therefore confirm that an ALK-positive lymphoma expresses NPM-ALK (rather than a variant ALK-fusion protein) and may also provide evidence for chromosomal anomalies involving the NPM gene other than the classical (2;5) translocation.
Assuntos
Linfoma Difuso de Grandes Células B/genética , Proteínas de Neoplasias/análise , Proteínas Nucleares/análise , Proteínas Nucleares/genética , Animais , Anticorpos Monoclonais , Western Blotting , Núcleo Celular/química , Cromossomos Humanos Par 2 , Cromossomos Humanos Par 5 , Citoplasma/química , Humanos , Imuno-Histoquímica , Linfoma Difuso de Grandes Células B/química , Camundongos , Nucleofosmina , Fragmentos de Peptídeos/análise , Proteínas Recombinantes de Fusão/análise , Translocação GenéticaRESUMO
A new monoclonal antibody (MUM1p) was used to study the cell/tissue expression of human MUM1/IRF4 protein, the product of the homologous gene involved in the myeloma-associated t(6;14) (p25;q32). MUM1 was expressed in the nuclei and cytoplasm of plasma cells and a small percentage of germinal center (GC) B cells mainly located in the "light zone." Its morphologic spectrum ranged from that of centrocyte to that of a plasmablast/plasma cell, and it displayed a phenotype (MUM1(+)/Bcl-6(-)/Ki67(-)) different from that of most GC B cells (MUM1(-)/Bcl-6(+)/Ki67(+)) and mantle B cells (MUM1(-)/Bcl-6(-)/Ki67(-)). Polymerase chain reaction (PCR) analysis of single MUM1(+ )cells isolated from GCs showed that they contained rearranged Ig heavy chain genes with a varying number of V(H) somatic mutations. These findings suggest that these cells may represent surviving centrocytes and their progeny committed to exit GC and to differentiate into plasma cells. MUM1 was strongly expressed in lymphoplasmacytoid lymphoma, multiple myeloma, and approximately 75% of diffuse large B-cell lymphomas (DLCL-B). Unlike normal GC B cells, in which the expression of MUM1 and Bcl-6 were mutually exclusive, tumor cells in approximately 50% of MUM1(+) DLCL-B coexpressed MUM1 and Bcl-6, suggesting that expression of these proteins may be deregulated. In keeping with their proposed origin from GC B cells, Hodgkin and Reed-Sternberg cells of Hodgkin's disease consistently expressed MUM1. MUM1 was detected in normal and neoplastic activated T cells, and its expression usually paralleled that of CD30. These results suggest that MUM1 is involved in the late stages of B-cell differentiation and in T-cell activation and is deregulated in DLCL-B. (Blood. 2000;95:2084-2092)
Assuntos
Anticorpos Monoclonais , Linfócitos B/metabolismo , Proteínas de Ligação a DNA/metabolismo , Plasmócitos/metabolismo , Linfócitos T/metabolismo , Fatores de Transcrição/metabolismo , Linfócitos B/imunologia , Biomarcadores Tumorais/metabolismo , Western Blotting , Linhagem Celular , Homólogo 5 da Proteína Cromobox , Epitopos , Células HeLa , Humanos , Região Variável de Imunoglobulina/metabolismo , Imuno-Histoquímica , Fatores Reguladores de Interferon , Células Jurkat , Linfonodos/metabolismo , Linfoma/metabolismo , Mieloma Múltiplo/metabolismo , Plasmócitos/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Linfócitos T/imunologia , Fatores de Tempo , TransfecçãoRESUMO
The RING-finger promyelocytic leukemia (PML) protein is the product of the PML gene that fuses with the retinoic acid receptor-alpha gene in the t(15; 17) translocation of acute promyelocytic leukemia. Wild-type PML localizes in the nucleus with a typical speckled pattern that is a consequence of the concentration of the protein within discrete subnuclear domains known as nuclear bodies. Delocalization of PML from nuclear bodies has been documented in acute promyelocytic leukemia cells and suggested to contribute to leukemogenesis. In an attempt to get new insights into the function of the wild-type PML protein and to investigate whether it displays an altered expression pattern in neoplasms other than acute promyelocytic leukemia, we stained a large number of normal and neoplastic human tissues with a new murine monoclonal antibody (PG-M3) directed against the amino-terminal region of PML. As the PG-M3 epitope is partially resistant to fixatives, only cells that overexpress PML are detected by the antibody in microwave-heated paraffin sections. Among normal tissues, PML was characteristically up-regulated in activated epithelioid histiocytes and fibroblasts in a variety of pathological conditions, columnar epithelium in small active thyroid follicles, well differentiated foamy cells in the center of sebaceous glands, and hypersecretory endometria (Arias-Stella). Interferons, the PML of which is a primary target gene, and estrogens are likely to represent some of the cytokines and/or hormones that may be involved in the up-regulation of PML under these circumstances. In keeping with this concept, we found that PML is frequently overexpressed in Hodgkin and Reed-Sternberg cells of Hodgkin's disease, a tumor of cytokine-producing cells. Among solid tumors, overexpression of PML was frequently found in carcinomas of larynx and thyroid (papillary), epithelial thymomas, and Kaposi's sarcoma, whereas carcinomas of the lung, thyroid (follicular), breast, and colon were frequently negative or weakly PML+. We did not observe any changes in the levels of PML expression as the lesion progressed from benign dysplasia to carcinoma. Our immunohistological data are consistent with the hypothesized growth suppressor function of PML and strongly suggest that PML expression levels are likely to be modulated by a variety of stimuli, including cytokines and hormones.
