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C Bernabeu

Publications and source records attributed to C Bernabeu.

At least 55 records · Page 3Linked to original sources
PubMed1989

Different functional domains on the transferrin receptor molecule defined by monoclonal antibodies.

Three monoclonal antibodies (mAb) FG 1/5, FG 1/6 and FG 2/12, specific for the human transferrin receptor molecule (TR), have been used to define epitopes on the TR molecule and to block natural killer lysis. FG 2/12 mAb but not FG 1/5 or FG 1/6 blocked [125I-] transferrin binding to the cellular receptor. Furthermore, FG 1/5 and FG 1/6 mAbs competed out the binding of each other to the cells but not significantly that of FG 2/12. As expected, the binding of F2/12 but not of FG 1/5 or FG 1/6 was inhibited by transferrin. In addition, FG 2/12 inhibited in a dose-dependent manner the NK activity of purified T3- large granular lymphocyte effector cells against HeLa or Molt-4 cells but not against K-562 or U937 cells. FG 1/5 preferentially inhibited NK activity against HeLa cells and FG 1/6 mAb was completely uneffective. These inhibitions were stronger at low effector to target cell (E:T) ratios than at high E:T ratios, suggesting that NK cells and anti-TR mAbs compete for the same site in the target cell. It was shown that FG 1/5 and FG 2/12 mAbs blocked cells' conjugate formation by acting at the target cell level. Our results confirm the role of TR as a one of the target structures in NK lysis and suggest that the epitope recognized by NK cells is close to but different from the transferrin binding site.

Antibodies, Monoclonal↗
PubMed1988

Adjuvant-induced polyarthritis. Synovial cell activation prior to polyarthritis onset.

We studied synoviocytes obtained from rats that had been injected with Freund's complete adjuvant 12 days prior to killing. We found that several activation parameters were affected in these synoviocytes, namely, the concanavalin A protein-binding pattern, the production of superoxide, and the appearance of the p77 polypeptide, which we have previously shown to be associated with the activation caused by the induction of polyarthritis. Our findings suggest that prior to the establishment of the inflammatory process, synoviocytes are in a state of activation, and may be a component of the molecular mechanism during the early stages of disease.

Animals↗
PubMed1988

Characterization of a CD11c-reactive monoclonal antibody (HC1/1) obtained by immunizing with phorbol ester differentiated U937 cells.

A mouse monoclonal antibody (HC1/1) specific for a differentiation marker of human monocytes and granulocytes has been generated by using as immunogen the monocytic cell line U937 differentiated with phorbol esters. This differentiation antigen has been characterized as the p150/95 member of the CD11 family of glycoproteins by cellular distribution studies, immunoprecipitation and competition experiments with MAb 3.9 specific for the CD11c antigen. Immuno-alkaline phosphatase staining of normal tissue sections with the HC1/1 MAb demonstrated that the CD11c antigen is a useful macrophage marker. Furthermore, the MAb HC1/1 stains specifically populations of macrophages on skin and lymph node sections from different pathologies such as Sarcoidosis, Granuloma annulare, Lepromatous leprosy and Toxoplasmosis.

Antibodies, Monoclonal↗
PubMed1988

Autoantibodies from rheumatoid arthritis patients recognize antigens on the synoviocyte surface.

We have found autoantibodies in the sera from rheumatoid arthritis (RA) patients which recognize two cell surface antigens of approximately 70 kDa and 28 kDa from synoviocyte extracts as detected by immunoprecipitation analysis. These polypeptides were immunoprecipitated from extracts containing mainly macrophage-like synoviocytes (type A) but not from extracts of homogeneous fibroblast-like synoviocytes (type B). These autoantigens are not selectively expressed by RA synoviocytes, since both RA and non-rheumatoid synovia were reactive for RA sera. From the panel of different RA sera tested, 64% immunoprecipitated the 70 kDa band, and 27% recognized the 28 kDa polypeptide. These differences in the specificity of the sera seemed to be related to the clinical state of the donor. The sera from patients suffering from other autoimmune diseases such as autoimmune thyroiditis and systemic lupus erythematosus (SLE) do not appear to be reactive for these specificities, but sera from patients with Sjögren's syndrome, psoriatic arthritis, and Crohn's disease showed a weak cross-reactivity with the 70 kDa polypeptide. This autoreactivity against synovial cells in RA supports the idea that these cells participate in the initial immune response of the disease.

Antigen-Antibody Reactions↗
PubMed1987

Biochemical analysis of synoviocytes from normal and arthritic rats. Evidence for an activated state associated with adjuvant polyarthritis.

