Biomedical subjects

B J Clarke

Publications and source records attributed to B J Clarke.

At least 37 records · Page 2Linked to original sources

PubMed1988

Potential application of a new screening test for alpha-thalassemia-1 carriers.

A murine hybridoma cell line secreting monoclonal anti-human embryonic zeta-globin chain antibody has been established. Using this monoclonal antibody, a slot blot immunobinding assay for the detection of zeta-globin chains in adult hemolysates has been developed. This simple test can identify individuals who are alpha-thalassemia-1 carriers due to the (-SEA/) deletion. It is proposed that this test should be made generally available in Southeast Asia and Southern China, in order to identify couples who are at risk of begetting fetuses afflicted with homozygous alpha-thalassemia.

Animals↗

PubMed1988

An immunoassay to detect human embryonic epsilon globin chains by a murine monoclonal antibody.

Human embryonic epsilon globin chains were isolated from lysates of cultured human leukemic K562 cells induced with hemin. Mice were immunized with epsilon globin chains, and a murine hybridoma cell line was established that secreted monoclonal anti-epsilon globin antibody. A sensitive and specific slot blot immunoassay for epsilon globin chains has been developed. epsilon Globin chains were shown to be present in hemolysates from fetuses of 20 weeks' gestation but were not detected in normal cord blood hemolysates. This immunoassay is useful for the study of epsilon globin ontogeny during embryonic and fetal development as well as for searching for inappropriate epsilon globin chain expression in hematologic disorders resulting from genetic abnormalities or neoplastic processes.

Animals↗

PubMed1988

A novel monoclonal antibody based diagnostic test for alpha-thalassemia-1 carriers due to the (-SEA/) deletion.

The presence of minute amounts of embryonic zeta-globin chains in adult hemolysates is a marker for carriers of alpha-thalassemia-1 resulting from (--SEA/) deletion. Recently, we developed a murine monoclonal antihuman embryonic zeta-globin chain antibody, 8E8. By using this antibody, we have now established a slot-blot immunobinding assay for the rapid detection of zeta-globin chains in adult hemolysates. zeta-globin chains were found to be present in 30 blood samples obtained from individuals who were carriers of alpha-thalassemia-1. In another 30 blood samples from individuals who were not carriers of the (--SEA/) deletion, zeta-globin chains were not detected. This simple diagnostic test can be used in appropriate populations to identify those couples at risk of conceiving fetuses afflicted with the Hb Bart's hydrops fetalis syndrome due to homozygous alpha-thalassemia.

Antibodies, Monoclonal↗

PubMed1987

Distribution of a hematopoietic-specific differentiation antigen of K562 cells in the human myeloid and lymphoid cell lineages.

BALB/c mice were immunized with uninduced K562 erythroleukemia cells and hybridomas were isolated after fusion of immune spleen cells to P3/NS1 murine myeloma cells. One selected hybrid, designated 10L-30, secreted an antibody of subclass immunoglobulin G2a which was specific for hematopoietic cells. Analysis of 10L-30 binding by complement-mediated cytotoxicity, indirect immunofluorescence, solid-phase radioimmunoassay, and mixed hemadsorption assay indicated that the 10L-30 antigen was expressed on the myeloid cell lines K562, KG-1A, KG-1, some B- and T-lymphoid cell lines, and all normal human peripheral blood T-lymphocyte samples tested, but was absent on the more differentiated myeloid cell lines HL-60, ML-2, ML-3, and normal blood granulocytes. Induction of erythroid differentiation in hemin-treated K562 cells caused a 10-fold reduction in 10L-30 binding. Human erythroid and granulocytic progenitor cells, platelets, erythrocytes, and reticulocytes were nonreactive, as were a variety of nonhematopoietic human tumor cell lines. Freshly isolated leukemic bone marrow samples from patients with M5 (2 of 5), M6 (2 of 2), acute lymphoid leukemia (9 of 14), and chronic myeloid leukemia in lymphoid blast crisis (1 of 1) were 10L-30 positive. The combined evidence indicates that the 10L-30 antigen is a normal, hematopoietic-specific differentiation antigen which is strongly expressed on both immature cells of the myeloid lineage and more generally in lymphoid ontogeny. The 10L-30 antigen may be a useful marker of both normal and leukemic hematopoietic differentiation.

Animals↗

PubMed1987

Elevated atrial natriuretic peptide after the Fontan procedure.

