RESUMEN
In vitro incubation of human cytomegalovirus (Towne strain) with 8 U/ml human recombinant myeloperoxidase plus sodium chloride and glucose nearly abolished viral infectivity. To assay the effect on intracellular infection, cell toxicity of the enzymes was first studied. Even the high dose of 16 U/ml of recombinant myeloperoxidase plus 10 mU/ml glucose oxidase did not decrease MRC5 cell growth. By contrast, this dose reduced proliferation of activated THP1 cells. Even half of the myeloperoxidase dose proved slightly toxic to these cells. Non-cytotoxic concentrations of the reagents were used to monitor their effect on cytomegalovirus infection. In MRC5 cells, even the low dose of 4 U/ml myeloperoxidase plus glucose oxidase inhibited synthesis of cytomegalovirus early antigens, as tested by immunofluorescence. Viral release in the supernatant was decreased by 4 logs. In THP1 cells, which produce endogenously hydrogen peroxide, myeloperoxidase alone (8 U/ml) decreased the formation of early and late antigens by 53 and 44%, respectively.
Asunto(s)
Antivirales/farmacología , Infecciones por Citomegalovirus/virología , Citomegalovirus/efectos de los fármacos , Peroxidasa/farmacología , Proteínas Recombinantes/farmacología , División Celular , Línea Celular , Citomegalovirus/patogenicidad , Fibroblastos/fisiología , Fibroblastos/virología , Humanos , Peróxido de Hidrógeno/metabolismo , Macrófagos/fisiología , Macrófagos/virología , Monocitos/fisiología , Monocitos/virología , Peroxidasa/genética , Proteínas Recombinantes/genética , Replicación Viral/efectos de los fármacosRESUMEN
Characterization of the spontaneous immune response that frequently occurs in tumor-bearing animals, as well as immunization using dendritic cells pulsed with tumor antigens, suggests that a limiting factor of the tumor-specific immune response may be a defect in the co-stimulatory signal that is required for optimal activation of T cells. In this work, we describe a new approach to improve the antigen-presenting capacity of tumor cells, which does not require a source of purified tumor-associated antigen. We fused P815 mastocytoma cells with bone marrow-derived dendritic cells. We obtained one hybrid that displayed the phenotypic and functional properties of dendritic cells and expressed mRNA coding for the tumor-associated antigen P815 A/B. Injections of irradiated hybrid cells prevented the growth of preimplanted mastocytoma and induced long-lasting tumor resistance.
Asunto(s)
Células Dendríticas/inmunología , Inmunoterapia Adoptiva , Sarcoma de Mastocitos/inmunología , Sarcoma de Mastocitos/prevención & control , Animales , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/inmunología , Fusión Celular , Células Dendríticas/citología , Femenino , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Sarcoma de Mastocitos/patología , Ratones , Ratones Endogámicos CBA , Ratones Endogámicos DBA , Fenotipo , ARN Mensajero/metabolismo , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
The human MAGE1 gene directs the expression of an antigen recognized on a melanoma by autologous cytolytic T lymphocytes. MAGE1 belongs to a family of genes that are expressed in a number of tumors of various histological types but not in normal tissues except testis. The MAGE genes are arranged in two groups that are located within two different regions of the human X chromosome (Xq26-qter and Xp21.3). By hybridizing mouse genomic libraries with a MAGE1 probe, we identified three homologous genes. Two of these mouse genes, Smage1 and Smage2, are more than 99% identical to each other and encode the same protein of 330 aa. The 5' noncoding region of Smage2 provides the potential for regulating the expression of the gene through several different promoters located in front of alternative first exons. The third gene, Smage3, has the structure of a processed transcript. It codes for a protein with only 11 aa substitutions with respect to the Smage1/2 product. Somatic cell hybrids and interspecific backcross analysis showed that Smage3 is autosomal and that Smage1 and Smage2 are located between the Dmd and the Ar loci on the mouse X chromosome. Since this region is syntenic to the human Xp21.1-p22.1 region, we conclude that Smage1 and Smage2 are homologous to the MAGE-Xp rather than to the MAGE-Xq genes. Smage1/2 transcripts were detected in several tumor and embryonal cell lines but not in normal mouse tissues with the exception of testis. Expression of Smage3 was found in embryos from Day 11 to Day 15.