Adjuvant-induced polyarthritis in rats is a common model system used for the study of the synovitis that occurs in rheumatoid arthritis. Synoviocytes A, the major cell type covering the internal surface of the joint, could be involved in the pathogenesis of rheumatoid arthritis because of their increased proliferation and the intraarticular manifestations of the disease. So far only a few molecular studies have been reported on synoviocytes upon arthritis induction. We report here changes in polypeptides, between control and arthritic synoviocytes, by using two different radiolabeling methods and two-dimensional gel electrophoresis analysis. Major differences were found using metabolic labeling on regions of tropomyosins, cyclin, tubulins and vimentin. In addition, external surface labeling of the cells with lactoperoxidase showed clear differences between control and arthritic synoviocytes in the region of 77-100-kDa proteins. Some of these differences can be reproduced by certain macrophage activators such as phorbol myristate acetate and lipopolysaccharide acting on synoviocytes in vitro and in vivo respectively. These results exclude the possibility that the changes observed were due to a possible infiltration of other cell types in the arthritic synovia and strongly support the existence of an activated state of synoviocytes associated with arthritis induction.

Animals↗
PubMed1987

Involvement of the CD4 molecule in a post-activation event on T cell proliferation.

A monoclonal antibody (mAb) directed to a human leukocyte 55-kDa cell surface molecule with identical cellular distribution and biochemical properties to the CD4 was able to inhibit T cell proliferation induced either in a mixed lymphocyte culture or by activation with mAb anti-CD3, anti-CD2 or phytohemagglutinin. The inhibitory effect of anti-CD4 was observed in the absence of monocytes and was directly exerted on T4+ cells. This effect on cellular proliferation appears to be due to an inhibition of a postactivation event since the rise of cytoplasmic Ca2+ after activation with anti-CD3 mAb is not affected by the presence of anti-CD4 and the proliferation that occurs after an activation pulse of 3 h with ionophore and phorbol myristate acetate can be inhibited when the anti-CD4 is added after the pulse period. Kinetic studies demonstrated that the inhibition of cellular proliferation by anti-CD4 mAb was observed even if the antibody was added as late as 18-24 h after the initiation of the culture. The effect of this blocking anti-CD4 mAb on the interleukin (IL) 2/IL 2 receptor signalling pathways was also examined. The presence of anti-CD4 slightly affected the production of IL2. In fact, addition of exogenous recombinant IL2 at the initiation of the cultures did not restore the proliferative response. However, anti-CD4 had a strong inhibitory effect on the expression of IL2 receptors as analyzed by direct immunofluorescence cytometry. Taken together, these results indicate that the binding of the anti-CD4 mAb to T cells interferes with a late metabolic step being capable of abolishing the proliferative activity of fully activated cells.

Animals↗
PubMed1987

Interaction between the CD45 antigen and phytohemagglutinin. Inhibitory effect on the lectin-induced T cell proliferation by anti-CD45 monoclonal antibody.

Immunoprecipitation studies from 125I-labeled T cells, previously activated with the lectin phytohemagglutinin (PHA) from Phaseolus vulgaris, showed a preferential association between the CD45 glycoprotein family containing members of 220, 205, 190 and 180 kDa, and a protein of 33 kDa. This 33-kDa protein, with an identical molecular mass as PHA, was not associated with other highly expressed molecules such as class I histocompatibility antigens (HLA-A,B,C), transferrin receptor, CDw44 and CD11a. Further immunoprecipitation analysis from peripheral blood lymphocytes incubated with 125I-labeled PHA confirmed the identification of the CD45-associated 33-kDa protein as the lectin PHA. These results prompted us to analyze the possible involvement of the CD45 molecules in the T cell activation process induced by PHA. Thus, two monoclonal antibodies specific for CD45 antigens were able to inhibit the T cell proliferation induced by the lectin. This inhibitory ability was exerted by affecting both the interleukin 2 production and the interleukin 2 receptor expression.

Antibodies, Monoclonal↗
PubMed1986

Quantitative measurement of human immunoglobulin E using monoclonal antibodies to distinct epitopes.

