Search PubMed⌕ Search

Biomedical subjects

M Prat

Publications and source records attributed to M Prat.

At least 91 records · Page 5Linked to original sources
PubMed1985

A novel H-2s class I molecule expressed on a B-cell leukemia from SJL/J mice.

Anti-H-2.33 [(B10.D2 X A)F1 anti-B10.A(5R)], which predominantly contains antibodies recognizing H-2Kb and IAb molecules, was found to be cytotoxic against DMLM 1678, a B-cell leukemia of SJL/J (H-2s) origin. The antiserum precipitated a typical class I (H-2-like) molecule from labeled tumor cell preparations as judged by molecular mass, papain susceptibility and association with beta 2-microglobulin. Sequential immunoprecipitation studies revealed that it was distinct from either H-2Ks or H-2Ds, the 2 molecules expressing the private antigens of the H-2s haplotype. Absorption analysis using congenic mice mapped the gene controlling the expression of the novel molecule telomeric to the S-region within the major histocompatibility complex.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1985

Monoclonal antibodies to murine interferon-gamma: affinity purification and molecular characterization of murine interferon-gamma.

Monoclonal antibodies (MAb), AN-18.17.24, obtained by fusing the murine myeloma cell line Ag8.653 with spleen cells from rats immunized with murine interferon gamma (IFN-gamma) produced by the phorbol myristic acetate(PMA)-stimulated T-cell lymphoma L12-R4, were shown to be specific for MuIFN-gamma. An immunoadsorbent column was prepared and used for large-scale purification of MuIFN-gamma produced either by PMA-stimulated L12-R4 cells or by normal T-lymphocytes stimulated with Con A and interleukin 2. The purified product from L12-R4 cells, which retained biologic activity, was made up of two proteins (Mr = 16,500 and 17,500) as determined by sodium dodecyl sulfate gel electrophoresis. By contrast MuIFN-gamma produced by normal T-lymphocytes resulted in two proteins with Mr of 20,600 and 21,800 as determined by sodium dodecyl sulfate gel electrophoresis. These results indicate that MuIFN-gamma produced by L12-R4 tumor cells or by normal T-lymphocytes differ in their molecular weight, probably because of different degrees of glycosylation of the same molecule.

Animals↗
PubMed1985

Monoclonal antibodies against mouse gamma-interferon inhibit tumoricidal macrophage activation by T lymphocytes.

A monoclonal antibody, AN-18.17.24, specific for murine interferon-gamma (IFN-gamma) was produced by immunizing Wistar rats with IFN-gamma secreted by a T-cell lymphoma, L12-R4, upon stimulation with phorbol myristic acetate (PMA). Antiviral activity as well as tumoricidal activation induced by PMA-stimulated L12-R4 cell supernatant or by Con A-stimulated normal spleen cells were neutralized at the same extent by AN-18 monoclonal antibody. Moreover, depletion experiments showed that inhibition of tumoricidal macrophage activation must be ascribed to the direct binding of the IFN-gamma molecule by AN-18 MAb and not to the interference of the monoclonal antibody with the cell surface IFN-gamma receptor. These studies conclusively demonstrate that in supernatants of T lymphocytes stimulated with polyclonal activators IFN-gamma was the only molecule responsible for macrophage activation in tumor cell killing.

Animals↗
PubMed1985

CAR-3, a monoclonal antibody-defined antigen expressed on human carcinomas.

Several monoclonal antibodies were raised against the human epidermoid carcinoma line A 431. The antibody produced by clone AR-3, when tested in enzyme-linked immunosorbent assay, was found to react with the cell line used as immunogen, the human gastric carcinoma line KATO III, the colon carcinoma line HT29, and the ovarian carcinoma line SW626. This monoclonal antibody was found unreactive when tested on human peripheral blood leukocytes or on a number of normal or neoplastic cell lines. The antibody precipitated a high-molecular-weight glycosylated component. When tested on paraffin sections by the avidin:biotin: peroxidase method, the AR-3 antibody stained pancreatic (6:7), gastric (11:14), ovarian (5:6), colon (4:8), endometrial (4:6), and cervical (4:7) carcinomas. A small minority of carcinomas of other organs was also stained. Sarcomas, lymphomas, and other tumors of nonepithelial origin were constantly negative. Staining of some normal epithelial cells was also observed. Among the fetal tissue tested, the antibody reacted with pancreatic ducts and the small intestine. The antibody recognized metastatic carcinoma cells in peritoneal effusions. On the basis of its tissue distribution, the antigenic determinant defined by the AR-3 monoclonal antibody was called CAR-3. The monoclonal AR-3 did not cross-react with partially purified preparations of carcinoembryonic antigen, gastrointestinal carcinoma antigen, or the human milk fat globule antigen. The AR-3 MAb appear, thus, to broaden the number of available reagents for histopathological diagnosis of carcinomas.