To assess the response of atrial natriuretic peptide (ANP) to rapidly changing right atrial pressures in vivo, we measured ANP levels in 15 patients undergoing the Fontan procedure and compared them with control levels in nine patients undergoing cardiac surgery for lesions not associated with atrial hypertension. There were no significant differences in preoperative ANP levels: 57 +/- 15 pg/ml for patients undergoing the Fontan procedure, 43 +/- 8 pg/ml for control patients. There was no significant change in ANP during surgery or in the postoperative period in control patients. In contrast, ANP increased significantly to 333 +/- 70 pg/ml (p less than .0025) after establishment of right atrial-pulmonary artery continuity with the Fontan procedure and was related to right atrial pressure, which increased from 5 mm Hg before to 14 mm Hg after the Fontan procedure (p less than .001). There was no significant change in left atrial pressure. During the first postoperative day, ANP levels fell to 141 +/- 34 pg/ml (p less than .01) but later increased to 290 +/- 80 pg/ml (p less than .025), a finding that may suggest depletion of a readily releasable intracellular pool of ANP before mobilization of a storage pool. There was no direct relationship between ANP levels and effusions in patients undergoing the Fontan procedure. Pharmacologically significant increases in ANP occur in patients undergoing the Fontan procedure, correlating with increased right atrial pressures, but the relationship between ANP and the fluid derangements after the procedure remains unclear.

Atrial Natriuretic Factor↗

PubMed1985

The immunodepletion of factor VII from human plasma using a monoclonal antibody.

A murine hybridoma cell line which secretes monoclonal antibody to factor VII has been prepared to facilitate the immunodepletion of this clotting factor from plasma. Specific monoclonal antibody was purified from mouse ascites tumours by protein A-Sepharose chromatography and shown to be of the IgG1 immunoglobulin subclass. On immunoblotting, this antibody reacted with a single protein band identical to purified factor VII. The purified monoclonal antibody was coupled to Sepharose 4B and was used to immuno-deplete factor VII from pooled normal human plasma. The prothrombin time of plasma immunodepleted in this way was 35 s compared to 12 s for the starting plasma. Specific factor assays of the immunodepleted plasma showed factor VII activity to be less than 1% while the levels of the other clotting factors were unchanged. The immunodepleted plasma was equivalent to severe congenital factor VII deficient plasma as a substrate for factor VII assays. Bound factor VII could be eluted from the immunoaffinity column with citrate buffer, pH 6.0, with good recovery.

Animals↗

PubMed1985

Monoclonal antibody recognizing human melanoma-carcinoma cross-reacting oncofetal antigen epitopically associated with carcinoembryonic antigen.

By fusion of mouse NS1 myeloma cells with splenocytes from a BALB/c mouse immunized with human melanoma cells, an IgG1 monoclonal antibody, designated as 140.72, was produced. By the mixed hemadsorption antibody binding assay, 140.72 was shown to react with 17 of 20 melanoma cell lines and with 5 of 14 carcinoma cell lines. This antibody also reacted with 3 of 3 normal melanocyte cultures in much lower titers. It did not react with any of 35 other normal and malignant lines, including neuroblastoma, glioblastoma, sarcoma, teratoma, fibroblast, and lymphoid cell lines. Absorption with fresh melanoma and carcinoma homogenates confirmed the results of direct tests. Fetal reactivity of antibody 140.72 was determined by positive absorption with 10 of 11 tissue homogenates derived from different fetuses of 10-16 weeks' gestation. The reactivity of this antibody was completely removed by absorption with a highly purified preparation of carcinoembryonic antigen (CEA) derived from a colon carcinoma. The antigenic activity was detected in the culture medium of reactive cell lines. Immunoprecipitation analyses of melanoma and carcinoma cells indicated that the antigenic determinant recognized by antibody 140.72 is on a glycoprotein with an apparent molecular weight of 95,000-150,000 common to both serologically reactive cell types. Additionally, a 200,000-molecular-weight glycoprotein corresponding to the CEA molecule was detected only on the reactive carcinoma cells. These data confirmed previous findings obtained with polyclonal anti-CEA antisera for the existence of shared CEA-related antigenic determinants on human carcinomas and melanomas and provided additional molecular characterization of these glycoproteins. Further characterization of the molecules bearing the antigenic determinant recognized by antibody 140.72 should be performed with a view to exploring its potential in the immunodiagnosis and immunotherapy of patients with melanoma.

Animals↗

PubMed1984

Human embryonic zeta-globin chains in adult patients with alpha-thalassemias.

Human embryonic zeta-globin chains are alpha-globin-like chains that are normally present during the first three months of gestation. In this investigation, zeta-globin chains measured by a specific and sensitive radioimmunoassay and by an electrophoretic technique were found to be present in all 7 patients studied with hereditary Hb H disease, and in 8 out of 24 patients with alpha-thalassemia trait. zeta-Globin chains were not detected in 20 other patients with beta-thalassemia trait. These results suggest that the deletion of two alpha-globin genes on the same chromosome is accompanied by the continued expression of embryonic zeta-globin genes in adult individuals.

