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1.
Hybridoma (Larchmt) ; 24(4): 182-8, 2005 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-16120023

RESUMEN

In order to generate an immune response against myeloma cells in an homogenous murine model, a stable hybrid cell line (DCSp) was established through the syngenic fusion between mouse dendritic cells (DC) and mouse Sp2/0 myeloma cells. DCSp cells behaved as potent T cell stimulators and were able to induce Sp2/0 specific cytotoxicity. When mice were immunized with irradiated hybrids before SP2/0 injection, they exhibited a significantly higher rate of survival as compared with controls. When tumors were detected, their emergence was not delayed, and time elapsed between tumor clinical perception and death remained unchanged. A humoral immune response was also always associated. We assume that this stable dendritoma cell line can be considered a valuable tool for myeloma studies in an homogenous mouse model. The efficiency of dendritoma as a weapon against tumor cells and the benefit of syngeny in experimental models are discussed.


Asunto(s)
Vacunas contra el Cáncer/inmunología , Células Dendríticas/inmunología , Hibridomas , Mieloma Múltiple/inmunología , Animales , División Celular , Ratones , Ratones Endogámicos BALB C , Microscopía Electrónica , Mieloma Múltiple/patología , Linfocitos T Citotóxicos/citología
2.
Parasitol Res ; 89(6): 487-94, 2003 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-12658461

RESUMEN

The behavior of microtubular structures during division was followed by immunofluorescence in Trichomonas vaginalis using an anti-alpha-tubulin monoclonal antibody together with nuclear staining by DAPI, allowing us to describe successive mitotic stages. In contrast to recent reports, we showed that: (1) the microtubular axostyle-pelta complex depolymerized during division, (2) the flagella were assembled during mitosis, and (3) the flagellar number was restored in each daughter kinetid before cytokinesis. Observation of griseofulvin-treated T. vaginalis cells revealed that the elongation of the mitotic spindle or paradesmosis was not the main motile force separating the daughter kinetids to opposite poles during division, suggesting the existence of other mechanisms and/or molecules involved in this morphogenetic event. Examination of treated cells re-incubated in fresh medium showed the nucleation of microtubules radiating from the perinuclear area, the origin of which is discussed. Finally, we confirm the effectiveness of griseofulvin against T. vaginalis and propose that this antifungal drug could be a promising antitrichomonal agent.


Asunto(s)
Antitricomonas/farmacología , Griseofulvina/farmacología , Microtúbulos/efectos de los fármacos , Trichomonas vaginalis/efectos de los fármacos , Trichomonas vaginalis/ultraestructura , Animales , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , División Celular , Técnica del Anticuerpo Fluorescente/métodos , Inmunohistoquímica/métodos , Microtúbulos/ultraestructura , Morfogénesis , Factores de Tiempo , Trichomonas vaginalis/citología , Trichomonas vaginalis/crecimiento & desarrollo
3.
Hybridoma ; 19(3): 229-39, 2000 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-10952411

RESUMEN

In this work, we have constructed two functional mouse/human chimeric antibodies (IgMkappa and IgG1kappa isotypes) by inserting genomic DNA fragments encoding VH and Vkappa variable regions of the murine monoclonal antibody IgMK-83D4 into mammalian expression vectors containing human mu, gamma1, and kappa constant exons, and by transfecting them into the nonsecreting mouse myeloma X-63 cell line. In previous works, we have demonstrated that 83D4 murine mAb reacts with Tn determinant (GalNAcalpha-O-Ser/Thr) expressed in 90% of breast, ovary, and colon carcinomas. Both expressed chimeric antibodies were purified from the transfected cell line supernatant by affinity chromatography, and their reactivities against Tn antigen were confirmed by ELISA on asialo ovine submaxilar mucin and immunofluorescence studies on MCF-7 breast carcinoma cell line. We have demonstrated by gel filtration chromatography, that the principal secreted forms were monomers for IgG1kappa and pentamers for IgMkappa. The binding affinities of these chimeric antibodies against synthetic Tn glycopeptides, were evaluated by surface plasmon resonance showing an affinity constant similar to that of 83D4 native antibody for IgMkappa and a lower affinity constant for IgG1kappa chimeric antibody. On the other hand, the replacement of mouse C regions with human C regions confers both chimeric antibodies the ability to activate human complement. These mouse/human chimeric antibodies should be much less immunogenic and could play an important role in the lysis of tumor cell expressing Tn-antigen. Therefore, these anti-Tn chimeric antibodies could be considered as potential tools for human in vivo studies.


