RESUMO
BACKGROUND: In addition to endogenous opioids, a number of peptide sequences, derived from endogenous (hemorphins, alphaS1-casomorphin), and exogenous proteins (casomorphins, exorphins) have been reported, possessing opioid activity. In the present work, we report the identification of a new peptide, receptorphin (Tyr-Ile-Phe-Asn-Leu), derived from the sequence of the second transmembrane loop of the opioid receptor. This sequence is unique for the opioid receptor, and conserved in all species and receptor-types. RESULTS AND DISCUSSION: Receptorphin competes for opioid binding, presenting a kappa-receptor interaction, while it binds equally to delta- and mu- opioid and somatostatin-binding sites, and inhibits the cell proliferation of a number of human cancer cell lines, in a dose-dependent and reversible manner, at the picomolar or the nanomolar range. Receptorphin shows a preferential action on prostate cancer cells. CONCLUSION: Our work identifies, for the first time a peptide, in a receptor sequence, possessing ligand-agonistic activities. A hypothesis, based on receptorphin liberation after cell death, is presented, which could tentatively explain the time-lag observed during opioid antiproliferative action.
Assuntos
Antineoplásicos/farmacologia , Oligopeptídeos/farmacologia , Receptores Opioides/química , Divisão Celular/efeitos dos fármacos , Humanos , Fragmentos de Peptídeos/farmacologia , Receptores de Superfície Celular/efeitos dos fármacos , Receptores de Superfície Celular/metabolismo , Receptores Opioides delta/efeitos dos fármacos , Receptores Opioides delta/metabolismo , Receptores Opioides kappa/efeitos dos fármacos , Receptores Opioides kappa/metabolismo , Receptores Opioides mu/efeitos dos fármacos , Receptores Opioides mu/metabolismo , Células Tumorais CultivadasRESUMO
BCMA (B cell maturation) is a nonglycosylated integral membrane type I protein that is preferentially expressed in mature B lymphocytes. Previously, we reported in a human malignant myeloma cell line that BCMA is not primarily present on the cell surface but lies in a perinuclear structure that partially overlaps the Golgi apparatus. We now show that in transiently or stably transfected cells, BCMA is located on the cell surface, as well as in a perinulear Golgi-like structure. We also show that overexpression of BCMA in 293 cells activates NF-kappa B, Elk-1, the c-Jun N-terminal kinase, and the p38 mitogen-activated protein kinase. Coimmunoprecipitation experiments performed in transfected cells showed that BCMA associates with TNFR-associated factor (TRAF) 1, TRAF2, and TRAF3 adaptor proteins. Analysis of deletion mutants of the intracytoplasmic tail of BCMA showed that the 25-aa protein segment, from position 119 to 143, conserved between mouse and human BCMA, is essential for its association with the TRAFs and the activation of NF-kappa B, Elk-1, and c-Jun N-terminal kinase. BCMA belongs structurally to the TNFR family. Its unique TNFR motif corresponds to a variant motif present in the fourth repeat of the TNFRI molecule. This study confirms that BCMA is a functional member of the TNFR superfamily. Furthermore, as BCMA is lacking a "death domain" and its overexpression activates NF-kappa B and c-Jun N-terminal kinase, we can reasonably hypothesize that upon binding of its corresponding ligand BCMA transduces signals for cell survival and proliferation.
Assuntos
Linfócitos B/metabolismo , Proteínas de Ligação a DNA , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Fatores de Transcrição , Sequência de Aminoácidos , Animais , Antígeno de Maturação de Linfócitos B , Linfócitos B/citologia , Diferenciação Celular/imunologia , Linhagem Celular , Membrana Celular/imunologia , Membrana Celular/metabolismo , Núcleo Celular/imunologia , Núcleo Celular/metabolismo , Citoplasma/imunologia , Citoplasma/metabolismo , Ativação Enzimática/imunologia , Vetores Genéticos/farmacologia , Humanos , Líquido Intracelular/imunologia , Líquido Intracelular/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno , Sistema de Sinalização das MAP Quinases/imunologia , Camundongos , Dados de Sequência Molecular , NF-kappa B/antagonistas & inibidores , Mapeamento de Peptídeos , Proteínas/genética , Proteínas/metabolismo , Proteínas/fisiologia , Receptores do Fator de Necrose Tumoral/antagonistas & inibidores , Receptores do Fator de Necrose Tumoral/genética , Receptores do Fator de Necrose Tumoral/fisiologia , Deleção de Sequência , Fator 1 Associado a Receptor de TNF , Fator 2 Associado a Receptor de TNF , Fator 3 Associado a Receptor de TNF , Células Tumorais Cultivadas , Proteínas Elk-1 do Domínio ets , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
The tumor necrosis factor (TNF) family member B cell activating factor (BAFF) binds B cells and enhances B cell receptor-triggered proliferation. We find that B cell maturation antigen (BCMA), a predicted member of the TNF receptor family expressed primarily in mature B cells, is a receptor for BAFF. Although BCMA was previously localized to the Golgi apparatus, BCMA was found to be expressed on the surface of transfected cells and tonsillar B cells. A soluble form of BCMA, which inhibited the binding of BAFF to a B cell line, induced a dramatic decrease in the number of peripheral B cells when administered in vivo. Moreover, culturing splenic cells in the presence of BAFF increased survival of a percentage of the B cells. These results are consistent with a role for BAFF in maintaining homeostasis of the B cell population.