Asunto(s)
Antígenos de Neoplasias/genética , Proteínas de Neoplasias , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , Regulación Neoplásica de la Expresión Génica , Humanos , Células Híbridas , Antígenos Específicos del Melanoma , Ratones , Datos de Secuencia Molecular , Alineación de SecuenciaRESUMEN
The bombesin-related peptides gastrin-releasing peptide (GRP) and neuromedin B (NMB) have been demonstrated in the anterior pituitary (AP) on an immunological basis. We studied the presence of mRNAs for these peptides and for their receptors by RNAse protection assay using fresh adult male rat AP, AP cell reaggregates cultured in the presence of estradiol and the rat AP derived GH3 cell line. In total RNA from fresh AP we detected high amounts of NMB mRNA and much smaller amounts of GRP mRNA, while finding a weak signal for GRP-receptor (GRP-R) and NMB-receptor (NMB-R) mRNAs. In total RNA from the reaggregate cell cultures we detected high levels of NMB mRNA as well as a strong signal for GRP-R mRNA. Finally, in GH3 cells, high levels of NMB mRNA and GRP-R mRNA were found, while GRP mRNA and NMB-R mRNA remained undetectable even in high amounts (200 micrograms) of total RNA. We conclude that mRNAs encoding both bombesin-related peptides and each of the mRNAs encoding their receptors are expressed in rat AP tissue. NMB mRNA is more prominent than GRP mRNA in all AP-like tissues examined (fresh AP, estradiol-treated reaggregate AP cell cultures and GH3 cells). NMB-R mRNA and GRP-R mRNA are both present in low levels in fresh AP whereas the GRP-R mRNA is predominant in GH3 cells and estradiol treated AP reaggregate cell cultures. Compared to fresh AP tissue, NMB mRNA and GRP-R mRNA expression is enhanced in estradiol-treated reaggregate cell cultures.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Regulación de la Expresión Génica , Neuroquinina B/análogos & derivados , Péptidos/genética , Adenohipófisis/metabolismo , Receptores de Bombesina/genética , Animales , Sondas de ADN , Estradiol/farmacología , Péptido Liberador de Gastrina , Masculino , Neuroquinina B/biosíntesis , Neuroquinina B/genética , Biosíntesis de Péptidos , Neoplasias Hipofisarias/química , Neoplasias Hipofisarias/patología , ARN Mensajero/análisis , Ratas , Ratas Wistar , Receptores de Bombesina/biosíntesis , Ribonucleasas/metabolismo , Células Tumorales CultivadasRESUMEN
Mutagen treatment of mouse P815 tumor cells produces immunogenic mutants that express new transplantation antigens (tum- antigens) recognized by cytolytic T cells. The gene encoding tum- antigen P91A comprises 12 exons and a mutation located in exon 4 is responsible for the production of a new antigenic peptide. Transfection experiments showed that the expression of the antigen could be transferred not only by the entire gene but also by gene segments comprising only the mutated exon and parts of the surrounding introns. This was observed with subgenic regions that were not cloned in expression vectors. Antigen expression did not require the integration of the transfected gene segment into a resident P91A gene by homologous recombination. It also occurred when the subgenic segment was transfected without the usual selective gene, which comprises an eucaryotic promoter, and also without plasmid sequences, which are known to contain weak promoters. When a stop codon was introduced at the beginning of exon 4, the expression of the antigen was maintained and evidence was obtained that an ATG codon located in this region served as initiation site for the translation of the antigenic peptide. But we have not obtained evidence indicating that antigenic peptides are direct translation products rather than degradation products of entire proteins.