Asunto(s)
Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/genética , Especificidad de Anticuerpos/genética , Antígenos de Carbohidratos Asociados a Tumores/inmunología , Antígenos de Carbohidratos Asociados a Tumores/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Animales , Anticuerpos Monoclonales/aislamiento & purificación , Fusión Celular , Vectores Genéticos , Humanos , Hibridomas/metabolismo , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/genética , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/aislamiento & purificación , Cadenas Ligeras de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/aislamiento & purificación , Inmunoglobulina M/biosíntesis , Inmunoglobulina M/genética , Cadenas kappa de Inmunoglobulina/genética , Cadenas kappa de Inmunoglobulina/aislamiento & purificación , Ratones , Ratones Endogámicos C57BL , Proteínas Recombinantes de Fusión/inmunología , Transfección , Células Tumorales Cultivadas
4.
Am J Clin Pathol ; 112(2): 171-8, 1999 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-10439796

RESUMEN

We compared the efficacy of 4 methods for isolating circulating tumor cells: immunocapture with Ber-EP4-coated magnetic beads, density gradient separation, ammonium chloride, and distilled water-mediated erythrocyte lysis. Human blood from healthy volunteers was mixed with serial dilutions of prostate (LNCaP) and liver (HepG2) derived tumor cells. Isolation of circulating tumor cells was followed by reverse transcriptase-polymerase chain reaction with primers specific for prostate-specific antigen and alpha-fetoprotein. Ber-EP4 antigen expression was evaluated by immunohistochemistry in 27 hepatocellular carcinomas and 34 prostate adenocarcinomas. Peripheral blood samples from 12 patients with hepatocellular carcinoma and 10 with prostate adenocarcinoma also were tested. Density gradient separation and Ber-EP4 immunocapture were the most sensitive techniques for isolating circulating tumor cells in in vitro tests. Isolation by density gradient separation was significantly more sensitive than Ber-EP4 immunocapture when applied to peripheral blood samples of patients with cancer, a result consistent with the variable expression of Ber-EP4 antigen that we found by immunohistochemistry in prostate adenocarcinomas and hepatocellular carcinomas.


Asunto(s)
Antígenos de Superficie/inmunología , Biomarcadores de Tumor/inmunología , Separación Inmunomagnética/métodos , Células Neoplásicas Circulantes , Adenocarcinoma/sangre , Cloruro de Amonio/farmacología , Anticuerpos Monoclonales , Carcinoma Hepatocelular/sangre , Centrifugación por Gradiente de Densidad/métodos , Cartilla de ADN/química , ADN de Neoplasias/análisis , Células Epiteliales/inmunología , Células Epiteliales/patología , Estudios de Evaluación como Asunto , Hemólisis/efectos de los fármacos , Humanos , Técnicas para Inmunoenzimas , Neoplasias Hepáticas/sangre , Masculino , Células Neoplásicas Circulantes/inmunología , Células Neoplásicas Circulantes/patología , Antígeno Prostático Específico/genética , Neoplasias de la Próstata/sangre , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sensibilidad y Especificidad , Células Tumorales Cultivadas , alfa-Fetoproteínas/genética
6.
Hybridoma ; 16(4): 317-24, 1997 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-9309422

RESUMEN

We report here the first amino acid sequence of an anti-Tn monoclonal antibody raised against human breast cancer cells and show that a single chain Fv fragment of this IgM retains the Tn-binding specificity as defined by functional assays with asialo-OSM and membrane extracts from MCF-7 cells. Sequence comparisons and molecular modeling of 83D4 indicate that the antibody combining site displays a cavity-like feature primarily defined by the CDR H1 and H2 loops. This pocket could accommodate a single Tn molecule, thus, suggesting a structural explanation for the predominant expression of a particular VH gene segment in a group of antibodies that recognize tumor-associated antigens arising from an aberrant O-glycosylation.


Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Antineoplásicos/inmunología , Antígenos de Carbohidratos Asociados a Tumores/inmunología , Fragmentos de Inmunoglobulinas/inmunología , Inmunoglobulina M/inmunología , Región Variable de Inmunoglobulina/inmunología , Cadenas kappa de Inmunoglobulina/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Antineoplásicos/genética , Especificidad de Anticuerpos , Antígenos de Carbohidratos Asociados a Tumores/genética , Secuencia de Bases , Neoplasias de la Mama/inmunología , Carcinoma/inmunología , Clonación Molecular , Amplificación de Genes , Humanos , Hibridomas , Fragmentos de Inmunoglobulinas/genética , Inmunoglobulina M/genética , Región Variable de Inmunoglobulina/genética , Cadenas kappa de Inmunoglobulina/genética , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión , Alineación de Secuencia , Análisis de Secuencia de ADN , Células Tumorales Cultivadas
7.
FEBS Lett ; 412(1): 190-6, 1997 Jul 21.
Artículo en Inglés | MEDLINE | ID: mdl-9257718

RESUMEN

The partial amino acid sequence of the tetrameric isolectin B4 from Vicia villosa seeds has been determined by peptide analysis, and its three-dimensional structure solved by molecular replacement techniques and refined at 2.9 A resolution to a crystallographic R-factor of 21%. Each subunit displays the thirteen-stranded beta-barrel topology characteristic of legume lectins. The amino acid residues involved in metal- and sugar-binding are similar to those of other GalNAc-specific lectins, indicating that residues outside the carbohydrate-binding pocket modulate the affinity for the Tn glycopeptide. Isolectin B4 displays an unusual quaternary structure, probably due to protein glycosylation.


Asunto(s)
Antígenos de Carbohidratos Asociados a Tumores/metabolismo , Fabaceae/química , Lectinas/química , Plantas Medicinales , Secuencia de Aminoácidos , Simulación por Computador , Cristalografía por Rayos X , Lectinas/metabolismo , Sustancias Macromoleculares , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Lectinas de Plantas , Estructura Secundaria de Proteína
8.
Anticancer Res ; 17(4A): 2639-46, 1997.
Artículo en Inglés | MEDLINE | ID: mdl-9252694

RESUMEN

BACKGROUND: The detection of occult carcinoma cells in patients with breast cancer may aid determination of prognosis and the development of new therapeutic approaches. In this study, we report a new method to detect rare human breast cancer cells, which combines an immunomagnetic separation (IMS) procedure with cytokeratin 19 (CK 19) immunostaining. MATERIALS AND METHODS: Four monoclonal antibodies (MAb) previously characterized against cell surface antigens (1BE12, ED8, 7B10 and 83D4), were evaluated for IMS optimization. Immunoseparated epithelial cells were identified using a MAb against CK 19. We compared the IMS procedure with the immunocytochemistry (ICC) and the RT-PCR for CK 19 on an "in vitro" experimental model. RESULTS: The best results in IMS procedures were obtained using MAbs 1BE12 (directed against Lewis y antigen) and ED8 (directed against MUC 1). In reconstitution experiments, using several ratios of T47D cells mixed with peripheral-blood mononuclear (PBMN) cells, the IMS procedure reliably detects one mammary carcinoma cell in 5 x 10(5) PBMN cells, whereas the ICC detects up to one T47D cell per 10(5) PBMN cells. The best sensitivity was observed with the RT-PCR (up to one T47D cell per 10(6) PBMN cells). We found the same high specificity with the three methods evaluated. CONCLUSIONS: The IMS procedure using MAbs 1BE12 or ED8 associated with CK 19 immunostaining is a specific, sensitive, and feasible method for the detection of rare human breast cancer cells. This method proved to be better than the ICC staining but its sensitivity was lower than that of RT-PCR for CK 19.


Asunto(s)
Neoplasias de la Mama/diagnóstico , Neoplasia Residual/diagnóstico , Anticuerpos Monoclonales , Neoplasias de la Mama/patología , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Inmunohistoquímica , Separación Inmunomagnética , Queratinas/genética , Queratinas/metabolismo , Reacción en Cadena de la Polimerasa , Células Tumorales Cultivadas
9.
J Clin Microbiol ; 34(12): 3002-6, 1996 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-8940438