Assuntos
Linfócitos B/imunologia , Ativação Linfocitária , Proteínas de Membrana/imunologia , Proteínas de Membrana/fisiologia , Receptores do Fator de Necrose Tumoral/imunologia , Receptores do Fator de Necrose Tumoral/fisiologia , Fator de Necrose Tumoral alfa , Animais , Fator Ativador de Células B , Antígeno de Maturação de Linfócitos B , Linhagem Celular , Sobrevivência Celular , Homeostase , Humanos , Imunoglobulina G/imunologia , Cadeias kappa de Imunoglobulina/genética , Cadeias kappa de Imunoglobulina/imunologia , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Tonsila Palatina/imunologia , Receptores do Fator de Necrose Tumoral/genética , Proteínas Recombinantes/imunologia , Baço/imunologia , TransfecçãoRESUMO
The BCMA gene is a new gene discovered by the molecular analysis of a t(4;16) translocation, characteristic of a human T cell lymphoma. It has no significant similarity with any known protein or motif, so that its function was unknown. This report describes the cloning of murine BCMA cDNA and its genomic counterpart. The mouse gene is organized into three exons, like the human gene, and lies in murine chromosome 16, in the 16B3 band, the counterpart of the human chromosome 16p13 band, where the human gene lies. Murine BCMA cDNA encodes a 185 amino acids protein (184 residues for the human), has a potential central transmembrane segment like the human protein and is 62% identical to it. The murine BCMA mRNA is found mainly in lymphoid tissues, as is human BCMA mRNA. Alignment of the murine and human BCMA protein sequences revealed a conserved motif of six cysteines in the N-terminal part, which strongly suggests that the BCMA protein belongs to the tumor necrosis factor receptor (TNFR) superfamily. Human BCMA is the first member of the TNFR family to be implicated in a chromosomal translocation.
Assuntos
Proteínas de Membrana/genética , Sequência de Aminoácidos , Animais , Antígeno de Maturação de Linfócitos B , Linfócitos B/metabolismo , Sequência de Bases , Mapeamento Cromossômico , Clonagem Molecular , DNA Complementar , Humanos , Hibridização in Situ Fluorescente , Proteínas de Membrana/química , Camundongos , Dados de Sequência Molecular , Receptores do Fator de Necrose Tumoral/química , Receptores do Fator de Necrose Tumoral/genética , Mapeamento por Restrição , Alinhamento de Sequência , Análise de Sequência de DNA , Translocação GenéticaRESUMO
Leukemic cells from a 45-year-old male patient with a CD3+, CD56+, CD57+, CD7+ acute lymphoblastic leukemia were cultured in vitro in the absence of any added growth factor for up to 6 years and a continuous lymphoblastoid cell line (NOI-90) was established. NOI-90 cells have the same phenotype and karyotype as initial leukemic cells. Southern blot of DNA from NOI-90 cells showed that TCRbeta, TCRgamma, and J(H) were in germ line. Two and 25% of NOI-90 cells were positive when stained with the IOT14 and 7G7/B6 moAbs, which recognize the CD25 molecule (IL-2R alpha chain); moreover, 4% and 13% of the cells were positive when stained with the TU-27 and mik beta3 moAbs which recognize the CD122 molecule (IL-2Rbeta chain). Equilibrium binding experiments with radiolabelled IL-2 revealed the presence of a small number of high affinity IL-2R on both fresh and continuously growing cells. Media conditioned by NOI-90 cells could induce proliferation of an IL-2-dependent cell line and this IL-2 activity could be detected by a sensitive immunoenzymatic assay using antibodies recognizing distinct epitopes of IL-2. Moreover, IL-2 activity could be adsorbed by immunoaffinity on anti-IL-2 polyclonal purified IgG and the retained molecule displayed a m.w. of 14.5 kDa in SDS-PAGE. In addition, IL-2 immunoreactive molecules could be revealed in the cytoplasm of the cells. Finally, IL-2 fixed on the cell membrane could be detected by indirect immunofluorescence. Although added IL-2 could not induce cell proliferation, monoclonal antibodies against CD25, CD122 and IL-2 could specifically inhibit spontaneous cell proliferation in a dose-dependent manner. NOI-90 cells failed to demonstrate any cytotoxic activity against the K-562, Raji or Daudi cells. These findings indicate that NOI-90 cells are of non-T, non-B, origin lacking NK activity but proliferate under an autocrine pathway which involves, at least partly, the IL-2/IL-2R system.