Asunto(s)
Antígenos de Histocompatibilidad/genética , Linfocitos T Citotóxicos/inmunología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Southern Blotting , Línea Celular , Clonación Molecular , Análisis Mutacional de ADN , Expresión Génica/genética , Ratones , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Recombinación Genética , Mapeo Restrictivo , Transfección/genéticaRESUMEN
Recent binding studies in the central nervous system and other tissues provide evidence that the mammalian bombesin-like peptides, gastrin-releasing peptide (GRP) and neuromedin-B (NMB), exert their numerous physiological effects through at least two different receptors. We describe the structure and expression of a cloned NMB-preferring bombesin receptor (NMB-R) with properties distinct from a GRP-preferring bombesin receptor (GRP-R) reported previously. In particular, the NMB-R shows higher affinity binding to NMB than to GRP in BALB 3T3 fibroblasts expressing the cloned NMB-R. The distinct regional distribution of NMB-R and GRP-R mRNA in the brain suggests that both bombesin receptor subtypes play independent roles in mediating many of the dramatic effects of bombesin-like peptides in the central nervous system.
Asunto(s)
ADN/genética , Neuroquinina B/análogos & derivados , Receptores de Neurotransmisores/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Encéfalo/citología , Encéfalo/metabolismo , Encéfalo/ultraestructura , Sistema Nervioso Central/citología , Sistema Nervioso Central/metabolismo , Sistema Nervioso Central/ultraestructura , ADN/análisis , ADN/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Fibroblastos/ultraestructura , Péptido Liberador de Gastrina , Expresión Génica , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Neuroquinina B/metabolismo , Neuroquinina B/fisiología , Hibridación de Ácido Nucleico , Péptidos/metabolismo , Péptidos/fisiología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Bombesina , Receptores de Neuroquinina-3 , Receptores de Neurotransmisores/efectos de los fármacos , Receptores de Neurotransmisores/genética , Receptores de Neurotransmisores/fisiología , TransfecciónRESUMEN
The bombesin-like peptides are a family of structurally related amidated peptide ligands that are known to have a variety of potent pharmacological actions on various cells, including neurons in the rat brain. Two mammalian representatives of the bombesin family of peptides have been identified, gastrin-releasing peptide (GRP) and neuromedin B (NMB). Previously, we cloned the rat preproGRP gene and determined the locations of neurons expressing this gene using in situ hybridization. In this study, we describe the structure and sequence of the rat preproNMB gene, and the first detailed cellular localization of preproNMB mRNA in rat brain using in situ hybridization. Nucleotide sequence analysis of cDNA and genomic clones reveals a 117 amino acid precursor whose overall structure is similar to that described for human preproNMB. Sequence similarity between the rat NMB and GRP genes is observed only over a limited 10 amino acid sequence encoding the carboxy termini of the GRP and NMB peptides, the region shown to be necessary and sufficient for high-affinity receptor binding. In situ hybridization studies performed with cRNA probes specific for NMB or GRP mRNA show that the distribution of cells expressing either mRNA in brain is very distinct. NMB mRNA is found most prominently in the olfactory bulb, dentate gyrus, and dorsal root ganglion. In contrast, the highest levels of GRP mRNA are observed in the forebrain (isocortex and hippocampal formation). This heterogeneity of mRNA distribution for these peptides suggests that these 2 structurally related peptides may have very distinct functions as neuropeptides in the rat nervous system.
