The mechanism of leukoregulin enhancement of target cell susceptibility to NK cell mediated cytotoxicity in humans.
Explore the source record for details and available documents.
Cell Membrane Permeability↗
Biomedical subjects
R P McCabe
Publications and source records attributed to R P McCabe.
Leukoregulin, a direct-acting anticancer immunological hormone that is distinct from lymphotoxin and interferon.
Human lymphokine preparations can directly lyse or suppress proliferation of human tumor cells or can enhance the susceptibility of human tumor cells to lysis mediated by natural killer lymphocytes. In the past, these antitumor activities were attributed to lymphotoxin. This study demonstrates, however, that these human lymphokine antitumor cell activities are biochemically separable from lymphotoxin and are properties of a lymphokine which was named leukoregulin because it is produced by lymphocytes and it regulates target cell physiology and growth. Leukoregulin obtained by high-performance liquid chromatography and isoelectric focusing was free of detectable lymphotoxin, interferon, interleukins 1 and 2, and macrophage-activating factor activities. Leukoregulin has an apparent molecular weight of 135,000 as measured by linear gradient polyacrylamide gel electrophoresis and gel filtration chromatography and has isoelectric pHs of approximately 5.3 and 7.5. The molecular weight of leukoregulin, determined in the dissociating conditions of sodium dodecyl sulfate polyacrylamide gel electrophoresis, was 32,000. Flow cytometric analysis showed that tumor cell lysis, growth inhibition, and enhancement of susceptibility to natural killer cell-mediated cytotoxicity by leukoregulin were accompanied by rapid alterations in tumor cell membrane permeability. Lymphotoxin from human peripheral blood leukocytes and highly purified lymphotoxin from RPMI 1788 human lymphoblastoid cells lysed murine alpha-L929 tumor cells but did not possess any of the direct acting antihuman tumor cell cytostatic, cytolytic, or natural killer cell enhancing activities that leukoregulin exhibited against a broad spectrum of human tumor cell lines. The dual modes of the anticancer actions of leukoregulin, direct cytotoxicity and indirect enhancement of natural killer cell cytotoxicity, make leukoregulin a unique-acting lymphokine and suggest several ways in which leukoregulin may be used as a therapeutic agent against cancer.
Cell Line↗
Generation of tumor cell-reactive human monoclonal antibodies using peripheral blood lymphocytes from actively immunized colorectal carcinoma patients.
The use of human monoclonal antibodies (MCA) in the detection and treatment of human cancer has been limited by the apparent scarcity of MCA to tumor cell surface antigens. Using peripheral blood lymphocytes from autologous tumor-immunized patients, we isolated 36 MCA that react to sections of colorectal carcinoma. Twenty of these human MCA appear to be directed against cell surface antigens. Two-thirds of the human MCA-producing cell lines were diploid human B-cells rather than human-mouse heterohybridomas. Direct antibody-binding assays performed with the MCA indicated that they recognized antigenic determinants preferentially expressed on tumor cells. Experiments with paired specimens of air-dried, dissociated colon tumor cells and normal colonic mucosa cells suggested that the MCA bound significantly more to the cell surfaces of tumor cells than to the surfaces of normal colonic mucosa cells. Similarly, tests with a panel of cryostat sections of paired colon tumor and normal colonic mucosa showed that MCA bound to the tumor cells and not to the normal colonic mucosa. None of the MCA bound to cells from frozen sections of normal breast, stomach, liver, skeletal muscle, or skin. Furthermore, the human MCA did not react with carcinoembryonic antigen and human erythrocyte antigens as measured by various techniques. Our data also demonstrated that these transformed B-cells and hybridomas were stable producers of human MCA. Thus, our studies show that these tumor-specific human MCA may have the specificity and stability necessary for in vivo evaluation of their use in the detection and treatment of cancer.
Animals↗
Mechanism of natural delayed-type hypersensitivity reactions to tumor cells in nonimmunized syngeneic guinea pigs.