Animals↗
PubMed1985

Enhancement of immunity against RSV-induced sarcomas by generation of hapten-reactive helper T lymphocytes.

Previous work from this laboratory has shown that preimmunization of syngeneic hosts with Rous sarcoma virus (RSV)-transformed cells elicits a strong immune response against the growth of transplantable RSV sarcomas, mediated by T lymphocytes expressing the surface phenotype of helper cell precursors (Prat, Di Renzo & Comoglio, 1983). This paper shows that anti-tumour immunity may be elicited in tumour-bearing animals by triggering an experimentally pre-amplified T-helper cell population at the site of tumour growth. Mice were treated with cyclophosphamide (which inactivates suppressor T cells) followed by skin sensitization to trinitrochlorobenzene (TNCB) according to a protocol that has been shown to induce an appreciably amplified generation of trinitrophenyl (TNP)-reactive helper T cells (Fujiwara et al., 1984). Five weeks after TNCB painting, mice were transplanted s.c. with a lethal dose of RSV-induced syngeneic sarcoma cells; the injection at the tumour site of TNCB induced the regression of the tumour in mice in which the TNP-helper cell population has been amplified, but not in controls, including those injected with a non-related hapten or sensitized to TNCB without inactivation of suppressors.

Animals↗
PubMed1985

Monoclonal antibodies against the human epidermoid carcinoma A 431.

About 2,000 antibody-producing hybrids were obtained by fusion of lymphocytes harvested from mice hyperimmunized with the human carcinoma line A 431 and P3-X63-Ag8-653 myeloma cells. Antibody specificity was screened in a radiobinding assay performed on glutaraldehyde-fixed cultured cells, and on paraffin-embedded sections stained with the method of avidin-biotin-peroxidase. Among the clones, 16 produced antibodies reacting with a variety of tumor lines but not with human fetal fibroblasts or peripheral blood leukocytes. On the basis of their specificity, monoclonal antibodies were classified into three groups. Some reacted only with the A431 line used as immunogen: One antibody reacted with an antigen preferentially expressed on cancer of the gastrointestinal tract and ovary; A group of monoclonal antibodies displayed a broader spectrum of reactivity defining a panel of antigens that could be tentatively classified as epithelial specific.

Animals↗
PubMed1984

Monoclonal antibodies against murine gamma interferon.

Monoclonal antibodies against murine immune interferon (IFN-gamma) were produced by fusing the murine nonsecreting myeloma cell line P3.X63.Ag8.653 with spleen cells from rats immunized with IFN-gamma-containing supernatants obtained by stimulating a T-cell lymphoma, L12-R4, with phorbol 12-myristate 13-acetate. Supernatants from a twice-cloned hybridoma (AN-18.17.24) were found to neutralize and to adsorb in depletion experiments up to 27 units of mouse IFN-gamma but not equivalent amounts of mouse leukocyte or fibroblast IFNs. The AN-18.17.24 monoclonal antibody neutralized to the same extent mouse IFN-gamma from different sources--namely, (i) concanavalin A-stimulated spleen cells, (ii) alloantigen-stimulated spleen cells, and (iii) monkey fibroblasts transfected with the cloned gene of murine IFN-gamma. Moreover, the monoclonal antibody displayed species specificity, since it did not neutralize IFN-gamma of human origin. Binding inhibition experiments with murine IFN-gamma preparations exposed to enzymatic or physicochemical degradation demonstrated that the protein moiety and not the carbohydrate residues were responsible for the binding to the AN-18.17.24 monoclonal antibody. Finally, this monoclonal antibody immunoprecipitated two molecular species of IFN-gamma of about 16.8 and 17.8 kilodaltons, respectively, from [35S]methionine- or [3H]glucosamine-labeled supernatants of stimulated L12-R4 cells.