Asunto(s)
Química Encefálica , Neuroquinina B/análogos & derivados , Péptidos/genética , ARN Mensajero/análisis , Médula Espinal/análisis , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN/genética , Diencéfalo/análisis , Péptido Liberador de Gastrina , Datos de Secuencia Molecular , Neuroquinina B/genética , Hibridación de Ácido Nucleico , Mapeo Peptídico , Precursores de Proteínas/genética , Sondas ARN , Ratas , Endonucleasas Específicas del ADN y ARN con un Solo Filamento , Telencéfalo/análisis , Distribución TisularRESUMEN
Expression of the mammalian prepro-gastrin-releasing peptide (preproGRP) gene has been shown to be restricted to neural and neuroendocrine cell types. In this paper, the structure and nucleotide sequence of the rat preproGRP gene coding regions and promoter is described and analyzed. The gene is divided into 3 exons, encoding a signal sequence, the 29 amino acid rat GRP, and a 92 amino acid extension peptide. While the overall prohormone structure is similar to that predicted from the sequence of the human gene, differences in transcription are apparent. Several forms of the rat preproGRP mRNA are found in brain: a 1.1 kb form which initiates in both brain and gut primarily from a TATAA-directed promoter, and less abundant forms of about 1.5 kb, whose initiation sites are heterogeneous, located 300-400 base pairs upstream of the 1.1 kb initiation site, and found only in spinal cord and a subset of brain nuclei expressing preproGRP mRNA. Comparison of the human and rat promoter region sequences identifies regions of high similarity upstream from both the 1.1 kb and 1.5 kb mRNA initiation sites, which may be important in the cell type-specific regulation of the preproGRP gene.
Asunto(s)
Encéfalo/metabolismo , Péptidos/genética , Regiones Promotoras Genéticas , Precursores de Proteínas/genética , ARN Mensajero/genética , Transcripción Genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Humanos , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Ratas , Ratas Endogámicas , Homología de Secuencia de Ácido NucleicoRESUMEN
There are two distinct mRNAs that encode the precursor to gastrin-releasing peptide (GRP) in rat brain. These two messages arise from separate transcription initiation sites located approximately 400 base pairs apart, which are presumably regulated by separate promoters. In the present study, we mapped the distribution of neurons containing GRP mRNAs by in situ hybridization using cRNA and synthetic DNA probes specific for the 1.5 kb GRP transcript and probes complementary to both the 1.5 kb and 1.1 kb transcripts. The distribution of neurons expressing GRP mRNA appears to be wider than that previously observed by immunohistochemical studies, suggesting an important functional role for this neuropeptide in a number of brain regions. We detected the 1.5 kb transcript only in cingulate cortex, Ammon's horn of the hippocampus and in subiculum. In contrast, the probe which hybridized to both GRP mRNAs labeled a broad range of brain areas, including those containing the 1.5 kb mRNA. These data suggest that the 1.5 kb mRNA encoding rat GRP is expressed only in specific parts of the limbic system, whereas the expression of the 1.1 kb GRP message is more widespread.
Asunto(s)
Encéfalo/metabolismo , Hormonas Gastrointestinales/genética , Péptidos/genética , ARN Mensajero/genética , Animales , Secuencia de Bases , Encéfalo/citología , Péptido Liberador de Gastrina , Masculino , Datos de Secuencia Molecular , Sondas de Oligonucleótidos , Especificidad de Órganos , ARN Mensajero/análisis , Ratas , Ratas Endogámicas , Transcripción GenéticaRESUMEN
Mammalian gastrin-releasing peptide (GRP) is found in cells of neuroendocrine and neural origin, and GRP mediates a variety of physiological and trophic responses when it binds to high affinity cell surface receptors on effector cells. Analysis of cDNA clones derived from prepro-GRP mRNAs predict the concurrent expression of a unique series of peptide hormones, the GRP gene-associated peptides (GGAPs). Alternative RNA splicing of the primary GRP gene transcript results in mRNAs that could encode 3 distinct forms of GGAPs. Using specific antisera directed against synthetic peptides representing portions of the predicted GGAPs, we found multiple GGAP forms in the endocrine cells of human fetal lung and in human small cell lung cancer cells (SCLC). Within a single pulmonary endocrine cell, at least 2 of these 3 predicted forms could be demonstrated using immunohistochemical techniques. In addition, concordant expression of GRP and GGAPs was found in 10 SCLC cell lines and 3 human SCLC tumors. These findings establish GGAPs as a novel peptide family in man and warrant further investigation into their potential role in normal and malignant growth.