Nonimmunized 2/N guinea pigs respond to the presence of chemical carcinogen-transformed syngeneic tumorigenic cells with a sustained (delayed-hypersensitivity-type) 4-day intradermal induration consisting of predominantly polymorphonuclear leukocytes on day 1 and mononuclear cells by day 4, which is independent of the presence of tumor-specific antigens on the tumorigenic cells. Chemical carcinogen-induced morphologically transformed but nontumorigenic cells also induce a polymorphonuclear response by day 1, but neither induration nor a mononuclear response is present on day 4, demonstrating the specificity of the 4-day sustained indurative response for tumorigenic cells. Induration and cellular infiltrates are unaltered if tumor cells are treated prior to injection with the cytostatic lymphokine lymphotoxin or with x-irradiation to inhibit cell proliferation. The intradermal polymorphonuclear leukocyte host response on day 1, but not the mononuclear response on day 4, is also induced by mitomycin C-treated cells or a cytokine culture medium from the cells. No response is present on day 1 or day 4 when cell membranes or lyophilized cells are injected. Thus natural delayed-hypersensitivity-type skin reactivity is a mononuclear leukocyte response specifically directed against intact and metabolically active but not necessarily proliferating tumor cells.
Animals↗
Secretion of proteinase inhibitors by tumorigenic and nontumorigenic guinea pig and Syrian hamster fibroblasts: evidence for autocrine regulation of local proteolysis.
A cytokine that inhibits fibrinolysis has been detected in the serum-free culture medium of guinea pig and hamster fibroblasts. This proteinase inhibitor was also present in Triton X-100 extracts of guinea pig cells. It was stable at pH 3.0 for 2 hr and was produced by cells rather than assimilated from serum in the culture medium as evidenced by: (a) an apparent molecular size (less than 45 kilodaltons) less than that of the principal serum-derived proteinase inhibitors; (b) its continued secretion after several passages of the cells in serum-free medium; and (c) the lack of inhibitory activity in the medium of mitomycin C-treated cells. The cytokine inhibited the proteinase activity of human urokinase, soluble TPA-stimulated guinea pig plasminogen activator, and the cell-associated plasminogen activator of tumorigenic guinea pig cells. Soluble plasminogen activator appeared to be inhibited to a greater degree than the cell-associated enzyme. The fluorogenic substrate (7-(N-carbobenzoxyglycylglycylargininamido)-4-methylcoumarin was used in a direct assay of proteinase activity and demonstrated that the cytokine inhibited both plasminogen activator and plasmin, the two proteinases of the fibrinolytic cascade. Tumorigenic guinea pig and hamster fibroblasts as well as nontransformed guinea pig fibroblasts were found to produce the inhibitory cytokine, and the amount of inhibitor secreted was independent of the tumorigenic potential of the cells. Production of the inhibitor by normal cells may be related to contact inhibition of growth, and this cytokine may contribute to the fine regulation of local proteolysis within tissues.
Animals↗
A diagnostic-prognostic test for bladder cancer using a monoclonal antibody-based enzyme-linked immunoassay for detection of urinary fibrin(ogen) degradation products.
An enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody was developed to determine the clinical value of urinary fibrinogen/fibrin degradation product levels for the identification and management of patients with bladder cancer. Assays were performed on 286 serial urine specimens from 56 bladder carcinoma patients. Specimens were grouped according to whether the patient had an evident tumor at the time of specimen collection (134 specimens, 41 patients) or was clinically disease-free following treatment (152 specimens, 38 patients). Many patients contributed specimens to both groups as determined by their clinical status at the time of collection. In addition, 45 specimens from 33 patients with inflammation of the urogenital tract and 81 specimens from 19 patients with renal or prostatic cancer were assayed for urinary fibrin degradation products. The ELISA, using a high-sensitivity procedure, identified 83% of the specimens from bladder cancer-positive patients with an overall accuracy with all specimens of 78% and a false-negative rate of 5% for all specimens tested. The high-sensitivity ELISA appeared most appropriate for monitoring bladder cancer patients for recurrence of tumor after surgery. The ELISA using a high-specificity procedure appeared most appropriate for screening. The high-specificity ELISA accurately identified 96% of urine specimens from non-bladder cancer patients with a false-positive rate of only 5%. These results demonstrate that the ELISA is an efficient, reliable, quantitative, and noninvasive immunoassay that can be useful both for the identification of bladder cancer patients and for monitoring the course of the disease.
Aged↗
Relationship of neoantigens induced by 3-methyl-cholanthrene treatment of Syrian hamster embryo cells to antigens expressed on fetal and 3-methyl-cholanthrene-transformed neoplastic cells.