RESUMEN

Thirteen laboratories participated in blind tests of a panel of 20 coded cerebrospinal fluid specimens (7 uninfected samples, 3 samples infected with 1 50% tissue culture infective dose [TCID50]/0.1 ml [nonenterovirus strains], and 10 samples infected with 10, 1, or 0.1 TCID50/0.1 ml [three different enterovirus serotypes]) on the Amplicor enterovirus PCR assay (Roche Diagnostic Systems). The panel was also evaluated by in-house PCR (two nested-PCR and three one-step PCR assay) or tissue culture (eight laboratories). The viral load was shown to influence greatly the sensitivity of the assay. The average sensitivity of the Amplicor test ranged from 67 to 98% for viral titers of 1 to 10 TCID50/0.1 ml, respectively; titers of 0.1 TCID50/0.1 ml resulted in a sensitivity of only 16%. The overall specificity of the Amplicor test was 98%. The Amplicor assay compared favorably to the five in-house PCR tests (no significant difference in either sensitivity or specificity) and was much more sensitive than tissue culture (P < 0.001), even for high viral loads. It was easy to perform, rapid (about 6 h), well-standardized, and appeared to be suitable for the diagnosis of enterovirus meningitis on a routine basis in laboratories trained in molecular biology techniques.


Asunto(s)
Infecciones por Enterovirus/diagnóstico , Enterovirus/genética , Meningitis/diagnóstico , Reacción en Cadena de la Polimerasa/métodos , Virología/métodos , Secuencia de Bases , Cartilla de ADN/genética , Enterovirus/aislamiento & purificación , Infecciones por Enterovirus/líquido cefalorraquídeo , Infecciones por Enterovirus/virología , Estudios de Evaluación como Asunto , Humanos , Meningitis/líquido cefalorraquídeo , Meningitis/virología , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa/normas , Reacción en Cadena de la Polimerasa/estadística & datos numéricos , ARN Viral/líquido cefalorraquídeo , ARN Viral/genética , Sensibilidad y Especificidad , Virología/normas , Virología/estadística & datos numéricos
10.
Int J Oncol ; 8(2): 401-6, 1996 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-21544376

RESUMEN

We report the development of an immuno-lectin-enzymatic assay (CA83.4) with the purpose of quantifying serum glycoproteins bearing Tn determinant (GalNAc alpha-O-Ser/Thr). An anti-Tn monoclonal antibody (83D4) is bound to the solid phase in order to capture glycoproteins. After the addition of a test sample, we used biotinylated isolectin B4 from Vicia villosa and avidin-peroxydase to act as a detection system. The linear relationship between CA83.4 determinations and the serum dilutions, the reproducibility of the dosage in intra- and inter-assay, and the specificity of the test for the N-acetylgalactosamine residue in a-glycosidic O-linkages, demonstrated the reliability of this trial. Self-recognition of Vicia villosa B4 molecules (K-D: 0.73x10(-6) M determined using biosensor technology) could determine an additional step of signal amplification in this assay. Using 0.25 units/ml of CA83.4 antigen as the cut-off level, higher values were found in 25/49 patients with breast cancer, 8/13 with colorectal carcinoma, 3/11 with lung cancer, but in none of the 49 patients with non-malignant diseases nor in 97 healthy controls. This first report on soluble Tn-glycoprotein detection assays suggests that Tn-glycoproteins are specific serological tumor markers and we believe that they could represent a valuable tool in the diagnosis of cancer.

11.
J Clin Pathol ; 48(5): 433-7, 1995 May.
Artículo en Inglés | MEDLINE | ID: mdl-7629290

RESUMEN

AIMS: To determine whether the monoclonal antibody (MoAb) 83D4, previously shown to be highly specific for carcinoma cells, can be used as an immunocytological marker to discriminate between benign and malignant cells in serous effusions; and to test for a correlation between expression of the antigen reacting with MoAb 83D4 on effusion cells and the amount of soluble 83D4 antigen in effusion fluids. METHODS: Thirty three pleural and 23 peritoneal effusions from 56 cancer patients with metastatic disease were tested for the presence of Tn associated 83D4 antigen by immunocytochemical staining, and for the presence of soluble antigen in supernatants. The patients had undergone various chemotherapy and radiation therapy protocols. RESULTS: As a result of the various types of treatment, the cytological characteristics of the cells were often modified and the antigenic epitopes may have been altered. Positive staining for 83D4 MoAb was obtained in 36 (97%) of the 37 malignant effusions, eight (73%) of 11 suspect effusions, and three (38%) of the eight apparently benign effusions (free of malignant cells). In these latter cases, cytological reassessment showed a few suspect cells in two cases. 83D4 soluble antigen was detected in 30 of 37 malignant effusions (81%), five of 11 suspected infusions (46%), and five of eight apparently benign effusions (63%). CONCLUSIONS: Immunocytochemical staining with anti-83D4 antibody is useful for differentiating reactive or atypical mesothelial cells from epithelial cells, especially in breast cancer effusions.