Asunto(s)
Carcinoma de Células Pequeñas/análisis , Neoplasias Pulmonares/metabolismo , Pulmón/metabolismo , Péptidos/genética , Secuencia de Aminoácidos , Línea Celular , ADN/análisis , Feto/metabolismo , Péptido Liberador de Gastrina , Regulación de la Expresión Génica , Humanos , Inmunoquímica , Neoplasias Pulmonares/análisis , Péptidos/metabolismo , ARN Mensajero/análisis , Células Tumorales CultivadasRESUMEN
The gastrin-releasing peptide (GRP) is a neuropeptide hormone and growth factor produced normally by neural and neuroendocrine cells, as well as by human small-cell lung cancer (SCLC) tumors and derived cell lines. This study compares the structure of the human prepro-GRP gene in four SCLC cell lines that express variable levels of steady-state GRP mRNA. The regulation of GRP gene expression appears to be at the level of primary transcription based on nuclear run on studies. In the two SCLC cell lines expressing GRP we find a single transcription start site for GRP mRNA, and near this site we find four DNase I hypersensitive sites. These hypersensitive sites are absent in the two cell lines that do not express GRP. The presence of DNase hypersensitive sites in the promoter region of the GRP gene is the structural feature that best correlates with transcriptional activation. These four DNase hypersensitive sites are candidates for cis acting regulatory regions, which may be important in determining the level of transcription of the human prepro GRP gene.
Asunto(s)
Desoxirribonucleasa I/genética , Gastrinas/genética , Regulación de la Expresión Génica , Péptidos/genética , Transcripción Genética , Secuencia de Bases , Carcinoma de Células Pequeñas/genética , Línea Celular , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Péptido Liberador de Gastrina , Amplificación de Genes , Humanos , Neoplasias Pulmonares/genética , Datos de Secuencia Molecular , Mapeo Nucleótido , Regiones Promotoras Genéticas , Precursores de Proteínas/genética , ARN Mensajero/metabolismoRESUMEN
The 27-amino-acid gastrin-releasing peptide (GRP1-27) is a neuropeptide and growth factor that is synthesized by various neural and neuroendocrine cells. The major pro-GRP hormone (isoform I) contains both GRP1-27 and a novel C-terminal extension peptide termed pro-GRP31-125. In order to define potentially active neuropeptides that could be generated from this novel protein domain, we analyzed the posttranslational processing of endogenous human pro-GRP1-125 in a small-cell lung cancer cell line. Because such studies are much easier in an overexpression system, we investigated at the same time the posttranslational processing of baculovirus-expressed human pro-GRP1-125 in an insect ovary cell line. In the small-cell lung cancer cell line, GRP1-27 was cleaved as expected from the endogenous prohormone at a pair of basic amino acids (29 and 30) and alpha-amidated at its C-terminal methionine; however, a number of novel peptides were generated by additional cleavages in the pro-GRP31-125 domain. In the insect ovary cell line, GRP1-27 was cleaved from the expressed prohormone by a different mechanism, as were a number of other peptides that appeared to be similar in size to those produced by the human neuroendocrine tumor cell line. These data show for the first time that an insect ovary cell line that is widely used to overexpress proteins can process a human neuropeptide precursor. They also reveal the existence of novel pro-GRP-derived peptides that are candidates for biologically active ligands.
Asunto(s)
Hormonas Gastrointestinales/genética , Péptidos/genética , Precursores de Proteínas/genética , Procesamiento Proteico-Postraduccional , Secuencia de Aminoácidos , Animales , Línea Celular , Clonación Molecular , Genes , Humanos , Virus de Insectos/genética , Datos de Secuencia Molecular , Mariposas NocturnasRESUMEN
The gastrin-releasing peptide (GRP) is a gastrointestinal hormone, a neuropeptide, and a growth factor. To study systematically GRP gene expression in embryonic and in adult mammals, we cloned the rat prepro-GRP gene from brain cDNA libraries. Analysis of these cDNA clones along with organ-specific mRNA studies show that there are prepro-GRP transcripts initiating in the brain, but not in the duodenum, from a novel promoter. The latter is located at least four hundred base pairs upstream from a second promoter that is active in both duodenum and in brain. In contrast to human prepro-GRP, there is no alternative processing of the 3'-region of rat brain and duodenum prepro-GRP transcripts; hence, these mRNAs encode a single 147 amino acid prepro-GRP hormone that is homologous to the third isoform of the human prepro-GRP hormone.