Animals↗
PubMed1983

Characterization of T lymphocytes mediating in vivo protection against RSV-induced murine sarcomas.

Tumors induced by Rous sarcoma virus (RSV) in different mouse strains share a common tumor-associated transplantation antigen, whose expression is controlled both by the viral transforming src gene and by cellular gene(s) (Prat et al., 1981). The mechanism(s) responsible for the in vivo protective immune response against two RSV-induced sarcomas has now been investigated. In the "Winn assay" the growth of both tumors was specifically prevented or significantly reduced by the simultaneous administration of lymphocytes isolated from immunized donors. The effector cells were radiosensitive (2,500 rad), Lyt-1+, Lyt-2,3+ Thy.1+ cells. Better protection was afforded by transfer of immune spleen cells from mice pretreated with cyclophosphamide, which is known to abrogate T-cell suppressor activity. In a 5-day mixed lymphocyte/tumor cell culture specific anti-RSV-induced sarcoma cytotoxic activity was barely detected, while a good production of interferon-gamma (IFN-y) was observed. The cells involved showed the same functional and surface phenotype as displayed by the effectors of the "Winn" assay. It is concluded that in the immune rejection of RSV-induced sarcomas, Lyt-1+, Lyt-2,3+ T cells, rather than cytotoxic T lymphocytes, and lymphokines such as IFN-gamma are involved.

Animals↗
PubMed1982

Dissection of the antigenic determinants expressed on the cell surface of RSV-transformed fibroblasts by monoclonal antibodies.

Fusion of P3/X63-Ag8 mouse myeloma cells with splenocytes obtained from mice hyperimmunized with a BHK hamster fibroblast line transformed by an env-strain of Rous sarcoma virus (RSV) resulted in the production of antibody-secreting hybridomas. Seven hybrid clones secreted antibodies binding to RSV-transformed BHK fibroblasts but not to the parental control non-transformed line. The antibodies produced by three of these clones did identify antigenic determinants expressed also on BHK cells transformed by SV 40. The antibodies produced by four other clones reacted specifically with cells transformed by RSV but not with cells productively infected by the transformation-defective Rous-associated virus-1; one of these reacted with RSV-transformed hamster cells only, three others identified antigenic determinants common to RSV-transformed cells of different animal species. These data give further support to the idea that RSV-transformed cells express a cell-surface antigen, specific for transformation, the expression of which is controlled by the transforming src gene, and that the specificities carried on it follow a complex pattern, arising from the interaction between antigenic determinants of cellular and viral origin.

Animals↗
PubMed1982

Cell surface molecules and fibronectin-mediated cell adhesion: effect of proteolytic digestion of membrane proteins.

Proteases have been used as a tool to investigate the role of surface molecules in fibronectin-mediated cell adhesion. Proteolytic digestion of membrane-proteins by pronase (1 mg/ml for 20 min at 37 degrees C) completely inhibited adhesion of baby hamster kidney (BHK) fibroblasts on fibronectin-coated plastic dishes. Various degrees of inhibition were also obtained after treatment with proteinase K, chymotrypsin, papain, subtilopeptidase A, and thermolysin. Protein synthesis was required to restore the adhesive properties of pronase-treated cells, showing the protein nature of the molecules involved in adhesion to fibronectin. A peculiar feature of these proteins was their resistance to cleavage by trypsin. After prolonged trypsin treatment (1 mg/ml for 20 min at 37 degrees C), cells adhered and spread on fibronectin-coated dishes, even when protein synthesis was inhibited by 4 microM cycloheximide. Under these conditions only three glycoproteins (gp) of molecular weight 130,000, 120,000, and 80,000 were left on the cell surface. These were precipitated by a rabbit antiserum against BHK cells that also inhibited adhesion of trypsin-treated cells. gp120 and gp80 were left at the cell surface after mild pronase digestion (0.2 mg/ml for 20 min at 37 degrees C), under conditions not affecting adhesion. These data suggest that these glycoproteins may be involved in fibronectin-mediated cell adhesion in some yet unknown way.