Assuntos
Interleucina-2/farmacologia , Células-Tronco Neoplásicas/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Complexo CD3/análise , Antígeno CD56/análise , Antígenos CD57/análise , Meios de Cultivo Condicionados/farmacologia , Células-Tronco de Carcinoma Embrionário , Evolução Fatal , Técnica Indireta de Fluorescência para Anticorpo , Rearranjo Gênico , Humanos , Cadeias Pesadas de Imunoglobulinas/análise , Imunofenotipagem , Masculino , Pessoa de Meia-Idade , Células-Tronco Neoplásicas/efeitos dos fármacos , Receptores de Antígenos de Linfócitos T alfa-beta/análise , Receptores de Antígenos de Linfócitos T gama-delta/análise , Receptores de IgG/análise , Células Tumorais CultivadasRESUMO
The human TEL gene is involved in several 12p13 chromosomal abnormalities present in various human hematological malignancies, the most frequent being the t(12;21)(p13;q22), specific for childhood acute lymphoblastic leukemia. The predicted product of TEL harbours an amino acid region similar to the ETS DNA binding domain. We now report the isolation of the murine TEL cDNA and the characterization of the human TEL proteins. Human and murine TEL proteins are particularly homologous within their aminoterminal regions and their ETS domains. TEL proteins are nuclear and display specific DNA binding activity toward classical ETS binding sites. In addition, we show that TEL mRNAs initiate translation at either of the two first inframe ATGs (codon 1 and 43) to encode 50 kDa and 57 kDa TEL proteins. In vivo, each of these primary translational products is modified by multiple phosphorylation events.
Assuntos
DNA Complementar/genética , Proteínas de Ligação a DNA/genética , Proteínas Nucleares/genética , Fosfoproteínas/genética , Proteínas Repressoras , Fatores de Transcrição/genética , Animais , Sequência de Bases , Western Blotting , Células COS , Cromossomos Humanos Par 12 , Cromossomos Humanos Par 21 , Clonagem Molecular , DNA/metabolismo , DNA Complementar/isolamento & purificação , Proteínas de Ligação a DNA/isolamento & purificação , Humanos , Hibridização in Situ Fluorescente , Leucemia de Células B/genética , Camundongos , Dados de Sequência Molecular , Proteínas Nucleares/isolamento & purificação , Fosfoproteínas/isolamento & purificação , Fosforilação , Proteínas Proto-Oncogênicas c-ets , RNA Mensageiro/isolamento & purificação , Fatores de Transcrição/isolamento & purificação , Translocação Genética , Células Tumorais Cultivadas , Variante 6 da Proteína do Fator de Translocação ETSRESUMO
BCMA is a human gene expressed preferentially in mature B lymphocytes as a 1.2 kb mRNA, which encodes a 184 amino acid peptide (BCMAp). The study of BCMA mRNA expression, using human malignant B cell lines characteristic of different stages of B lymphocyte differentiation, demonstrated that the BCMA mRNA is absent in the pro-B lymphocyte stage. It is expressed faintly at the pre-B cell stage and its expression increases with B lymphocyte maturation. Polyclonal antibodies were used to show, by cellular fractionation and immunoprecipitation, that BCMAp is a non-glycosylated integral membrane protein. Furthermore, BCMAp inserts, in vitro, into canine microsomes, as a type I integral membrane protein. Cell surface labeling showed that BCMAp is not expressed in the plasma membrane of mature B lymphocytes. Immunofluorescence studies revealed that BCMAp lies in a cap-like structure near the nucleus, that was identified as the Golgi apparatus by co-localization of BCMAp with CTR433, a marker of the medial cisternae of the Golgi apparatus. Confocal scanning laser microscopy of U266 plasma cells labeled with markers of various Golgi apparatus subcompartments strongly suggests that BCMAp is located in the cis part of the Golgi apparatus. Thus, BCMAp is the first Golgi resident protein with a tissue specificity and whose expression is linked to the stage of differentiation of B lymphocytes. The location of BCMAp in the Golgi apparatus and its high expression in plasmocytes (secreting large amounts of Ig) suggest that BCMAp is implicated in the intracellular traffic of Ig.