Assuntos
Núcleo Celular/química , Núcleo Celular/patologia , Transformação Celular Neoplásica/patologia , Proteínas de Neoplasias , Proteínas Nucleares , Fatores de Transcrição/biossíntese , Sequência de Aminoácidos , Carcinoma/química , Carcinoma/patologia , Transformação Celular Neoplásica/química , Epitélio/metabolismo , Humanos , Leucemia Promielocítica Aguda/genética , Tecido Linfoide/química , Tecido Linfoide/patologia , Linfoma/química , Linfoma/patologia , Dados de Sequência Molecular , Especificidade de Órgãos , Proteína da Leucemia Promielocítica , Sarcoma/química , Sarcoma/patologia , Proteínas Supressoras de TumorRESUMO
The anti-CD30 immunotoxin (IT) Ber-H2/saporin is effective in patients with refractory Hodgkin's disease. However, responses are short and partial, one of the main reasons being the inability to repeat IT doses because of formation of human antibodies against the murine antibody and/or the toxin. To overcome this problem, we constructed two new anti-CD30 ITs by covalently linking the mouse monoclonal antibody Ber-H2 to the type 1 ribosome-inactivating proteins (RIPs) momordin (MOM) and pokeweed antiviral protein from seeds (PAP-S), which do not cross-react with each other or with saporin. Both ITs inhibited protein synthesis by Hodgkin's disease and anaplastic large-cell lymphoma (ALCL)-derived CD30+ target cell lines with a very high efficiency (IC50 ranging from < 5 x 10(-13) M to 2.75 x 10(-11) M, as RIP). In a SCID mouse model of xenografted CD30+ human ALCL, a 3d treatment with non-toxin doses of Ber-H2/MOM (50%LD50), started 24 h after transplantation, prevented tumour development in about 40% of the animals and significantly delayed tumour growth rate in the others. Main toxicity signs in mice and rabbits were dose-related increase of serum transaminases (AST and ALT) and creatine phosphokinase (CPK). LD50 (as RIP) in Swiss mice was 7 mg/kg for Ber-H2/MOM and 0.45 mg/kg for Ber-H2/PAP-S. Sequential administration of two anti-CD30 ITs (Ber-H2/MOM and Ber-H2/saporin) was well tolerated and did not result in formation of antibodies cross-reacting and with the two plant toxins. The results presented in this paper suggest that in the future, sequential administration of anti-CD30 humanized antibodies linked to antigenically distinct type 1 RIPs (saporin, MOM, PAP-S) should be feasible.
