Functional and immunological characterization of the stimulation of human lymphocytes with a mitogen from group A streptococci (SM).
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B-Lymphocytes↗
Biomedical subjects
S Ferrone
Publications and source records attributed to S Ferrone.
An antiglobulin microcytotoxicity assay to analyze non-complement fixing monoclonal antibodies to human histocompatibility antigens.
An antiglobulin microcytotoxicity assay has been used to analyze non-complement fixing monoclonal antibodies to human histocompatibility antigens. The assay utilizes methodology similar to that of the widely used microcytotoxicity assay for HLA typing, requires low numbers of target cells and is suitable to test large numbers of samples. The sensitivity of the assay is influenced by the anti-mouse Ig antiserum used, by the sequence of addition of the various reagents and by the incubation time. The assay is suitable to screen supernatants of clones derived from hybridization experiments and to characterize the serological specificity of anti-HLA monoclonal antibodies.
Animals↗
Immunochemical and functional analysis of Ia-like antigens on human monocyte-macrophages with monoclonal antibodies.
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Antibodies, Monoclonal↗
Heterogeneity of human melanoma-associated antigens defined by monoclonal antibodies and conventional xenoantisera.
Immunochemical analysis of cultured human melanoma cell detergent extracts and spent culture medium with conventional xenoantisera and monoclonal antibodies identified four types of 94,000 (94K) dalton molecules and two types of high-molecular-weight melanoma-associated antigens by the following characteristics: (1) association with other components, (2) mobility in SDS-PAGE under reducing and nonreducing conditions, (3) antigenicity, and (4) presence in spent culture medium. Conventional xenoantisera were found to contain antibody populations to antigenically distinct structures, some of which have similar apparent molecular weights. Immunodepletion studies showed that the antigenic determinant detected by the monoclonal antibody 225.28S to a high-molecular-weight melanoma-associated antigen was expressed on a subpopulation of the antigens defined by the conventional xenoantiserum #8995. These data prove that antibodies reactive with antigens of similar molecular weight cannot be assumed to identify the same structures, and indicate that tumor-associated antigens may be heterogeneous in the expression of antigenic determinants defined by monoclonal antibodies.
Animals↗
Characterization of antigens on human lymphocytes coded by messenger RNA translated in vitro.
Translation and immunoprecipitation were used to identify messenger RNAs (mRNAs) coding for surface antigens expressed on human lymphoblastoid cells. The mRNAs were extracted from several human lymphoid cell lines as well as from fibroblastoid lines. These mRNAs were translated in vitro, and the translation products were reacted with xenoantisera raised against the antigens on human lymphoid cells. Products immunoprecipitated by these antisera were analysed by electrophoresis and fluorography. Four antisera immunoprecipitated a polypeptide with a mol. wt (MW) of approximately 32,000 (p32) from translations programmed with mRNA extracted from all the cell lines. Two antisera immunoprecipitated, in addition to p32, another polypeptide with a MW of approximately 25,000 (p25) only from translations programmed with RNA from lymphoid cell lines. p25 mRNA in the different lymphoid cell lines fell into three basic abundance classes as determined by in vitro translation and immunoprecipitation. Cells from two Burkitt's lymphomas (Raji and Daudi) did not express detectable p25 mRNA. Two T-lymphoblastoid lines (Molt-4 and 1301) contained five- to 10-fold less p25 mRNA than the B-lymphoid cell lines (Victor, RPMI-8866, RPMI-6410, RPMI-8226 and RPMI-1788). Both p32 and p25 were expressed on the cell surface inasmuch as lymphoblastoid cells adsorbed antibodies to both polypeptides. Human fibroblast, Raji or Daudi cells adsorbed anti-p32 antibodies from the antiserum but not anti-p25. Quantitative absorptions of the antiserum with T- or B-lymphoblastoid cells was used to determine the relative amounts of p32 and p25 expressed on the cell surface. B-lymphoblastoid cells were found to express two- to five-fold more p25 on the cell surface then T-lymphoblastoid cells. p25 does not represent an immunoglobulin light-chain precursor inasmuch as a 1000-fold excess of unlabeled human Ig did not compete with p25 translated in vitro for binding by its respective antibody.