Asunto(s)
Antígenos de Carbohidratos Asociados a Tumores/análisis , Líquido Ascítico/inmunología , Biomarcadores de Tumor/análisis , Derrame Pleural Maligno/inmunología , Adenocarcinoma/secundario , Anticuerpos Monoclonales , Neoplasias de la Mama/patología , Femenino , Humanos , Neoplasias Ováricas/patología , Derrame Pleural Maligno/patología , Solubilidad
13.
Appl Biochem Biotechnol ; 47(2-3): 229-37; discussion 237-9, 1994.
Artículo en Inglés | MEDLINE | ID: mdl-7944340

RESUMEN

Since the two reports published in 1986 by the laboratories of R. Lerner and P. G. Schultz, it has been clearly established that antibodies may be induced to act as catalysts in numerous chemical reactions. In all cases, catalytic antibodies were elicited using a substrate-based approach. In the present article, we propose an alternative and complementary enzyme-based approach to generate catalytic antibodies. This approach uses the properties of anti-idiotypic antibodies to generate internal images of enzyme active sites. Experimental results are discussed for polyclonal and monoclonal anti-idiotypic antibodies.


Asunto(s)
Anticuerpos Antiidiotipos/biosíntesis , Anticuerpos Catalíticos/biosíntesis , Acetilcolinesterasa/química , Acetilcolinesterasa/inmunología , Animales , Anticuerpos Antiidiotipos/inmunología , Anticuerpos Catalíticos/inmunología , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Humanos , Ratones
14.
J Acquir Immune Defic Syndr (1988) ; 7(2): 116-23, 1994 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-7507991

RESUMEN

One mechanism for expanding the cellular tropism of human immunodeficiency virus (HIV) in vitro is through formation of phenotypically mixed particles (pseudotypes) with human T lymphotropic virus type I (HTLV-I). In this study we found that pseudotypes allow penetration of HIV particles into CD4-negative cells, previously nonsusceptible to HIV infection. The infection of CD4-negative cells with pseudotypes could be blocked with anti-HTLV-I serum but failed to be significantly inhibited with anti-HIV serum or a V3-neutralizing anti-gp120 monoclonal antibody. This may represent a possibility for pseudotypes to escape neutralization by the immune system in vivo. Previous reports have suggested that carbohydrate structures may be conserved neutralization epitopes on retroviruses. In this study, the neutralizing capacity of lectins and anti-carbohydrate monoclonal antibodies was found to block infection by cell-free pseudotypes in CD4-negative cells. We suggest that although viral cofactors might expand the tropism of HIV in vivo, HIV and HTLV-I seem to induce common carbohydrate neutralization epitopes.


Asunto(s)
Carbohidratos/inmunología , Antígenos VIH/inmunología , VIH/inmunología , Virus Linfotrópico T Tipo 1 Humano/inmunología , Anticuerpos Monoclonales/inmunología , Línea Celular , Epítopos/análisis , Epítopos/inmunología , Células Gigantes/inmunología , VIH/fisiología , Antígenos VIH/análisis , Humanos , Sueros Inmunes/inmunología , Lectinas/inmunología , Pruebas de Neutralización , Reacción en Cadena de la Polimerasa , Provirus/inmunología , Provirus/fisiología , Ensayo de Radioinmunoprecipitación
15.
Arch Virol ; 134(1-2): 179-84, 1994.
Artículo en Inglés | MEDLINE | ID: mdl-8279954

RESUMEN

In combination with HIV gp120 V3-loop antibody, two carbohydrate specific neutralizing antibodies (83D4 and 2G12) had a synergistic neutralizing effect on HIV infection. However, sCD4 and an antibody which blocks gp 120/CD4 binding (1B1) both displayed antagonism.