Animals↗
PubMed1981

Mouse fibroblasts transformed by Rous sarcoma virus express a virus-specific non-virion transplantation antigen.

Rous sarcoma virus (RSV)-transformed fibroblasts from different animals species a serologically detectable virus-induced non-virion cell surface antigen (VCSA), whose expression is controlled by the transforming viral src gene and by a cellular gene. Now, by in vivo immunization, we have found that RSV-transformed fibroblasts from different mouse strains share a virus-specific transplantation antigen. In fact, only animals immunized with irradiated syngeneic or allogeneic fibroblasts transformed by RSV, but not animals immunized with cells transformed by different oncogenic agents, rejected a lethal dose of syngeneic RSV-induced tumor cells. Immunoprecipitation tests with monospecific antisera showed that the expression of this antigen did not correlate with the presence of intracellular viral proteins other than the src gene product pp60src. However, hyperimmunization of mice with hamster or quail fibroblasts transformed by RSV, that express a high level of pp60src, did not induce transplantation resistance. It is concluded that the expression of both the serologically-defined VCSA and the transplantation antigen is the result of the interaction of pp60src with host cell gene product(s) rather than the simple exposure of pp60src at the outer cell surface.

Animals↗
PubMed1981

Transformation-sensitive protein with molecular weight of 45,000 secreted by mouse fibroblasts.

The [35S]methionine-labeled proteins released in the medium conditioned by normal and transformed mouse fibroblasts have been analyzed by sodium dodecyl sulfate-polyacrylamide electrophoresis. Three major proteins, fibronectin, procollagens, and a protein with a molecular weight of 45,000 (45K protein) have been identified. The 45K protein, which has not yet been described, accounts for about 30% of the proteins released by control 3T3 fibroblasts or mouse embryo cultures. Quantitation of the radioactivity incorporated by the 45K protein indicated a 10- to 15-fold decrease in 3T3 fibroblasts transformed by Kirsten, Abelson, or Rous sarcoma viruses. The amounts of fibronectin and procollagens released in the medium by transformed cells were also reduced by factors of 3- and 5-fold, respectively. Pulse chase experiments have shown that the decreased level of the 45K protein in the medium of transformed cells cannot be explained by a reduced rate of secretion or by extracellular proteolytic degradation. It is not known, however, whether decreased synthesis is responsible for this alteration. The "tumor promoter" phorbol myristate acetate, which is known to induce many of the alterations associated with neoplastic cells, also induced 3T3 fibroblasts to release the 45K protein in amounts comparable to that of transformed cells. Thus, this protein represents a new molecular marker of oncoviral transformation.

Animals↗
PubMed1980

Identification and partial characterization of five major membrane glycoproteins of BHK fibroblasts.

Five major membrane glycoproteins of the BHK-B4 hamster fibroblast plasma membrane have been identified by binding specific rabbit antibodies to the cell surface and by recovering the detergent solubilized immunocomplexes with Protein A-Sepharose immunoadsorption. These glycoproteins, designated as gp45, gp65, gp95, gp130 and gp140, are exposed at the cell surface since: (i) they were accessible to antibodies in intact viable cells; (ii) they were radioiodinated by the lactoperoxidase-glucose oxidase procedure; and (iii) they were cleaved by proteolytic enzymes in conditions affecting only the cell surface. Among these glycoproteins the gp130 is the predominant component and its exposed portion is characterized by lack of sensitivity to trypsin cleavage. Glycoproteins of different molecular weight, but immunologically related to the major hamster membrane glycoproteins, have been detected at the surface of both rat and mouse fibroblasts.

Animals↗
PubMed1980

Interaction between cellular and viral genes in the expression of the RSV-induced transformation-specific cell-surface antigen VCSA.