Assuntos
Linfócitos B/química , Complexo de Golgi/química , Proteínas de Membrana/química , Receptores do Fator de Necrose Tumoral , Antígeno de Maturação de Linfócitos B , Compartimento Celular , Imunofluorescência , Complexo de Golgi/imunologia , Humanos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Proteínas de Membrana/genética , Microscopia Confocal , Microssomos/química , Conformação Proteica , RNA Mensageiro/química , Células Tumorais CultivadasRESUMO
The expression and function of IL-2R chains expressed on B-cell chronic lymphocytic leukemia (B-CLL) cells were analyzed. IL-2R alpha was expressed in 31 out of 34 studied cases; in 17 cases more than 50% of the cells were positive whereas in three cases the proportion of IL-2R alpha+ cells was less than 10%. In two patients, 6 and 13% of the cells were IL-2R beta+, in six other cases only 2-3% of the B-CLL cells could be stained with the TU-27 moAb whereas in all other cases no positive cells could be detected. Equilibrium binding experiments using 125I-rIL2 revealed high (seven out of 15 studied cases), intermediate (four out of 15 cases) and low (five out of 15 cases) affinity IL-2R. The number of high and intermediate affinity IL-2R was low (range: 145-800 and 40-2800 binding sites/cells, respectively). In all cases investigated, both IL-2R alpha and IL-2R beta chain mRNA could be detected, although their quantity was variable from patient to patient. Exogenous recombinant IL-2 induced, in a dose-response manner, cell proliferation in ten out of 23 cases and this effect was independent of the expression of IL-2R alpha; however, only cells expressing high affinity IL-2R could respond to exogenous rIL-2. Moreover, anti-IL-2R alpha moAb could inhibit both spontaneous (in three out of five cases) and IL-2-induced (in five out of five cases) B-CLL cell proliferation. These findings demonstrate that in a subgroup of B-CLL, leukemic cells are dependent on the IL-2/IL-2R system whereas in another group, although cells expressed functional IL-2 binding sites, they could not respond to the mitogenic signal of IL-2.
Assuntos
Leucemia Linfocítica Crônica de Células B/metabolismo , Receptores de Interleucina-2/metabolismo , Northern Blotting , Southern Blotting , Divisão Celular , Imunofluorescência , Rearranjo Gênico de Cadeia Pesada de Linfócito B , Humanos , Interleucina-2/farmacologia , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/patologia , RNA Mensageiro/metabolismo , Receptores de Interleucina-2/genética , Proteínas Recombinantes/farmacologiaRESUMO
In a previous study of a t(4;16)(q26;p13) translocation, found in a human malignant T-cell lymphoma the BCMA gene, located on chromosome band 16p13.1, has been characterized. In this study we show that the BCMA gene is organized into three exons and its major initiation transcription site is located 69 nucleotides downstream of a TATA box. RNase protection assays demonstrated that the BCMA gene is preferentially expressed in mature B cells, suggesting a role for this gene in the B-cell developmental process. A cDNA complementary to the BCMA cDNA was cloned and sequenced and its presence was assessed by RNase protection assay and anchor-PCR amplification. This antisense-BCMA RNA is transcribed from the same locus as BCMA, and exhibits mRNA characteristic features, e.g. polyadenylation and splicing. It also contains an ORF encoding a putative 115 aa polypeptide, presenting no homology with already known sequences. RNase protection assays demonstrated the simultaneous expression of natural sense and antisense-BCMA transcripts in the majority of human B-cell lines tested.