Assuntos
Antineoplásicos Fitogênicos/uso terapêutico , Doença de Hodgkin/tratamento farmacológico , Imunotoxinas/uso terapêutico , Antígeno Ki-1/imunologia , N-Glicosil Hidrolases , Proteínas de Plantas/farmacologia , Proteínas de Plantas/uso terapêutico , Animais , Anticorpos Monoclonais/uso terapêutico , Humanos , Camundongos , Camundongos SCID , Proteínas Ribossômicas/uso terapêutico , Proteínas Inativadoras de Ribossomos Tipo 1 , Proteínas Inativadoras de Ribossomos Tipo 2 , Células Tumorais CultivadasRESUMO
The human BCL-6 gene, which is rearranged in approximately 30% of diffuse large B cell lymphomas, encodes a 706-amino-acid nuclear protein of the Kruppel-type zinc finger transcription factors mainly expressed in normal germinal center B cells and related lymphomas. Four monoclonal antibodies (PG-B6, PG-B6a, PG-B6p, and PG-B6m), specifically directed against the human BCL-6 protein, were generated by immunizing BALB/c mice with a recombinant protein corresponding to the BCL-6 amino-terminal region (amino acids 3 to 484). The PG-B6 monoclonal antibody reacted with a BCL-6 epitope sensitive to fixatives and preserved in all mammalian species. PG-B6a (a is for avian) recognized the most evolutionarily conserved BCL-6 epitope (expressed in all animal species including avian). PG-B6p (p is for paraffin) recognized a fixative-resistant epitope of BCL-6 that was detectable on paraffin sections after microwave heating in 1 mmol/L EDTA buffer. PG-B6m (m is for mantle) was the least specific monoclonal antibody as, in addition to BCL-6, it reacted with a yet undefined antigen selectively located in the cytoplasm of mantle and marginal zone B cells. All monoclonal antibodies detected strong nuclear expression of BCL-6 in follicular lymphomas, diffuse large B cell lymphomas, Burkitt's lymphomas, and nodular, lymphocyte-predominance Hodgkin's disease. In diffuse large B cell lymphomas, BCL-6 expression was independent of BCL-6 gene rearrangements and did not correlate with expression of other markers or the proliferation index. BCL-6 was not expressed in B-CLL, hairy cell leukemia, mantle-cell- and marginal-zone-derived lymphomas. Labeling of paraffin sections with PG-B6p proved useful for differentiating proliferation centers in B-CLL (BCl-2+/BCL-6-) from trapped germinal centers in mantle cell lymphomas (BCL-2-/BCL-6+) and for identifying neoplastic cells in cases of nodular, lymphocyte-predominance Hodgkin's disease. Because of their high specificity, wide reactivity in humans and animal species including avians (PG-B6a), and suitability for labeling routine paraffin sections (PG-B6p), the reagents described in this paper should prove valuable in both research and diagnostics.
Assuntos
Anticorpos Monoclonais/imunologia , Proteínas de Ligação a DNA/análise , Epitopos/análise , Proteínas Proto-Oncogênicas/análise , Fatores de Transcrição/análise , Sequência de Aminoácidos , Animais , Bovinos , Galinhas , Columbidae , Proteínas de Ligação a DNA/imunologia , Proteínas de Ligação a DNA/metabolismo , Epitopos/imunologia , Humanos , Técnicas Imunológicas , Tecido Linfoide/química , Tecido Linfoide/metabolismo , Tecido Linfoide/patologia , Linfoma/química , Linfoma/diagnóstico , Linfoma/patologia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Tonsila Palatina/química , Tonsila Palatina/patologia , Proteínas Proto-Oncogênicas/imunologia , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-6 , Coelhos , Ratos , Ovinos , Especificidade da Espécie , Baço/química , Baço/patologia , Suínos , Fatores de Transcrição/imunologia , Fatores de Transcrição/metabolismoRESUMO
The BCL-6 gene encoding a nuclear-located Kruppel-type zinc finger protein is rearranged in about 30% diffuse large B-cell lymphomas and is expressed predominantly in normal germinal center B cells and related lymphomas. These findings suggest that BCL-6 may play a role in regulating differentiation of normal germinal center B cells and that its deregulated expression caused by rearrangements may contribute to lymphomagenesis. This prompted us to investigate the expression of the BCL-6 protein in Hodgkin's disease (HD), focusing on the nodular lymphocyte predominance subtype (NLPHD), which differs from classical HD by virtue of the B-cell nature of the malignant cell population (so-called L&H cells) and its relationship with germinal centers. Forty-one HD samples (19 NLPHD, 12 nodular sclerosis, and 10 mixed cellularity) were immunostained with the monoclonal antibodies PG-B6 and PG-B6p that react with a fixative-sensitive and a formalin-resistant epitope on the aminoterminal region of the BCL-6 gene product, respectively. Strong nuclear positivity for the BCL-6 protein was detected in tumor (L&H) cells in all cases of NLPHD. In contrast, BCL-6 was expressed only in a small percentage of Hodgkin and Reed-Sternberg cells in about 30% of classical HD cases. Notably, the nuclei of reactive CD3+/CD4+ T cells nearby to and rosetting around L&H cells in NLPHD were also strongly BCL-6+, but lacked CD40 ligand (CD40L) expression. This staining pattern clearly differed from that of classical HD, whose cellular background was made up of CD3+/CD4+ T cells showing the BCL-6-/CD40L+ phenotype. These results further support the concept that NLPHD is an histogenetically distinct, B-cell-derived subtype of HD and suggest a role for BCL-6 in its development.