Monoclonal antibodies (MAb) which recognize distinct epitopes on human immunoglobulin E have been used to develop two-site sandwich radio- and enzyme-linked immunoassays for the quantitation of human IgE. In the first step, a purified anti-IgE MAb coated to polyvinyl or polystyrene microtiter plates specifically bound the IgE contained in the samples. In the second step, another anti-IgE MAb (either iodinated or conjugated to beta-galactosidase) directed to a different antigenic determinant was used to estimate the amount of bound IgE. This simple method permitted the determination of IgE concentrations of 10 ng/ml and greater in about 3 h. Coefficients of variation on a single day did not exceed 7.5% for IgE levels, covering a wide range of the standard curve. The values obtained on serum samples showed a good correlation with those obtained using the paper radioimmunosorbent test (PRIST).

Antibodies, Anti-Idiotypic↗
PubMed1986

VLA-3: a novel polypeptide association within the VLA molecular complex: cell distribution and biochemical characterization.

The family of human T cell activation-associated proteins, named VLA complex, is formed by the molecular association of cell surface glycoproteins of 210, 165, 130 and 80 kDa. In this report, we describe eight different monoclonal antibodies (HP mAb) specific for the 80-kDa polypeptide which preferentially associates with the 165-kDa member. Comparative immunoprecipitation and cell-binding studies demonstrated that the HP mAb recognize an epitope(s) on the 165/80 kDa subset different from those recognized by other anti-VLA mAb previously described. Furthermore, cellular and tissue distribution studies by flow cytometry and peroxidase staining showed that the HP reactivity pattern is different from other VLA specificities. The 165/80-kDa complex defined by HP mAb is present on human thymocytes, peripheral blood lymphocytes as well as on T, B and myelomonocytic cell lines. However, only the 80-kDa subunit was precipitated by HP mAb from activated T lymphocytes. These results suggest that the association between the 165- and 80-kDa subunits diminishes during the activation process, and that the epitopes recognized by the HP mAb are located on the 80-kDa protein. The novel polypeptide association of 165/80 kDa has been termed VLA-3.

Antibodies, Monoclonal↗
PubMed1985

beta 2-Microglobulin from serum associates with several class I antigens expressed on the surface of mouse L-cells.

Bovine beta 2-microglobulin (beta 2-m) present in fetal calf serum (FCS) is able to replace endogenous beta 2-m associated with several class I antigens from human and mouse cells maintained in culture [Bernabeu et al. (1984) Nature, Lond. 308, 642-645]. Here we show that human HLA-A2 and HLA-B7, as well as mouse H-2Ld and H-2Dd heavy chains expressed after gene transfer in mouse L-cells, associate to a large extent with bovine beta 2-m. We also demonstrate that bovine beta 2-m associated with the endogenous H-2Kk/Dk heavy chains generates an antibody response when L-cells are injected into syngeneic C3H mice.

Animals↗
PubMed1984

Recognition of HLA-A2 by cytotoxic T lymphocytes after DNA transfer into human and murine cells.

A gene coding for the major histocompatibility antigen HLA-A2 was transferred into human HLA-A2 negative M1 cells and murine L cells. Following transfection, these cells expressed molecules at the cell surface that are biochemically indistinguishable from HLA-A2 antigens on the human cell line JY from which the HLA-A2 gene was isolated. The M1A2 cells were recognized and lysed by a cytolytic T-cell clone specific for HLA-A2. The transfected L cells which express HLA-A2 in association with human beta 2-microglobulin were not lysed by this T-cell clone. The specific cytolysis of M1A2 cells could be inhibited by monoclonal antibodies to HLA-A2, and monoclonal antibodies to T3, T8, and LFA-1 on cytotoxic T lymphocytes. These results suggest that killing by allospecific T cells requires HLA-A2 antigens as well as other species-specific structures on the target cell surface.

Animals↗
PubMed1984

Coexpression of the human HLA-A2 or HLA-B7 heavy chain gene and human beta 2-microglobulin gene in L cells.

L cells expressing human HLA-A2 or HLA-B7 class I antigen heavy chains are not recognized by human cytotoxic T lymphocytes directed at HLA-A2 or HLA-B7 antigens. To test whether the absence of human beta 2-m was the cause of the lack of recognition by the human cytotoxic T lymphocytes, coexpression of the human beta 2-m gene and the HLA-A2 or HLA-B7 heavy chain in L cells ("double transfectants") was obtained. In addition, L cells expressing HLA-A2 or HLA-B7 antigens in association with human beta 2-m were obtained by an exchange reaction, in which human beta 2-m from serum replaced the endogenous murine beta 2-m. Both types of transfectant cells were used in 51Cr-release assays and cold target inhibition assays for human cytotoxic T cell clones which were directed at HLA-A2 or HLA-B7. Neither human CTL clones nor a mixture of CTL specific for HLA-A2 and HLA-B7 were able to recognize these cells. Several alternative explanations for these observations are discussed.