Assuntos
Linfócitos B/metabolismo , Proteínas/genética , Receptores do Fator de Necrose Tumoral , Transcrição Gênica , Sequência de Aminoácidos , Antígeno de Maturação de Linfócitos B , Sequência de Bases , Southern Blotting , Diferenciação Celular , Células Cultivadas , Cromossomos Humanos Par 16 , Clonagem Molecular , DNA , Éxons , Humanos , Interleucina-2/genética , Linfoma de Células T , Dados de Sequência Molecular , Poli A , Proteínas/metabolismo , RNA Mensageiro/metabolismo , Ribonucleases , TATA Box , Translocação GenéticaRESUMO
BACKGROUND: Primary T-cell lymphoma of the small intestine is rare, and most cases have proved rapidly fatal. METHODS: We describe a case of lymphoma involving the small intestine uniformly and extensively in a 28-year-old man on initial examination seen with long-standing diarrhea, malabsorption, and recurrent episodes of intestinal obstruction. Clinical remission was obtained with pentostatin (2'-deoxycoformycin, supplied by Professor Catovsky, London UK) after the patient had failed to improve under conventional chemotherapy. Tumor specimens as well as mesenteric lymph node, liver, and bone marrow specimens were studied with conventional pathology and immunochemistry. Additionally, mesenteric lymph nodes and peripheral blood cells were studied for T-cell receptor (TCR) gene rearrangement and karyotype. RESULTS: Lymphoma cells were small T-lymphocytes with irregular pleomorphic nuclei, bearing the CD3, CD4 and TCR alpha-beta phenotype. Peripheral-blood cytology and bone marrow biopsy were normal. Southern blot analysis of the TCR beta-chain gene revealed the same monoclonal rearrangement in the mesenteric lymph nodes and peripheral blood lymphocytes. An as yet undescribed t(4;16)(q26;p13) translocation, involving the region where the interleukin-2 (IL-2) gene has been mapped, was present in the mesenteric lymph nodes and peripheral blood lymphocytes. CONCLUSION: We believe this is the first description of an extensive, small intestinal lymphoma of low-grade malignancy made up of monoclonal T-cells with a TCR alpha-beta and helper/inducer phenotype, associated with a novel chromosomal translocation.
Assuntos
Neoplasias Intestinais/genética , Linfoma não Hodgkin/genética , Linfoma de Células T/genética , Translocação Genética , Adulto , Biomarcadores/análise , Cromossomos Humanos Par 16 , Cromossomos Humanos Par 4 , Rearranjo Gênico da Cadeia beta dos Receptores de Antígenos dos Linfócitos T , Humanos , Imuno-Histoquímica , Neoplasias Intestinais/imunologia , Neoplasias Intestinais/patologia , Intestino Delgado , Cariotipagem , Linfoma não Hodgkin/imunologia , Linfoma não Hodgkin/patologia , Linfoma de Células T/imunologia , Linfoma de Células T/patologia , Masculino , Fenótipo , Translocação Genética/genéticaRESUMO
Nucleotide sequences of variable regions of autoantibodies may help to understand the origin of B cells secreting autoantibodies, both in the context of monoclonal lymphoid proliferations and polyclonal autoimmune diseases. We established the nucleotide sequence of variable genes of four monoclonal IgM secreted by lymphoplasmacytic proliferations and directed to myelin-associated glycoprotein, of five anti-lamin B autoantibodies in patients with a lupus like vasculitis, and of one monoclonal IgM secreted in a chronic lymphocytic leukemia patient and directed to the cardiolipin/beta 2 glycoprotein I complex. A selection process (antigen-driven?) was probably implicated in the origin of autoantibodies in the first two situations although a random process occurred in the last one.
Assuntos
Autoanticorpos/genética , Doenças Autoimunes/imunologia , Linfócitos B/imunologia , Anticorpos Monoclonais , Doenças Autoimunes/fisiopatologia , Sequência de Bases , Divisão Celular/fisiologia , HumanosRESUMO
The nodular lymphocyte predominance form of Hodgkin's disease (NLPHD) is considered as a B cell derived lymphoproliferative disease. A patient with NLPHD presented with an absolute increase in blood B cells with cytological features of mantle zone cells; these B cells were polyclonal, did not exhibit bcl-2 gene rearrangement, and some of them displayed non-clonal chromosomal aberrations. EB virus genome was not detected by Southern analysis. Thus, this study, taking advantage of the presence of an unusual population of blood atypical B cells, confirms data obtained on lymph nodes where, however, malignant cells may be scarce, indicating that NLPHD is a polyclonal B cell lympho-proliferative disease of mantle zone origin.