Asunto(s)
Encéfalo/metabolismo , Péptidos/genética , Precursores de Proteínas/genética , Transcripción Genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN/genética , Duodeno/metabolismo , Endonucleasas , Humanos , Ratones , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Regiones Promotoras Genéticas , ARN Mensajero/genética , Ratas , Endonucleasas Específicas del ADN y ARN con un Solo FilamentoRESUMEN
A prepro-gastrin-releasing peptide (GRP) gene was introduced into Swiss 3T3 mouse embryo fibroblasts by DNA transfection in an attempt to establish autocrine growth stimulation. Clonal transfectants expressed varying amounts of GRP encoding mRNA. They synthesized and secreted a approximately 15-kd pro-GRP hormone but not fully processed 2.8-kd GRP. Accordingly, no changes in growth properties were associated with GRP gene expression. We postulate that Swiss 3T3 fibroblasts lack the enzymes necessary to process significantly pro-GRP into biologically active peptides and that this deficiency may be responsible for the failure to establish autocrine growth stimulation in the transfected cells.
Asunto(s)
Biosíntesis de Péptidos , Precursores de Proteínas/biosíntesis , Transfección , Animales , Línea Celular Transformada , Fibroblastos/metabolismo , Péptido Liberador de Gastrina , Vectores Genéticos , Ratones , Péptidos/genética , Precursores de Proteínas/genéticaRESUMEN
Three rat monoclonal antibodies were selected for the immunodetection of small cell lung cancer metastases in bone marrow and other hematologic samples. By membrane immunofluorescence, they define three distinct surface antigens here termed lung cancer-associated antigens or LCAs. The latter are widely expressed on small cell lung cancer and non-small cell lung cancer cells/cell lines, but not detectable on a variety of normal and transformed bone marrow, blood and lymphoid cells. Anti-LCA1 (IgM) is similar to the many anti-lacto-N-fucopentaose III IgM antibodies rasied against human tumors. In contrast, anti-LCA2 (IgG2b) and anti-LCA3 (IgG2a) define surface proteins of 29, 32, 41 and 98 kilodaltons, respectively, that have not been reported earlier. These three reagents have immunodiagnostic potential, since in combination they label all 49 lung cancer cell lines tested. Their ability to detect lung cancer metastases in patient's bone marrow samples is documented in an accompanying paper.
Asunto(s)
Anticuerpos Monoclonales , Enfermedades de la Médula Ósea/diagnóstico , Carcinoma de Células Pequeñas/secundario , Neoplasias Pulmonares/patología , Animales , Antígenos de Neoplasias/análisis , Carcinoma de Células Pequeñas/diagnóstico , Técnica del Anticuerpo Fluorescente , Humanos , RatasRESUMEN
Detection of bone marrow metastases by indirect immunofluorescence methods was investigated using three monoclonal antibodies (MoAbs) raised against small cell lung cancer (SCLC). These antibodies, designated anti-LCA1, -LCA2 and -LCA3, recognize three different antigens on the surface of SCLC cells. Eighty-four bone marrow samples from 74 different patients were studied. Whereas tumor cells were found in 32 (38%) by MoAb staining, only 10 (12%) were positively identified using conventional morphological methods. Nine out of the morphologically positive specimens showed reactivity with at least two monoclonal antibodies. Among the 32 samples proven positive by immunofluorescence, an important antigenic variability was noted. Anti-LCA1 recognized tumor cells in 62%, anti-LCA2 and anti-LCA3 in 53%. Due to the recognition of bone marrow involvement by fluorescence methods in 26% of the 34 patients classified as limited disease, a new subgroup of limited disease patients was defined whose prognosis remains undetermined. Our results confirm the utility of immunodetection in the diagnosis of SCLC bone marrow metastases and emphasize the advantage of using a panel of MoAbs with different antigenic specificities. Further study is needed to determine the prognostic significance of bone marrow involvement established by immunodetection.