Asunto(s)
Antígenos CD4/inmunología , Anticuerpos Anti-VIH/inmunología , Proteína gp120 de Envoltorio del VIH/inmunología , VIH/inmunología , Fragmentos de Péptidos/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Línea Celular , Humanos , Ratones , Pruebas de Neutralización , Conejos
16.
Breast Cancer Res Treat ; 32(2): 139-52, 1994.
Artículo en Inglés | MEDLINE | ID: mdl-7532464

RESUMEN

The Tn determinant (GalNAc alpha-O-Ser/Thr) is expressed by about 90% of human carcinomas, but is cryptic in most normal human tissues. A murine monoclonal antibody (MAb) 83D4, developed following immunization with human breast carcinoma cells, reacts with a Tn-related epitope. In the present study we characterized the glycoprotein antigen identified by 83D4 in the human breast carcinoma cell line MCF-7. We further showed that the 83D4 antigenic determinant is masked in human milk fat globule membranes (HMFGM), and can be exposed upon mild m-periodate treatment after desialylation. Western-blot analysis resolved the 83D4 antigen from MCF-7 into two main components of 120-190 kD and > 500 kD respectively. Non equilibrium pH gradient electrophoresis/SDS PAGE revealed the acidic nature of the reactive glycoproteins (pI 4.43-4.70). 83D4 antigenic activity resolved by CsCl gradient ultracentrifugation layered on a wide range of densities (1.30-1.46 g/ml) including typical densities of mucin-like glycoproteins but also lower densities. The amino acid composition of the antigen, relatively rich in serine but poor in threonine and proline, confirmed the divergence from other mucin-like carcinoma-associated glycoproteins. Dicarboxylic amino acids were abundant, accounting in part for the acidic nature of the molecules. ELISA and Western-blot analysis of the subcellular fractions from MCF-7 cells revealed that the 83D4 antigen is mainly contained in plasma membranes (85%) from which it may be resolved into two broad bands (slow and fast migrating components). These results provide information on a group of breast carcinoma associated glycoproteins related to but different from typical mucins, and provide data on alteration of O-glycosylation in tumor cells.


Asunto(s)
Adenocarcinoma/química , Antígenos de Neoplasias/análisis , Antígenos de Carbohidratos Asociados a Tumores/análisis , Neoplasias de la Mama/química , Glicoproteínas/análisis , Leche Humana/química , Proteínas de Neoplasias/análisis , Adenocarcinoma/inmunología , Aminoácidos/análisis , Animales , Anticuerpos Monoclonales/inmunología , Antígenos de Neoplasias/inmunología , Antígenos de Neoplasias/aislamiento & purificación , Antígenos de Carbohidratos Asociados a Tumores/inmunología , Neoplasias de la Mama/inmunología , Secuencia de Carbohidratos , Epítopos/inmunología , Femenino , Glicoproteínas/inmunología , Glicoproteínas/aislamiento & purificación , Glicosilación , Humanos , Lectinas/metabolismo , Glicoproteínas de Membrana/análisis , Glicoproteínas de Membrana/inmunología , Glicoproteínas de Membrana/aislamiento & purificación , Ratones , Datos de Secuencia Molecular , Peso Molecular , Mucina-1 , Mucinas/análisis , Mucinas/inmunología , Proteínas de Neoplasias/inmunología , Proteínas de Neoplasias/aislamiento & purificación , Neuraminidasa/metabolismo , Unión Proteica , Procesamiento Proteico-Postraduccional , Fracciones Subcelulares/química , Células Tumorales Cultivadas
17.
Proc Natl Acad Sci U S A ; 90(19): 8876-80, 1993 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-8415624

RESUMEN

Monoclonal antibody 9A8 was selected by immunizing mice with AE-2, a monoclonal antibody directed against the active site of acetylcholinesterase. In accordance with the idiotypic network theory, monoclonal anti-idiotypic antibody 9A8 displayed internal-image properties of the original immunogen, the acetylcholinesterase active site. Hydrolysis of acetylthiocholine and related esters of thiocholine by 9A8 follows saturation kinetics and kinetic parameters were determined. The hydrolytic activity is characterized by a lowered kcat value (81 s-1) and an increased Km value (0.6 mM) when compared with the original enzyme. However, the rate acceleration (kcat/kuncat = 4.15 x 10(8) remains higher than for the esterase activities usually described for catalytic antibodies directed against transition-state analogs. The 9A8 activity exhibits a relaxation of specificity toward both substrates and inhibitors. This specificity does not correspond to a known enzymatic activity. The anti-idiotypic approach should be valuable for producing different structural and functional copies of the same enzyme active site. This should allow further insights into structure-activity relationships. Furthermore, use of chemically modified enzymes as immunogens may result in anti-idiotypic antibodies with catalytic activities not found in the native enzymes.