Assuntos
Linfócitos B/patologia , Doença de Hodgkin/sangue , Adulto , Linfócitos B/imunologia , Southern Blotting , Doença de Hodgkin/genética , Doença de Hodgkin/imunologia , Humanos , Imunofenotipagem , Linfonodos/imunologia , Linfonodos/patologia , Subpopulações de Linfócitos , Linfocitose/imunologia , Linfocitose/patologia , MasculinoRESUMO
OBJECTIVE: To gain insight into the genetic origin of human antilamin autoantibodies, we determined the nucleotide sequence of the light and heavy chain variable region (VL and VH) domains of 5 IgM antibodies directed to lamin B. These antibodies represent a distinct subset of antinuclear antibodies, and their presence is associated with a particular lupus-like syndrome. METHODS: We derived and cloned lymphoblastoid cell lines from peripheral blood B cells of 3 patients, selected anti-lamin B-producing subclones, and sequenced the messenger RNA coding for Ig heavy and light chains. RESULTS: We isolated 2 subclones (1 IgM kappa, 1 IgM lambda) from one patient (FUR) and 2 subclones (both IgM lambda) from another (HER). In contrast, all 8 lines derived from B cells isolated from the third patient (BEN) synthesized identical anti-lamin B IgM kappa antibodies: All VL and VH domains from these 5 IgM were encoded by different VL or VH genes. DH regions were all different, and there was no restriction in the use of JL or JH segments. Analysis of the nucleotide sequence of the VL domains allowed the identification of the putative germinal gene in 3 instances (V kappa IV, Humkv325, and V lambda III.1); the overall ratios of replacement:silent mutations (R:S) were 6.5 and 1.2 in the complementarity-determining regions (CDRs) and framework regions (FRs), respectively. The 2 other lambda sequences belonged to the V lambda III family. With regard to VH domains, 3 of 5 derived from previously identified germline genes (VHIV 4.19, VHIV 4.22, and VHIII 9.1); the overall R:S ratio for these genes was 8 and 1.5 in CDRs and FRs, respectively. CONCLUSION: Taken together, these data provide evidence that the repertoire of human antilamin autoantibodies is not restricted and that the antigen (or another kind of selective pressure) plays a role in the generation of autoantibodies to lamin B. This hypothesis is in accordance with the reactivity of these antibodies to discrete epitopes of lamin B.
Assuntos
Antígenos/imunologia , Autoanticorpos/imunologia , Imunoglobulina M/genética , Região Variável de Imunoglobulina/genética , Proteínas Nucleares/imunologia , Sequência de Bases , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/genética , Cadeias mu de Imunoglobulina/genética , Lamina Tipo B , Laminas , Linfócitos/metabolismo , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Sondas de Oligonucleotídeos/genética , Células-Tronco/metabolismoRESUMO
The NH2-terminal structure of serum abnormal protein, as well as the sequence of the corresponding mRNA, were determined in a new case of alpha heavy chain disease. The patient presented with typical clinical features of the disease. Intestinal and mesenteric lymphoplasmic infiltration was monoclonal as assessed by the study of the configuration of heavy and light chain genes. The serum abnormal alpha chains included two molecular species: one starting at the beginning of the hinge region and the other being two amino acids shorter, missing the two first amino acids of the hinge region. The sequence of the mRNA displayed a leader exon, a 93 bp sequence of unknown origin and the second and third constant exons of human alpha 1 chain. These data are discussed in the light of previously reported molecular studies in heavy chain diseases.