Adolescent↗

PubMed1984

Monocytes direct T lymphocyte stimulation of human peripheral blood granulocyte-macrophage colony formation.

The committed granulocyte-macrophage progenitor cell (or colony-forming unit, CFU-GM) differentiates in vitro under the influence of the soluble glycoprotein factor(s) termed colony stimulating activity (GM-CSA), which can be derived from several cellular sources, including normal human peripheral blood mononuclear cells (PBMNC). We have investigated the respective roles of the monocyte and T lymphocyte components of PBMNC in generating GM-CSA using as an assay CFU-GM colony formation by purified peripheral blood null cells. Highly purified unstimulated T-lymphocytes did not themselves stimulate null cell CFU-GM colony formation, but when monocytes were also included, large numbers of colonies were observed, far greater than those stimulated by monocytes alone. This synergism was independent of HLA phenotype, occurring with T-lymphocyte and monocyte fractions from unrelated donors. Media conditioned by unstimulated purified monocytes promoted CFU-GM colonies from null cells only in the presence of T-lymphocytes, suggesting that monocytes produce a factor directing the synthesis and/or release of GM-CSA by T cells.

Cells, Cultured↗

PubMed1984

Quantitative analysis of the role of accessory cells in the development of human blood BFU-E-derived erythroid colonies.

A simplified method for the purification of human peripheral blood erythroid progenitor cells (BFU-E) using standard immunological techniques is described. Following removal of platelets, erythrocytes, nylon-wool-adherent cells, and sheep erythrocyte rosette-forming cells (RFC), BFU-E are routinely concentrated tenfold in the null cell fraction. Null cells plated at low density in erythroid cell cultures containing optimal amounts of methylcellulose, erythropoietin, and fetal calf serum did not give rise to spontaneous erythroid colonies. Coculture of null cells with highly purified, autologous RFC at a ratio of 1:25 yielded well-hemoglobinized erythroid colonies which were noticeably smaller than those found in cultures containing unfractionated peripheral blood mononuclear cells. However, further addition of very low numbers of purified adherent cells to null plus RFC dramatically increased the total hemoglobin content as well as the size and number of BFU-E-derived erythroid colonies. Addition of adherent cells alone to null cells had virtually no effect. Under conditions of optimal stimulation by adherent cells and RFC, the number of erythroid bursts was linearly related to null cells plated over an eightfold range. The synergism exhibited between adherent cells and RFC was not restricted by mismatched histocompatibility antigens. This system should be generally useful in quantitating the roles of more highly purified cellular and molecular populations in human erythropoiesis.

Cell Adhesion↗

PubMed1983

Anemia associated with rheumatoid disease. Inverse correlation between erythropoiesis and both IgM and rheumatoid factor levels.

Cell culture techniques were used to evaluate the number of erythroid colonies formed by circulating progenitor cells from 24 patients with rheumatoid disease and controls. A highly significant inverse correlation was demonstrated between erythroid colony counts and serum IgM and rheumatoid factor concentrations in the rheumatoid patients. The potential role of these factors in the pathogenesis of the anemia of rheumatoid disease is discussed.

Adolescent↗

PubMed1983

Adult hemoglobins are synthesized in murine fetal hepatic erythropoietic cells.

The hemoglobins present in murine fetal hepatic erythroblasts on days 12-15 of gestation were studied by biochemical and immunocytologic techniques. In addition, fetal hepatic hemopoietic progenitor cells obtained from normal and mutant f/f mouse fetuses on days 11-13 of gestation were cultured in vitro with added erythropoietin and adult spleen cell conditioned medium to form large erythroid colonies. In all instances, adult hemoglobin synthesis was detected in the fetal hepatic erythroblasts and in the erythroid cell cultures in vitro. The tumor promoter, 12-O-tetradecanoylphorbol 13-acetate, enhanced the fetal hepatic erythroid colony growth in vitro, but did not alter the hemoglobin phenotypic expression.

Aging↗

PubMed1982

Human melanoma-specific oncofetal antigen defined by a mouse monoclonal antibody.

140.240, an IgG2a mouse monoclonal antibody raised against a cultured human melanoma cell line, was highly specific for melanoma cells as determined by direct and absorption analyses in a mixed hemadsorption assay. Supernatants of doubly cloned hybridomas producing antibody 140.240 reacted with all cultured and fresh melanomas tested but failed to react with a variety of carcinomas, sarcomas, lymphomas, leukemias and other tumors of neuroectodermal origin. This antibody did not react with B-lymphoid cell lines, ruling out HLA-DR specificity. Non-reactivity of antibody 140.240 with peripheral blood lymphocytes obtained from the donor of the immunizing melanoma line excluded the possibility of detecting histocompatibility antigens. Nevus cells were also non-reactive. However, antibody 140.240 did identify an antigenic determinant on tissue homogenates prepared from fetuses of 10-14 weeks' gestation. The antigen involved was shed by cultured melanoma lines and, by immunoprecipitation analysis of radiolabelled lysates, had a molecular weight of approximately 87kdal. Thus, the structure identified by monoclonal antibody 140.240 is a melanoma-specific oncofetal antigen.