Asunto(s)
Anticuerpos Antiidiotipos/metabolismo , Anticuerpos Monoclonales/metabolismo , Colinesterasas/metabolismo , Acetilcolinesterasa/metabolismo , Animales , Anticuerpos Antiidiotipos/aislamiento & purificación , Anticuerpos Monoclonales/aislamiento & purificación , Sitios de Unión , Butirilcolinesterasa/metabolismo , Catálisis , Inhibidores de la Colinesterasa/farmacología , Células Clonales , Hibridomas , Sueros Inmunes/metabolismo , Fragmentos Fab de Inmunoglobulinas/aislamiento & purificación , Fragmentos Fab de Inmunoglobulinas/metabolismo , Cinética , Ratones , Especificidad por Sustrato
18.
J Mol Biol ; 230(2): 670-2, 1993 Mar 20.
Artículo en Inglés | MEDLINE | ID: mdl-8464073

RESUMEN

Isolectin B4 isolated from Vicia villosa seeds is specific for the Tn antigen, a carcinoma-associated molecular marker. Crystals of the isolectin grown in the presence of carbohydrate are tetragonal, space group P4(1) (or P4(3), with a = 91.3 A, c = 151.7 A and one tetramer in the asymmetric unit. The crystals diffract X-rays to 2.8 A resolution and are suitable for high-resolution structural analysis.


Asunto(s)
Antígenos de Carbohidratos Asociados a Tumores , Lectinas/química , Acetilgalactosamina , Cristalización , Humanos , Lectinas/aislamiento & purificación , Sustancias Macromoleculares , Difracción de Rayos X/métodos
20.
In Vitro Cell Dev Biol ; 28A(11-12): 716-24, 1992.
Artículo en Inglés | MEDLINE | ID: mdl-1282913

RESUMEN

The growth and differentiation of normal human mammary epithelial cells (HMEC) were studied after propagation of serial cultures from breast tissue biopsies from 42 mammoplasty patients. Cells were grown for up to 7 mo. in low calcium medium. HMEC cultures displayed heterogeneous growth patterns, according to the average doubling time of 44 +/- 6 h for 32 generations. Proliferation peaked at Day 30. HMEC maintained a normal karyotype and were organized in ductlike structures when cultured in collagen gel matrix. The cultures retained several phenotype traits of the epithelial lineage, including the expression of cytokeratins 18 and 19, specific mammary gland antigens, as shown by indirect HMEC immunostaining by the monoclonal antibodies DF3, EMA, 7B10, and 1BE12. Estrogen receptors were undetectable, whereas progesterone receptors were present at very low density. High-affinity cell surface receptors for epidermal growth factor (EGF) (Kd = 1.1 x 10(-10) M) were observed at a density of 50,000 to 100,000 sites per cell. Accordingly, [3H]thymidine incorporation in HMEC was optimally stimulated by EGF at concentrations of 10(-11) to 10(-10) M. HMEC were also seen to possess functional VIP receptors linked to the adenylate cyclase system, as we previously observed in seven human breast cancer cell lines. These results show that long-term cultures of HMEC provide useful models for studying the growth and differentiation of the normal human mammary gland, and the role of growth factors and hormones in these functions.


Asunto(s)
Mama/citología , Receptores ErbB/análisis , Adolescente , Adulto , Animales , Anticuerpos Monoclonales , Mama/química , Mama/ultraestructura , Diferenciación Celular , División Celular , Células Cultivadas , Células Epiteliales , Epitelio/química , Epitelio/ultraestructura , Femenino , Humanos , Cariotipificación , Queratinas/análisis , Ratones , Persona de Mediana Edad , Fenotipo , Receptores de Estrógenos/análisis , Receptores de la Hormona Gastrointestinal/análisis , Receptores de Progesterona/análisis , Receptores de Péptido Intestinal Vasoactivo , Factores de Tiempo
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