Antibodies, Monoclonal↗
A radioimmunoassay and biological profile of Ia-like antigens on human melanoma cells.
Human melanoma cells have unexpectedly been found to express Ia-like antigens, histocompatibility type antigens which are believed to be the gene products of the immune response region. Melanoma derived Ia-like antigens are immunologically functional. Monoclonal antibodies have been produced to these antigens. The level of Ia-like antigens on melanoma cells is significantly lower than on B lymphoid cells, and this is more likely to reflect a reduced synthesis than an increased shedding. We have employed radioimmunometric and immunofluorescent procedures in this investigation, Melanoma cell-derived Ia-like antigens have a structure similar to that of B lymphoid cell-derived Ia-like antigens. The antigens are composed of two non covalently associated glycoproteins. One chain, referred to as the alpha chain, has an approximate molecular weight of 34,000 and the other one, referred to as the beta chain, has an approximate molecular weight of 29,000. The expression of Ia-like antigens is restricted to malignant melanoma and not on benign nevi.
B-Lymphocytes↗
Analysis of the NIH workshop monoclonal antibodies to human melanoma antigens.
Reagents exchanged at the 2nd workshop on monoclonal antibodies (MoAb) to human melanoma antigens were analyzed using both serological and immunochemical assays. The analysis by laboratories participating in the workshop of our MoAb 225.28S, 345.134S, 376.96S, 465.12S, and 763.24TS reaffirms our own analysis of these reagents in that (1) they all react with the majority of melanoma cell lines tested and (2) the reactivity of MoAb 225.28S and 763.24TS is much more restricted than that of MoAb 345.134S 376.96S, and 465.12S. Our serological analysis revealed that the majority of workshop reagents reacted with cultured melanoma cells. Immunochemical analysis of these monoclonal antibodies allowed for their division into three groups according to the molecular weights of the antigens recognized in immunoprecipitation experiments, greater than 100 kd, 80-100 kd, and DR antigens. Further analysis of the first two groups of monoclonal antibodies by immunodepletion and antibody binding inhibition assays revealed that MoAb 9.2.27, 225.28S, and 763.24TS recognize distinct determinants with a heterogeneous distribution on subpopulations of a high molecular weight melanoma associated antigen. MoAb 376.96S and 705.F6 recognize either the same or spatially close determinant(s).
Antibodies, Monoclonal↗
Immunogenicity of serum HLA antigens in allogeneic combinations.
Immunization of patients with chronic renal insufficiency with plasma from selected donors elicited lymphocytotoxic antibodies. Analysis of these antibodies with Fab2 blocking assays showed that they are directed to HLA-A,B and to Ia-like antigens. These results indicate that serum HLA antigens are immunogenic in allogeneic combinations.
Animals↗
Heterogeneous distribution of the determinants defined by monoclonal antibodies on HLA-A and B antigens bearing molecules.
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Antibodies, Monoclonal↗
Expression of the H-2 antigenic complex on murine haematopoietic stem cells.
We used hybridoma-derived monoclonal antibody to H-2Kk antigens and xenoantibodies to beta 2-microglobulin (beta 2 m) to study the expression of the H-2 antigenic molecular complex on murine hematopoietic stem cells and the effect of long-term culture in vitro on the expression of these antigens. Monoclonal anti-H2Kk antibody produced potent complement-dependent inhibition of pluripotent (CFU-S), myeloid (CFU-C), and erythroid (CFU-E) stem cells from the bone marrow of C3H and AKR(H-2k) mice and was without effect on stem cells from Balb/C (H-2d) mice. Anti-beta 2m xenoantibodies inhibited stem cells from all three strains. Stem cells could not be 'rescued' from the inhibitory effects of the antibodies by the addition of thymocytes to marrow cells after antibody treatment. Both the anti-H-2Kk monoclonal antibody and the anti-beta 2m xenoantibodies produced potent inhibition of AKR (H-2k) CFU-C that had been maintained in culture for up to 6 weeks. These results indicate that murine CFU-S, CFU-C, and CFU-E express H-2 antigens and that the expression of these antigens by CFU-C is not altered during long-term culture.