Asunto(s)
Anticuerpos Monoclonales , Enfermedades de la Médula Ósea/diagnóstico , Carcinoma de Células Pequeñas/secundario , Neoplasias Pulmonares/patología , Carcinoma de Células Pequeñas/diagnóstico , Carcinoma de Células Pequeñas/inmunología , Técnica del Anticuerpo Fluorescente , HumanosRESUMEN
It is well known that small cell lung cancer (SCLC) has a high propensity to metastasize to the bone marrow and that such involvement has a prognostic significance. A more accurate detection of these bone marrow metastases is thus mandatory. In this study, we analysed the results of the detection of these metastases using an indirect immunofluorescence test. For this purpose, 3 anti-SCLC rat monoclonal antibodies (MoAbs) specific for 3 different antigens (LCA1, LCA2, LCA3) have been utilized to examine 59 bone marrow samples from patients at time of diagnosis and 20 samples from chemotherapy treated patients. Eight patients had bone marrow clearly involved by morphological analysis. They all had fluorescent cells recognized at immunodetection with at least 2 MoAbs positive for 7 out of the 8 samples. Fifteen samples, negative by morphological analysis, were proven positive by immunofluorescence. In 12 cases, involvement was detected only by 1 MoAb (6 anti-LCA1, 2 anti-LCA2, 4 anti-LCA3). A correlation was found between the number of samples proven positive by morphological analysis and the number of positive MoAbs for these samples (p less than 0.005). Among the bone marrow samples provided by the 32 limited disease patients, LCA positive cells were detected in 9 (28%) compared to 14 out of the 27 (52%) samples from extensive disease patients (p less than 0.05). We concluded that the indirect immunofluorescence with a panel of MoAbs increases the rate of detection of bone marrow SCLC metastases.
Asunto(s)
Anticuerpos Monoclonales , Médula Ósea/patología , Carcinoma de Células Pequeñas/patología , Neoplasias Pulmonares/patología , Anticuerpos Monoclonales/inmunología , Antígenos de Neoplasias/análisis , Carcinoma de Células Pequeñas/inmunología , Técnica del Anticuerpo Fluorescente , Humanos , Metástasis de la NeoplasiaRESUMEN
The hemagglutinin-neuraminidase (HN) gene of human type 3 parainfluenza virus has been inserted into a baculovirus expression vector under the control of the polyhedrin promoter. HN protein produced in insect cells by the recombinant baculovirus appeared to be glycosylated, was transported to the cell surface, and was biologically active. All of the HN epitopes previously mapped functionally to a region(s) involved in neuraminidase and/or hemagglutination activities were conformationally unaltered on the recombinant protein. The HN produced in this system also induced a protective immune response in immunized cotton rats. From these studies we conclude that the HN expressed in insect cells represents a source of authentic HN glycoprotein suitable for structural analysis and immunization.
Asunto(s)
Antígenos Virales/genética , Hemaglutininas Virales/genética , Virus de Insectos/genética , Neuraminidasa/genética , Virus de la Parainfluenza 3 Humana/genética , Respirovirus/genética , Proteínas del Envoltorio Viral/genética , Animales , Células Cultivadas , ADN Recombinante , Regulación de la Expresión Génica , Glicoproteínas/genética , Proteína HN , Insectos , Proteínas de la Membrana/genética , Vacunas Sintéticas/inmunología , Vacunas Virales/inmunologíaRESUMEN
A complementary DNA clone encoding human pre-pro gastrin-releasing peptide, a 27-amino acid neuropeptide and putative growth factor, was used to determine the chromosomal location of this gene. Southern blot hybridization to genomic DNA isolated from a panel of human-rodent somatic cell hybrids unambiguously maps this gene to human chromosome 18. In situ chromosomal hybridization confirms the hybrid data and further localized the gene to chromosome band 18q21. Karyotypic abnormalities in tumors and inherited disease states which involve chromosome band 18q21 may now be studied for correlated changes in the structure and expression of the human GRP gene.