Assuntos
DNA/ultraestrutura , Doença das Cadeias Pesadas , Cadeias alfa de Imunoglobulina/ultraestrutura , RNA Mensageiro/ultraestrutura , Adulto , Sequência de Aminoácidos , Sequência de Bases , Elementos de DNA Transponíveis , Deleção de Genes , Rearranjo Gênico , Doença das Cadeias Pesadas/sangue , Doença das Cadeias Pesadas/genética , Humanos , Masculino , Dados de Sequência MolecularRESUMO
We determined the nucleotide sequence of the VL and VH regions of four human monoclonal IgM directed to myelin-associated glycoprotein (MAG) and a nerve glycolipid, the sulfated glucuronic paragloboside (SGPG). Clonal lymphoblastoid cell lines (three cases) and an heterohybridoma (one case) secreting anti-MAG IgM were derived from patients' blood B cells. V kappa genes derived from the single germinal V kappa IV (two cases), the V kappa Id and the V kappa IIIa Humkv328h5 genes. VH genes derived from the VHIII 9.1 germinal gene (or a closely related gene) in two cases, whereas two others possibly represent new members of the VHIII or VHI variability subgroups. There was no obvious restriction in the use of J kappa, JH and DH segments. Somatic mutations were predominantly found in the CDR3 of the V kappa IV genes with an overall ratio of replacement over silent mutations of 7/0. The sequence of two VHIII genes exhibited five replacement mutations in CDR in comparison to that of the germ-line 9.1 gene. Although some V genes are likely to be overrepresented among anti-MAG IgM, the diversity of the immune repertoire for MAG and SGPG explains the lack of easily detectable public idiotopes among these IgM. This last finding, as well as a high ratio of replacement versus silent nucleotide mutations in the CDR of VL and probably VH genes, suggest that the pathogenesis of these monoclonal antibodies (and of the associated lymphoplasmocytic disorder) differs from that of other previously characterized monoclonal autoantibodies such as rheumatoid factors and cold agglutinins.
Assuntos
Genes de Imunoglobulinas , Imunoglobulina M/genética , Região Variável de Imunoglobulina/genética , Cadeias kappa de Imunoglobulina/genética , Proteínas da Mielina/imunologia , Proteínas do Tecido Nervoso/imunologia , Sequência de Aminoácidos , Anticorpos Monoclonais/imunologia , Sequência de Bases , Células Clonais , Glicoproteínas/imunologia , Humanos , Cadeias mu de Imunoglobulina/genética , Dados de Sequência Molecular , Glicoproteína Associada a Mielina , Oligodesoxirribonucleotídeos/química , Alinhamento de SequênciaRESUMO
We studied two patients with a leukaemic T cell lymphoma who presented with a marked increase in blood eosinophilia. To investigate the mechanism of the eosinophilia, supernatants of peripheral blood cells containing more than 80% lymphoma cells were tested by biological assays for the presence of colony stimulating factors (CSF). In one case supernatants stimulated the growth of granulocyte-macrophage (GM), erythroid and eosinophil colonies. These effects were neutralized by anti-GM-CSF antibodies; anti-IL5 antibodies slightly decreased eosinophil colony formation. Supernatants derived from the second patient cells stimulated the same lineages. Neutralizing experiments demonstrated that in addition to GM-CSF it contained interleukin 3 (IL-3) and interleukin 5 (IL-5). In agreement with the biological data, RNA studies using the polymerase chain reaction showed that cells from the first patient expressed GM-CSF transcripts; IL-5 transcripts were also detected in very low amounts. GM-CSF, IL-3 and IL-5 transcripts were detected in cells from the second patient. Thus eosinophilia associated with some T cell lymphoma is likely due to secretion of different combinations of cytokines by malignant cells.
Assuntos
Eosinofilia/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/sangue , Interleucina-3/sangue , Interleucina-5/sangue , Linfoma de Células T/imunologia , Adulto , Sequência de Bases , DNA de Neoplasias/análise , Eosinofilia/genética , Feminino , Humanos , Neoplasias Intestinais/genética , Neoplasias Intestinais/imunologia , Linfoma de Células T/genética , Linfoma Cutâneo de Células T/imunologia , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Neoplásico/análise , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/imunologiaRESUMO
Human alpha heavy chain disease is characterized by the production of abnormally short alpha IgH chains. In previously published cases it has been found that the malignant cells produce abnormal alpha mRNA, lacking VH and CH1 sequences and composed of a leader sequence peptide, sequences of variable length (69 to 84 bp) and of unknown origin, followed by normal CH2 and CH3 sequences. In this study we established the nucleotide sequence of alpha mRNA for six cases of alpha heavy chain disease. We observed that all six alpha mRNA lack the VH and CH1 sequences as do those previously described. They also contain in-frame inserts of unknown origin between the leader peptide and the normal CH2 and CH3 coding sequences. These inserts are of variable length (42 to 105 bp) and they are unrelated. These results suggest the existence of a common mechanism defect leading to deletions/insertions in alpha heavy chain disease rather than a specific interaction between alpha 1 IgH gene with a unique defined molecular species.