Animals↗
PubMed1983

Nascent polypeptide chains exit the ribosome in the same relative position in both eucaryotes and procaryotes.

We located the polypeptide nascent chain as it leaves cytoplasmic ribosomes from the plant Lemna gibba by immune electron microscopy using antibodies against the small subunit of the enzyme ribulose-1,5-bisphosphate carboxylase. Similar studies with Escherichia coli ribosomes, using antibodies directed against the enzyme beta-galactosidase, show that the polypeptide nascent chain emerges in the same relative position in plants and bacteria. The eucaryotic ribosomal exit site is on the large subunit, approximately 75 A from the interface between subunits and nearly 160 A from the central protuberance, the presumed site for peptidyl transfer. This is the first functional site on both the eucaryotic and procaryotic ribosomes to be determined.

Escherichia coli↗
PubMed1983

Expression of the major histocompatibility antigens HLA-A2 and HLA-B7 by DNA-mediated gene transfer.

Genes coding for the heavy chain of the class I antigens HLA-A2 or HLA-B7 of the human major histocompatibility complex have been introduced into mouse LtK- cells by cotransfection with the herpes simplex virus thymidine kinase gene. HAT-resistant colonies were isolated expressing either HLA-A2 or HLA-B7 as monitored by indirect immunofluorescence. Immunoprecipitation analysis of both antigens by either sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) or isoelectric focusing (IEF) showed that they were identical to the HLA-A2 and HLA-B7 expressed in the human lymphoblastoid cell line JY (homozygous HLA-A2, HLA-B7). However, human cytotoxic T lymphocytes (CTL) generated against JY and CTL clones specific for HLA-A2 or HLA-B7 were unable to recognize the transfectants as targets. These results indicate that the human HLA-A2 (or B7) complexed with the murine beta 2-microglobulin could be an inappropriate target structure for the CTL. However, because the transfectants are not killed by human CTL even in the presence of lectins, it is suggested that other molecules that are not able to overcome the human-mouse species barrier may be involved in the killing mechanism.

Animals↗
PubMed1982

Binding of human immunoglobulin M to Staphylococcus aureus bearing protein A.

Human immunoglobulin M binds to protein A-bearing Staphylococcus aureus. This binding is specific for protein A since it is inhibited by rabbit Fab against Staphylococcal protein A. Analysis of the IgM binding data gives an apparent KD of 0.94 nM and a maximum capacity of approximately 2 X 10(3) binding sites of IgM per bacteria.

Animals↗
PubMed1982

Nascent polypeptide chains emerge from the exit domain of the large ribosomal subunit: immune mapping of the nascent chain.

The site of the nascent polypeptide chain as it leaves the ribosome has been localized on the "exit domain" of the Escherichia coli ribosome by using IgG antibodies directed against the enzyme beta-galactosidase (EC 3.2.1.23). Thus, a functional site has been mapped on intact 70S ribosomes. The exit site is on the large subunit, approximately 70 A from the interface between subunits and nearly 150 A from the central protuberance, the likely site of peptide transfer. It is adjacent to the region corresponding to the rough endoplasmic membrane binding region of the eukaryotic ribosome but distant from ribosomal components participating in mRNA recognition and polypeptide elongation (i.e., distant from the "translational domain"). These results, together with the protease protection experiments of others, provide evidence that the nascent protein chain probably passes through the ribosome in an unfolded, fully extended conformation.

Binding Sites↗
PubMed1980

Recovery of pure ribosomal proteins from stained gels. A fast method of purification of active proteins.

A simple technique has been developed for eluting ribosomal proteins from stained gels in the presence of an acetic acid solution. The ribosomal proteins are then separated from the dye by anion-exchange chromatography under dissociating conditions. Ribosomal proteins purified by these methods give total cross-reaction with proteins obtained by standard procedures, when tested by immunodiffusion against their corresponding antibodies, and show the same electrophoretic mobility as standard proteins in bidimensional polyacrylamide gel systems. Ribosomal proteins L7/L12, recovered from stained gels and purified by these methods, are able to reconstitute the elongation-factor-G-dependent GTPase activity of ribosomal particles deprived of these proteins. Radioactive protein L1, recovered in the same way, is incorporated into a total reconstituted 50-S subunit, competing with an excess of standard L1 present in the pool of total proteins from 50-S subunits used for reconstitution. These results suggest that bidimensional electrophoresis can be considered an alternative system of purification of active proteins from complex mixtures.

Chromatography, Ion Exchange↗