Assuntos
Linfócitos B/metabolismo , Proteínas de Ligação a DNA/biossíntese , Regulação Neoplásica da Expressão Gênica , Doença de Hodgkin/metabolismo , Proteínas de Neoplasias/biossíntese , Células-Tronco Neoplásicas/metabolismo , Proteínas Proto-Oncogênicas/biossíntese , Fatores de Transcrição/biossíntese , Animais , Anticorpos Monoclonais/imunologia , Antígenos de Neoplasias/análise , Linfócitos B/patologia , Biomarcadores Tumorais/análise , Complexo CD3/análise , Antígenos CD4/análise , Ligante de CD40 , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/imunologia , Células-Tronco de Carcinoma Embrionário , Doença de Hodgkin/genética , Doença de Hodgkin/patologia , Humanos , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Glicoproteínas de Membrana/análise , Camundongos , Camundongos Endogâmicos BALB C , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/imunologia , Células-Tronco Neoplásicas/patologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/imunologia , Proteínas Proto-Oncogênicas c-bcl-6 , Células de Reed-Sternberg/metabolismo , Células de Reed-Sternberg/patologia , Formação de Roseta , Subpopulações de Linfócitos T/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/imunologiaRESUMO
The t(2;5)(p23;q35) translocation associated with CD30-positive anaplastic large cell lymphoma results in the production of a NPM-ALK chimeric protein, consisting of the N-terminal portion of the NPM protein joined to the entire cytoplasmic domain of the neural receptor tyrosine kinase ALK. The ALK gene products were identified in paraffm sections by using a new anti-ALK (cytoplasmic portion) monoclonal antibody (ALKc) that tends to react more strongly than a previously described ALK1 antibody with the nuclei of ALK-expressing tumor cells after microwave heating in 1 mmol/L ethylenediaminetetraacetic acid buffer, pH 8.0. The ALKc monoclonal antibody reacted selectively with 60% of anaplastic large cell lymphoma cases (60 of 100), which occurred mainly in the first three decades of life and consistently displayed a T/null phenotype. This group of ALK-positive tumors showed a wide morphological spectrum including cases with features of anaplastic large cell lymphoma "common" type (75%), "lymphohistiocytic" (10%), "small cell" (8.3%), "giant cell" (3.3%), and "Hodgkin's like" (3.3%). CD30-positive large anaplastic cells expressing the ALK protein both in the cytoplasm and nucleus represented the dominant tumor population in the common, Hodgkin's-like and giant cell types, but they were present at a smaller percentage (often with a perivascular distribution) also in cases with lymphohistiocytic and small cell features. In this study, the ALKc antibody also allowed us to identify small neoplastic cells (usually CD30 negative) with nucleus-restricted ALK positivity that were, by definition, more evident in the small cell variant but were also found in cases with lymphohistiocytic, common, and "Hodgkin's-like" features. These findings, which have not been previously emphasized, strongly suggest that the neoplastic lesion (the NPM-ALK gene) must be present both in the large anaplastic and small tumor cells, and that ALK-positive lymphomas lie on a spectrum, their position being defined by the ratio of small to large neoplastic cells. Notably, about 15% of all ALK-positive lymphomas (usually of the common or giant cell variant) showed a cytoplasm-restricted ALK positivity, which suggests that the ALK gene may have fused with a partner(s) other than NPM. From a diagnostic point of view, detection of the ALK protein was useful in distinguishing anaplastic large cell lymphoma cases of lymphohistiocytic and small cell variants from reactive conditions and other peripheral T-cell lymphoma subtypes, as well as for detecting a small number of tumor cells in lymphohemopoietic tissues. In conclusion, ALK positivity appears to define a clinicopathological entity with a T/null phenotype ("ALK lymphomas"), but one that shows a wider spectrum of morphological patterns than has been appreciated in the past.