Animals↗
Tissue distribution, molecular profile, and shedding of a cytoplasmic antigen identified by the monoclonal antibody 465.12S to human melanoma cells.
The mouse immunoglobulin G2 monoclonal antibody (MoAb) 465.12S reacts with a cytoplasmic antigen present in human melanoma cells but not detectable in melanocytes. Indirect immunofluorescent staining of a large number of surgically removed normal adult and fetal tissues with the MoAb 465.12S detected the cytoplasmic antigen in epithelial cells from several organs. The intensity of staining was greater in adult tissues than in the corresponding fetal tissues. Furthermore, the MoAb 465.12S stained nearly all of the surgically removed tumors tested but did not stain many of the normal tissues from which they originated. In almost all cases, the intensity and frequency of staining wa greater for tumor cells than for corresponding normal tissues. From cultured carcinoma and melanoma cells, the MoAb 465.12S immunoprecipitated four glycopolypeptides with molecular weights of 94,000, 75,000, 70,000, and 25,000. Incorporation of 3H-labeled sugars into the various components of the cytoplasmic antigen revealed that the M.W. 75,000 component was unusual in that it contained only glucosamine and mannose. The antigenic determinant defined by the MoAb 465.12S appears to be protein rather than carbohydrate in nature since it is heat sensitive and is expressed on the antigens synthesized by cells in presence of tunicamycin. Analysis of the spent culture medium of carcinoma and melanoma cell lines revealed that the cytoplasmic antigen is readily shed by these cells and consists of a major M.W. 94,000 and a minor M.W. 72,000 component. Treatment of cultured melanoma cells with the antibiotic tunicamycin showed that glycosylation of the cytoplasmic antigen is required for its shedding and/or stability in the spent culture medium.
Antibodies, Monoclonal↗
Molecular heterogeneity and shedding of a high-molecular-mass melanoma-associated antigen identified with monoclonal antibodies.
This review briefly discusses some recent developments in the use of monoclonal antibodies to study antigens of high molecular mass associated with melanomas, including information on their molecular profile, their heterogeneity, and the effect of interferon on their shedding by melanoma cells.
Antibodies, Monoclonal↗
Expression of Ia-like and HLA-A,B antigens on human multipotential hematopoietic progenitor cells.
Human multipotential hematopoietic progenitor cells can be assayed by their ability to form colonies of mixed cell lineages in vitro. These cells display la-like antigens and HLA-A,B,C antigens as evidenced by inhibition of colony formation by specific monoclonal antibodies and complement.
Antibodies, Monoclonal↗
A 94,000-dalton glycoprotein expressed by human melanoma and carcinoma cells.
The IgG2a monoclonal antibody (MoAb) 376.96S, secreted by a hybridoma derived from a mouse immunized with cultured human melanoma cells COLO 38, reacts with a single 94,000-dalton glycoprotein that is peripherally associated with the plasma cell membrane of cultured melanoma cells. Indirect immunofluorescence analysis with cryostat thin sections of human tissues showed that this antigen is absent from a large variety of normal tissues but is readily detectable on melanomas, nevi, and several different carcinomas. The MoAb 376.96S binds with cultured melanoma and carcinoma cell lines to a similar extent and can mediate both complement-dependent and cell-dependent lysis of these cells. The 94,000-dalton glycoprotein detected by MoAb 376.96S is distinct in its tissue distribution, antigenicity, and molecular profile from several structures previously identified with monoclonal antibodies that have similar molecular weights.
Antibodies, Monoclonal↗
Imaging of human melanoma xenografts in nude mice with a radiolabeled monoclonal antibody.