Animals↗

PubMed1982

Adult hemoglobins are synthesized in erythroid colonies in vitro derived from murine circulating hemopoietic progenitor cells during embryonic development.

Circulating peripheral blood cells and disaggregated yolk sac cells were obtained from normal mouse embryos as early as day 9 of gestation, prior to the formation of the fetal liver. These were cultured in vitro in plasma clots or methylcellulose, in the presence of either embryonic fluid or adult spleen cell-conditioned medium, with or without added erythropoietin. Large erythroid colonies were observed by the sixth day of culture. In all instances, these erythroid colonies synthesized adult hemoglobins. These results indicate that erythroid progenitor cells committed to adult hemoglobin synthesis are present in early embryonic circulation.

Animals↗

PubMed1982

Abnormal erythroid progenitor cells in human preleukemia.

Ten patients with preleukemia were studied by the erythroid cell clonal culture technique. In nine of these patients, erythroid colonies derived from peripheral blood BFU-E were not observed, while the other patient had markedly decreased peripheral blood BFU-E-derived erythroid colonies in vitro. In three patients, marrow cells were also cultured and no BFU-E-derived erythroid colonies were detected. These studies indicate that immature erythroid progenitor cells, BFU-E, in patients with preleukemia are either markedly decreased in number or grossly defective in their proliferative or differentiative capacities.

Adult↗

PubMed1982

Effect of modulators of erythropoiesis on the hemoglobinization of human erythroid cell cultures.

A rapid spectrophotometric assay capable of detecting the hemoglobin content of 1000 mature erythrocytes has been utilized to quantitate the total hemoglobin synthesized by the progeny of circulating human erythroid progenitors in both the plasma clot and methylcellulose culture systems. The pronounced variation in the effect of different erythropoietin preparations on the hemoglobin content of cultured human peripheral blood bursts, previously described in a subjective manner, has been objectively quantitated. Further experiments demonstrated that both lymphocyte conditioned media and dexamethasone substantially increased the total hemoglobin synthesized by the progeny of cultured erythroid progenitors. The elevated amount of hemoglobin present in erythroid cultures containing either lymphocyte conditioned media and/or dexamethasone was due to both increased colony numbers and colony size. Assay of the total hemoglobin content per erythroid culture is an accurate, sensitive, measure of erythropoiesis in vitro and should be a valuable adjunct to the enumeration of BFU-E-derived erythroid colonies.

Cells, Cultured↗

PubMed1981

Common neuroectodermal antigens on human melanoma, neuroblastoma, retinoblastoma, glioblastoma and fetal brain revealed by hybridoma antibodies raised against melanoma cells.

The hybridoma system has been utilized to produce antibodies to characterize the cell surface antigens on human melanoma cells. On initial screening, two antibodies derived by the fusion of mouse myeloma cell (SP2/0-Ag14) and splenocytes from a mouse immunized with a melanoma cell line (CaCL 78-1) showed cross-reactivity with 10 melanoma cell lines and did not react with any of 4 epithelial cancer lines, or 4 normal adult fibroblast lines. However, because of reactivity with 2 neuroblastoma cell lines, additional testing with other neuroectodermal derivatives was carried out and revealed a broad cross-reactivity among melanomas, neuroblastomas, retinoblastomas and glioblastomas and against antigens shared by fetal but not adult brain. Thus, these results indicate the existence of common neuroectodermal antigens on melanoma cells. Before melanoma specificity can be claimed for, an antibody reactivity with nonmelanoma cells bearing these neuroectodermal antigen should be excluded.

Absorption↗

PubMed1981

Identification of immunoglobulin class and subclass of mouse monoclonal antibodies to human cell surface antigens by mixed hemadsorption assay.

A microassay is described to determine immunoglobulin Ig class or IgG subclass of mouse monoclonal antibodies by mixed hemadsorption assay. Monoclonal antibody bound to adherent target cells is reacted with serial dilutions of a panel of class or subclass specific rabbit anti-mouse Ig antisera and binding of the latter is traced by anti-rabbit globulin-coated indicator erythrocytes. The class or subclass of the bound monoclonal antibody is revealed by preferential binding of the corresponding rabbit antibody. Unlike gel immunodiffusion analysis, the mixed hemadsorption assay may be performed with unconcentrated hybridoma culture supernatants.

Animals↗