Transformation of BHK hamster fibroblasts by an env- strain of Rous sarcoma virus (RSV) leads to the appearance at the cell surface of a virus-induced nonvirion antigen (VCSA), specific for transformation, whose expression is controlled by the transforming src gene. Previous work has shown that a rabbit anti-VCSA serum lyses specifically, in the presence of complement, 51Cr-labelled RSV-transformed cells from different animal species. Now, by competition experiments with a panel of different unlabelled cells we show that the VCSA expressed on RSV-transformed hamster fibroblasts is a complex of at least three distinct antigenic specificities: (1) one expressed on all RSV-transformed fibroblasts, regardless their species and the subgroup or strain of the transforming virus; (2) one cross-reacting with a cell-surface antigen (CSA) expressed at various degrees on untransformed avian fibroblasts, but not on mammalian fibroblasts; (3) one species-specific, present only on RSV-transformed hamster fibroblasts. It is concluded that VCSA is a complex of several antigenic determinants, and that some of these differ in different cells transformed by RSV. This observation indicates that VCSA expression at the cell surface is likely to be the result of the interaction between the viral src gene product pp60src with host cell gene(s) or gene product(s), rather than the simple expression of this molecule at the cell surface.

Animals↗
PubMed1980

Target antigens for antibodies and complement at the cell surface of RSV-transformed fibroblasts.

Using cells transformed by Rous sarcoma virus (RSV), an RNA tumour virus whose genetic and structural composition is fully known, the virus-induced surface antigens acting as targets for antibodies and complement were studied. Among the virus structural proteins, only the envelope antigen gp85, but not the core group-specific proteins or reverse transcriptase, were able to mediate immune lysis in the 51Cr-release assay. The group-specific antigenic determinants of gp85 were predominantly involved. The virus-induced cell surface antigen (VCSA), specific for transformation, was the only other molecule effective. Since different cells express either of these antigens, further support is given to the non-identity of virus structural antigens and VCSA.

Animals↗
PubMed1979

Relationship between Rous sarcoma virus-induced expression of membrane antigen and phenotypic transformation.

Rous sarcoma virus-transformed hamster BHK fibroblasts express a virus-induced cell surface antigen undetectable in cells either transformed by unrelated viruses or infected by transformation-defective strains of Rous sarcoma virus. To clarify whether this antigen plays any role in the process of malignant transformation or is expressed at the cell surface only as a consequence of the acquisition of the transformed phenotype, we have investigated its expression at the cell surface of Rous sarcoma virus-transformed BHK cells treated with dibutyryl cyclic adenosine 3':5'-monophosphate and at the surface of parental BHK cells transiently transformed by the tumor promoter phorbol myristate acetate. In the dibutyryl cyclic adenosine 3':5'-monophosphate-treated cells, in which most of the parameters of the transformed phenotype are reverted to normality, but the product of the transforming gene is still present, virus-induced cell surface antigen is expressed. In the mirror experiment, this antigen is not expressed by phenotypically transformed but genetically normal phorbol myristate acetate-treated cells. It is concluded that the tumor membrane antigen studied is intimately associated with the expression of the function(s) controlled by the transforming gene.

Animals↗
PubMed1978

Tumor-specific and tumor-associated membrane antigens of Rous sarcoma virus transformed hamster fibroblasts.

Hamster fibroblasts transformed by an env- strain of Rous sarcoma virus (RSV) express at their surface tumor-associated antigens of unknown origin and a tumor-specific antigen (VCSA) which is not expressed by hamster fibroblasts transformed by unrelated DNA or RNA oncogenic viruses. This antigen was detectable by rabbit antibodies and a complement-dependent 51Cr-release cytotoxicity assay and is common to RSV-transformed cells of different animal species. By comparing the anti-VCSA serum which antisera directed against purified gp85, gs-proteins, reverse transcriptase or detergentlysed virus particles, it was shown that VCSA is not a known virion structural protein. Moreover, VCSA expression does not correlate with viral replication since it is not detectable in chick embryo fibroblasts productively infected with the transformation-defective virus RAV-1 which shares virus structural genes with RSV. Finally, in hamster cells transformed by an RSV mutant, temperature-sensitive for the ability to transform the host cell, VCSA expression at the cell surface correlates with the expression of the transforming gene.

Animals↗