Assuntos
Doença das Cadeias Pesadas/genética , Cadeias alfa de Imunoglobulina/genética , RNA Mensageiro/química , Sequência de Aminoácidos , Sequência de Bases , Northern Blotting , Southern Blotting , DNA/química , Éxons , Humanos , Regiões Constantes de Imunoglobulina/genética , Dados de Sequência MolecularRESUMO
A t(4;16)(q26;p13.1) chromosome translocation found in tumour cells from a patient with a T cell lymphoma was shown to rearrange the interleukin 2 gene, normally located on chromosome band 4q26, with sequences from chromosome band 16p13.1. A cDNA library of tumour cells was screened with an interleukin 2 gene-specific probe. Three clones were isolated, which consisted, from 5' to 3', of the three first exons of the interleukin 2 gene followed by a 16p13 in-frame sequence encoding 181 amino acids. A probe derived from this sequence detected a 1.2 kb transcript in various cell lines exhibiting mature B lymphoid cell features, but this was not detected in other cell lines representative of other haematopoietic lineages, or in other organs. For this reason, the novel gene was termed BCM for B cell maturation. The open reading frame of BCM normal cDNA predicted a 184 amino acid protein with a single transmembrane domain which had no homology with any protein sequence stored in data banks. Our data indicate that BCM is a new gene whose expression coincides with B cell terminal maturation.
Assuntos
Cromossomos Humanos Par 16 , Cromossomos Humanos Par 4 , Rearranjo Gênico , Interleucina-2/genética , Linfoma de Células T/genética , Translocação Genética , Sequência de Aminoácidos , Sequência de Bases , Northern Blotting , Southern Blotting , Bandeamento Cromossômico , Clonagem Molecular , DNA/genética , DNA de Neoplasias/genética , DNA de Neoplasias/isolamento & purificação , Éxons , Biblioteca Gênica , Humanos , Hibridização In Situ , Cariotipagem , Dados de Sequência Molecular , RNA Neoplásico/genética , Valores de Referência , Mapeamento por Restrição , Homologia de Sequência do Ácido NucleicoRESUMO
Adherent cells from HIV-infected subjects as well as in vitro HIV-infected normal adherent cells produce spontaneously a 29-kD (p29) factor that inhibits mitogen-induced proliferation of normal T cells. p29 mediates a partial dose-dependent inhibition of total protein synthesis in both nonstimulated and PHA-activated cells that is associated with impaired PHA-induced expression of IL-2 receptor (IL-2R)alpha chain, HLA-class II molecules, and production of IL-2 by these cells; conversely, p29 does not modify the expression of IL-2R beta chain, 4F2, CD9, or transferrin receptor, or the production of IL-1 and TNF alpha by the cells. 1 h preincubation of the cells with p29 is sufficient to detect its biologic activity and added rIL-2 abrogates p29-induced inhibition of IL-2R alpha chain expression; however, p29 does not display any biologic effect on already expressed IL-2R alpha chains. The impaired expression of IL-2R alpha chain mediated by p29 is not due to a decreased accumulation of the corresponding mRNA transcripts, but is associated with a two-fold increase of intracellular cAMP. Binding experiments with 125I-rIL-2 reveals that p29 induces a 50% decrease in the number of both high and low affinity IL-2R per cell. p29 also inhibits alloantigen-induced proliferation of PBMC, whereas it does not modify IL-2-dependent proliferation of 48-h PHA-blasts that already express high affinity IL-2R. These findings indicate that p29 mediates its biologic activity during early stages of T cell activation affecting the expression of high affinity IL-2R and production of IL-2, through a nontranscriptional mechanism involving an increase of intracellular cAMP.
Assuntos
Infecções por HIV/metabolismo , Interleucina-2/biossíntese , Ativação Linfocitária/efeitos dos fármacos , Proteínas/farmacologia , Receptores de Interleucina-2/biossíntese , Proteínas dos Retroviridae/farmacologia , Linfócitos T/efeitos dos fármacos , AMP Cíclico/metabolismo , Humanos , Leucócitos Mononucleares/metabolismo , Biossíntese de Proteínas , Linfócitos T/imunologiaRESUMO
During the last few years, high-dose therapy with hemopoietic stem cell support has become a well-admitted therapeutic option for young patients with MM. The role of allogeneic or autologous graft and of blood rather than bone marrow as the source of hemopoietic stem cells must be further investigated. Autologous PBSC transplantation has, however, both practical and theoretic advantages over allogeneic and autologous BMT: (1) It can be applied to most patients, especially if blood stem cells are collected early in the course of therapy. (2) It usually induces relatively rapid hematologic reconstitution. (3) In comparison with autologous BMT, it appears to minimize the hazard of the reinfusion of malignant cells.