External photoscanning with display of radioactivity data as a color-scaled image detected xenografts of human melanoma in male nude inbred mice of BALB/c background 48 hours after injection of 131I-labeled monoclonal IgG 225.28S that is specific for human melanoma. A 131I-labeled polyclonal goat IgG against human melanoma-associated antigens could also image the tumor, but with this preparation there was considerable localization of radioactivity in normal tissues, resulting in less satisfactory tumor definition. Labeled normal mouse IgG did not image the melanoma grafts. Assay of radioactivity in tissues of melanoma-grafted mice confirmed tumor-specific localization of the antimelanoma antibodies. The tumor:blood ratio of radioactivity was 6.55 with the monoclonal antimelanoma IgG and 0.45 with the polyclonal IgG.
Animals↗
Differential segregation patterns of human chromosomes in somatic cell hybrids constructed with human B-lymphocytes and human melanoma cells.
Hybrids constructed with the murine fibroblasts IT22 and either human lymphoid cells or human melanoma cells retain the human chromosomes 1, 14, and 21 and lose the human chromosome 2. The human lymphocyte-mouse fibroblast hybrids preferentially retain the human chromosome 11, while the human melanoma-murine fibroblast hybrids preferentially retain the human chromosome 8 and lose the human chromosome X. These data suggest that the type of human parental cell used to construct human-rodent hybrids can influence the segregation of human chromosomes.
B-Lymphocytes↗
Heterogeneity of HLA-A,B, Ia-like, and melanoma-associated antigen expression by human melanoma cell lines analyzed with monoclonal antibodies and flow cytometry.
With the use of monoclonal antibodies, indirect immunofluorescence, and flow cytometry, human melanoma cell lines (Colo 38, M-16, and M-21) were examined for the quantitative level of expression of HLA-A,B antigens, Ia-like antigens, and two human melanoma-associated antigens (MAA) referred to as 280K and 94K MAAs. Each of the melanoma cell lines examined showed tremendous heterogeneity with regard to antigen expression. A detailed study of the heterogeneity of the four antigens listed revealed that variation in cell size could, in part, account for the large differences in antigen expression observed. Cell surface density of HLA-A,B antigens, Ia-like antigens, and the 280K and 94K MAAs remained relatively constant over a wide range of cell sizes that were examined, with the exception that small melanoma cells showed a slightly lower mean surface density of MAA expression than did large cells. A novel method was used to detect the expression of the two MAAs as a function of the Colo 38 cell cycle using dual-laser beam flow cytometry. The results of these experiments show that both the 280K MAA and the 94K MAA are differentially expressed during various stages of the cell cycle, with each antigen being maximally detected during G2 + M.
Antibodies, Monoclonal↗
Distribution and molecular characterization of a cell-surface and a cytoplasmic antigen detectable in human melanoma cells with monoclonal antibodies.
The monoclonal antibodies 225.28S and 465.12 to human melanoma-associated antigens have been tested with a large variety of surgically removed skin lesions and malignant tumors as well as with a panel of cultured cell lines in serological and immunochemical assays. The antibody 225.28S reacts with a plasma membrane antigen while the antibody 465.12 detects a cytoplasmic antigen. Both antibodies fail to react with melanocytes from normal skin as well as benign skin lesions but react with nevi, melanoma cells and some skin carcinomas. Analysis with surgically removed tumors and cultured human cell lines indicated that the plasma membrane antigen is restricted to skin lesions whereas the cytoplasmic antigen is synthesized by tumor cells of various histological origins. The plasma membrane antigen is composed of two glyco-polypeptides of 280,000 and greater than 440,000 daltons, while the cytoplasmic antigen consists of 4 glycopolypeptides of 94,000, 75,000, 70,000 and 25,000 daltons. None of these components are bridged by disulfide bonds. The cytoplasmic antigen was readily detected in the spent culture medium of melanoma cell lines in the form of a major 94,000 dalton and a minor 72,000 dalton structure, while the plasma membrane antigen was detectable only after vastly increasing the sensitivity of the assay system.
Antibodies, Monoclonal↗