Neoantigen(s) induced on Syrian hamster cells during chemical carcinogenesis are also found on fetal and neoplastic hamster cells. 46 neoplastic cell lines independently isolated from colonies of 3-methylcholanthrene (3-MCA) in vitro-transformed hamster cells growing in semi-solid agar medium were assayed for expression of neoantigens recognized by hamster antisera to primary cultured late-term (15 days) hamster embryo cells treated for 18 h with 10 micrograms 3-MCA/ml. Ratios of the binding of this sera compared to solvent control sera ranged from 0.7 to 2.1 in terms of cpm bound. Only four of the 46 neoplastic cell lines exhibited significant (P less than 0.05) neoantigen expression. No correlation existed between the concentration of 3-MCA used to establish the neoplastic cell line and expression of the neoantigen(s). Absorption of the sera with these four highly reactive neoplastic cell lines and mid-term (10 days) embryo cells indicated that the neoantigen(s) recognized were common to the four reactive neoplastic cell lines and the mid-gestation fetal cells. The occurrence of early persistent immunogenic cell-surface alterations during in vitro carcinogenesis provides an approach to isolation of preneoplastic populations and provides potential target structures for the inhibition of carcinogenesis.
Animals↗
Plasminogen activator, fibronectin, lymphotoxin sensitivity, and natural skin reactivity relationships to guinea pig cell tumorigenicity.
The quantitative expression of five properties of chemical carcinogen-induced, neoplastically transformed NIH strain 2 guinea pig fibroblasts was compared in cells possessing thousandfold differences in tumorigenicity. Plasminogen activator synthesis, sensitivity to lymphotoxin inhibition of cell proliferation, and the ability to induce a natural delayed tuberculin-type skin reaction in nonimmune syngeneic guinea pigs correlated directly with the number of cells required to produce a tumor. The most tumorigenic cells (10(2)-cell threshold dose) produced the most plasminogen activator, were most sensitive to lymphotoxin, and produced the greatest skin reactivity. Cells with a threshold tumor dose of 10(5)-10(7) cells exhibited the lowest expression of these properties. Fibronectin incorporation into an extracellular matrix was diminished in tumorigenic cells, as was anchorage-dependent growth; but neither diminished fibronectin incorporation nor the decreased anchorage requirement correlated quantitatively with the number of cells required to produce a tumor. The present investigation indicates that plasminogen activator synthesis, sensitivity to lymphotoxin, and the capacity of tumorigenic cells to induce natural delayed-type skin reactivity are among the factors that influence initial tumor growth. Plasminogen activator, an extracellular protease, may aid in the growth and spread of tumor cells in vivo by interfering with host fibrin deposition and by inactivating other host proteins such as lymphotoxin.
Animals↗
Immunogenicity of guinea pig cells transformed in culture by chemical carcinogens.
The immunogenicity of inbred strain 2/N guinea pig fibroblasts transformed to the malignant state in vitro by chemical carcinogens was evaluated with the use of a variety of in vivo and in vitro methods including delayed-type hypersensitivity skin and tumor transplantation tests and analysis of antibody production by immunofluorescence, complement fixation, and staphylococcal protein A binding tests. Neoplastic transformation was induced by direct treatment of cells in culture with benzo[a]pyrene, 3-methylcholanthrene, or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or by the host-mediated method by which fetuses were exposed to diethylnitrosamine or MNNG in vivo prior to cell culture. Rabbits and syngeneic guinea pigs were inoculated with unirradiated and X-irradiated clonally derived cells. Delayed hypersensitivity skin reactions to immunizing or other cells were equivalent in immunized or control guinea pigs, and no protection to tumor outgrowth from a challenge inoculum of immunizing cells was observed. Antibody activity induced in the sera of immunized guinea pigs was cross-reactive and removed by absorption with nontumorigenic cells. Rabbit antisera after absorption with fetal guinea pig cells were nonreactive with the specific immunizing or other culture cells. Chemical carcinogen-induced neoplastic transformation of guinea pig cells can, therefore, occur without formation of detectable, individually distinct cell surface tumor-specific neoantigens.
Animals↗
Tumor-associated antigens in spent medium of human melanoma cells: immunochemical characterization with xenoantisera.
Xenoantisera to human melanoma cells and to partially purified melanoma-associated antigens were coupled to protein A-bearing Staphylococcus Aureus or protein A-Sepharose and used as immunoadsorbents for the indirect immunoprecipitation of intrinsically radiolabeled proteins released into culture medium from various cultured human tumor and nontumor cell lines. These radiolabeled immunoprecipitates when analyzed by SDS-PAGE revealed highly reproducible molecular profiles of proteins and glycoproteins released by various cultured tumor lines and control cells into their spent culture media. A comparison of molecular profiles together with data indicating the binding specificity of known xenoantisera produced against human melanoma cells or their extracts led to the discovery of 2 macromolecules that are associated with human melanoma cells; a glycoprotein with a subunit m.w. of 240,000 (240K) and a single-chain glycoprotein of 94,000 (94K) m.w. also found in association with human carcinoma cells are described in this report.
Animals↗
Lack of association of serologically detectable human melanoma-associated antigens with beta 2 microglobulin: serologic and immunochemical evidence.
Serologic and immunochemical assays showed that human melanoma-associated antigens (MAA) identified with operationally specific xenoantisera were neither spatially nor structurally associated with beta 2-microglobulin (beta 2-mu), the light chain of the HLA-A,B antigen molecular complex; i.e., cultured melanoma cells coated with a specific anti-beta 2-mu xenoantiserum maintained their reactivity with anti-MAA xenoantisera. Furthermore, soluble MAA were not bound by a beta 2-mu immunoadsorbent. Finally, MAA were shed into the culture medium of melanoma cells and then were immunoprecipitated with specific anti-MAA xenoantisera, analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, they appeared as two distinct structures with molecular weights of 240,000 and 94,000 but comprised no structure with the characteristic 12,000 molecular weight of beta 2-mu. Conversely immunoprecipitates obtained by the reaction of spent culture medium of [3H]valine-labeled melanoma cells with anti-beta 2-mu xenoantiserum had the 12,000-molecular-weight component but no structures with the molecular weights established for MAA. Thus the data refute the contention that serologically detectable MAA have a molecular structure similar to that of HLA antigens.
Antibodies, Neoplasm↗
A radioimmunometric antibody-binding assay for evaluation of xenoantisera to melanoma-associated antigens.
A radioimmunometric antibody-binding assay was developed with the use of 125I-labeled protein A of Staphylococcus aureus (SpA) for the evaluation of xenoantisera to human melanoma-associated antigens. Antisera were produced in New Zealand male albino rabbits by the injection of cultured human melanoma cells or soluble, partially purified melanoma-associated antigens isolated from these cells. Xenoantisera were rendered operationally specific for melanoma-associated antigens by absorption with human red cells and cultured lymphoblasts. The methodologic parameters and the quantitative relationships among xenoantisera, cultured melanoma target cells, and 125I-labeled SpA and their effect on the measurement of xenoantibody binding were critically evaluated. Data indicated the usefulness of the radioimmunometric assay in monitoring the efficacy of absorption and in characterizing the specificity of xenoantisera to melanoma-associated antigens. The radioimmunometric binding assay when modified and used as a binding inhibition assay was effective in the assessment of the serologic activity of soluble melanoma-associated antigens and thus may be used to monitor the progress of antigen purification.
Animals↗
Purification and immunologic evaluation of human melnoma-associated antigens.
Melanoma-associated antigens (MAA) were isolated and their functional immunologic properties were evaluated. Spent fetal calf serum-free culture media and 3-m KCI extracts of cultured human melanoma cells grown in this medium were used as antigen sources. Ultracentrifugal flotation on KBr was used to separate MAA and HLA antigens present in the extracts or spent culture media; thus interference by histocompatibility antigens was prevented in subsequent tests of tumor antigenic activity. MAA purified in this manner retained their immunologic functions as evidenced by their ability to produce delayed cutaneous hypersensitivity reactions in patients with melanoma, specifically combine with antimelanoma xenoantibody, and elicit production of functionally specific xenoantibody. Possible structural differences between HLA antigens and MAA were considered in evaluation of the data.
Animals↗
Carcinoembryonic antigen (CEA) activity in pancreatic juice of patients with pancreatic carcinoma and pancreatitis.
Carcinoembryonic antigen (CEA) activity was assayed in plasma and in pure pancreatic juice from eight patients with carcinoma of the pancreas, 28 patients with pancreatitis, and 13 controls with no demonstrable pancreatic disease. Juice specimens were obtained via direct transduodenal cannulation of the pancreatic duct. The mean pancreatic juice CEA activities in controls, pancreatitis, and pancreatic carcinoma were 8.1 ng/ml, 18.6 ng/ml, and 309 ng/ml, respectively. Pancreatic juice CEA activity in patients with cancer of that organ was significantly higher than in those with pancreatitis or in controls. None of the 32 subjects with both pancreatic juice CEA activity less than 30 ng/ml and plasma CEA less than 2.5 ng/ml had pancreatic cancer. Three of the four patients with CEA elevations in both fluids above these levels harbored this malignancy. These findings suggest that combined measurement of CEA activity in plasma and pancreatic juice may help in diagnosing pancreatic disease.
Carcinoembryonic Antigen↗
Demonstration and immunochemical characterization of carcinoembryonic antigen in human pancreatic juice.
Pancreatic juice collected from 10 patients without evidence of malignant disease of the pancreas or other organs was pooled, extracted, and fractionated by Sepharose 6-B and Sephadex G-200 gel filtration. The carcinoembryonic antigen (CEA) activity in the material was demonstrated and studied by: a) radioimmunoassay, b) competitive binding to antibodies against CEA, c) precipitin inhibition, and d) Ouchterlony analysis. The immunochemical identity of the active material to CEA purified from liver metastases of colon cancer was demonstrated.
Binding, Competitive↗
Solid-phase radioimmunoelectrophoresis: an assay for carcinoembryonic antigen.
Explore the source record for details and available documents.
Adenocarcinoma↗
New chelating agent for attaching indium-111 to monoclonal antibodies: in vitro and in vivo evaluation.
111In possesses excellent radiophysical properties suitable for use in immunoscintigraphy of cancerous tissues when attached to an antitumor antibody. However, 111In has a tendency to accumulate in normal tissues such as liver. Instability of the linkage between 111In and antibody may contribute to this problem. To avoid this, we developed a new bifunctional chelating agent, 1,3-bis[N-[N-(2-aminoethyl)-2-aminoethyl]-2-aminoacetamido]-2-(4- isothiocyanatobenzyl)propane-N,N,N',N'',N''',N'''',N''''',N'''''- octaacetic acid (LiLo), that forms a kinetically stable chelate with metal ions such as indium. Using LiLo, indium-111 was conjugated to a human monoclonal antibody, 16.88. Competitive binding analysis revealed that the 16.88-LiLo conjugate is as immunoreactive as the unconjugated native antibody. This conjugate was compared with 111In-16.88, where diethylenetriaminepentaacetic acid dianhydride (DTPAa) was used as the chelating agent. In vitro stability studies showed that 111In was more stably bound to 16.88-LiLo than to 16.88-DTPA. Biodistribution studies in athymic mice bearing colorectal tumor xenografts indicated less liver retention with 16.88-LiLo than with 16.88-DTPA. These results demonstrate that LiLo is superior to DTPAa for attachment of 111In to the monoclonal antibodies.
Animals↗
Cobra venom factor immunoconjugates: effects of carbohydrate-directed versus amino group-directed conjugation.
Human IgM monoclonal antibody 16-88, derived from patients immunized with autologous colon carcinoma cells, was derivatized with two different cross-linkers, S-(2-thiopyridyl)-L-cysteine hydrazide (TPCH), which is carbohydrate-directed, and N-succinimidyl-3-(2- pyridyldithio)propionate (SPDP), which is amino group-directed. Two antibody functions, antigen binding and complement activation, were assayed upon derivatization with TPCH and SPDP. TPCH allowed for extensive modification (up to 17 TPCH molecules per antibody) without impairment of antigen binding activity, while this function was significantly compromised upon derivatization with SPDP. Antibody molecules derivatized with 16 SPDP residues showed almost complete loss of their antigen binding function. The complement activating ability of antibody 16-88 was significantly decreased after derivatization with TPCH or SPDP. In the case of SPDP derivatization, this decrease of the complement activating ability is predominantly a consequence of the impaired binding function. Upon conjugation of cobra venom factor (CVF), a nontoxic 137-kDa glycoprotein which is capable of activating the alternative pathway of complement, the antigen binding activity of SPDP-derivatized antibody was further compromised, whereas that of TPCH-derivatized antibody remained unaffected even after attachment of three or four CVF molecules per antibody. In both conjugates CVF retained good functional activity. CVF was slightly more active when attached to SPDP-derivatized antibody, suggesting a better accessibility of amino group-coupled CVF for its interaction with other complement proteins. These results indicate that carbohydrate-directed conjugation compromises the antibody function of complement activation, but allows for the generation of immunoconjugates with unimpaired antigen binding capability.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