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Biomedical subjects

J Bubeník

Publications and source records attributed to J Bubeník.

At least 91 records · Page 5Linked to original sources
PubMed1987

Cancer immunotherapy using local interleukin 2 administration.

Peri-tumoural administration of human recombinant interleukin-2 (RIL-2) into C57BL/10ScSnPh (B10) mice carrying subcutaneous transplants of syngeneic methylcholanthrene (MC)-induced sarcomas substantially inhibited tumour growth. Experiments were designed to compare the tumour-inhibitory effect of highly purified RIL-2 with that of unpurified human and rat lymphoid interleukin-2 (IL-2) preparations. It was found that the effect of RIL-2 was significantly lower than that of the lymphoid IL-2 preparations. These findings indicate that other lymphokines may participate in the positive results of local IL-2 immunotherapy using unpurified lymphoid IL-2 preparations. However, the admixture of human recombinant interleukin-1 (RIL-1) did not potentiate the immunotherapeutic effects of RIL-2. Sensitivity of MC-induced sarcomas to local RIL-2 immunotherapy was a general phenomenon. The growth of approximately eighty percent (5/6) of the MC-induced sarcomas could be inhibited with local RIL-2 administration. Moreover, direct correlation between the sensitivity of tumours to the tumour-inhibitory effect of RIL-2 in vivo and their susceptibility to the cytolytic effect of RIL-2-activated syngeneic killer spleen (LAK) cells in vitro was observed. This correlation indicates that LAK cells represent the effector cell mechanism responsible for the anti-tumour efficacy of local RIL-2 immunotherapy and that in vitro testing of sensitivity to the LAK cell-mediated cytolysis may be used to detect tumours responding to the local RIL-2 immunotherapy in vivo.

Animals↗
PubMed1987

Localization of a receptor binding site on the IL-2 molecule.

The present study was performed to localize in the IL-2 molecule the active site responsible for interaction with the IL-2 receptor. To predict the receptor binding site on the IL-2 molecule, a computer programme based on the hypothesis that the active site will contain parts of the protein molecule having a high tendency to form a bend was utilized. The tendency to form a bend was evaluated by assessing the probability of beta-turn occurrence; the highest probability was found in the tetrapeptide Asn-Pro-Lys-Leu, occupying the positions 33-36 of the IL-2 molecule. Accordingly, the hexadecapeptide H-Cys-Nle-Gly-Ile-Asn-Asn-Tyr-Lys-Asn-Pro-Lys-Leu-Thr-Arg-Met-Leu-NH2 that spans over the predicted tetrapeptide Asn-Pro-Lys-Leu and comprises the region 27-40 from the IL-2 amino acid sequence was synthesized. This synthetic (I-16) peptide was found to selectively inhibit the IL-2-dependent uptake of 3H-TDR by CTLL cells, apparently by competing with IL-2 for the IL-2 receptor. The synthetic I-16 hexadecapeptide was conjugated to carrier (BSA) protein and used for immunization of rabbits. Resulting I-16 antibodies were capable of binding specifically to the I-16 hexadecapeptide in indirect ELISA test; they reacted substantially with IL-2-producing but not with IL-2-non-producing Jurkat cells in indirect cell membrane immunofluorescence, and inhibited activation of killer spleen cells with human recombinant IL-2 as detected by 51Cr microcytotoxicity assay. Taken together, these results suggest that at least one of the receptor contact sites of the IL-2 is localized within the N-terminal part of the molecule in the region defined by amino acids 27-40 and coded for by the exon 1 and 2.

Antibodies↗
PubMed1987

Expression of IL-2 receptors on peripheral blood lymphocytes from young adult and aged human donors.

Peripheral blood mononuclear cells from young adult and aged human donors were activated with PHA and the expression of IL-2 receptors was examined. For the detection of IL-2 receptor-positive cells, flow cytofluorometry using monoclonal antibody Clonab IL-2R, fluorescein-conjugated swine anti-mouse IgG and fluorescence activated cell sorter FACS 440 were utilized. The age-related decrease in the expression of IL-2 receptors was observed: the percentage of IL-2 receptor-positive PBMC was substantially higher in the group of young adult donors than in the group of the aged individuals.

Adult↗
PubMed1986

The presence of retroviral particles in hybridoma cell lines.

The presence of very high numbers of type C and type A retroviral particles was repeatedly confirmed not only in myeloma cells NSI, Ag 8 and in thymoma cells BW5147, EL 4 but also in B and T cell hybridomas constructed from the myeloma and thymoma cells used. Retroviral particles were demonstrated by current electron-microscopic and physicochemical methods. Biological tests for the induction of possible malignant or any pathological changes in the artificially infected sensitive cells during long-term cultivations in vitro or in vivo gave negative results. Nevertheless, on account of the hitherto unknown action of retroviruses one can suppose that monoclonal products from B and T cell hybridomas, particularly when used for practical purposes, should be purified because of their infectious nature.

Animals↗
PubMed1986

Activation of killer cells from blood of urinary bladder carcinoma patients by short-term treatment with recombinant interleukin 2.

Highly purified recombinant human interleukin 2 induced cytotoxicity of lymphocytes from urinary bladder carcinoma patients and from control healthy donors when added during an 18-h 51Cr microcytotoxicity assay against bladder carcinoma (T24) target cells. Similar levels of killer cell activation were detected in mononuclear cell preparations from bladder carcinoma patients and control healthy donors; hence, no defect in the responsiveness of bladder carcinoma patients' lymphocytes to interleukin 2 could be observed. The effect of the recombinant interleukin 2 was dose-dependent. Addition of monoclonal antibody 7E9 directed against cell-type restricted antigen associated with the T24 target cells and capable of inducing antibody-dependent cellular cytotoxicity could not increase the cytotoxicity-inducing effects of interleukin 2.

Antibodies, Monoclonal↗
PubMed1986

Local treatment with human recombinant interleukin 2 inhibits growth of MC-induced sarcomas in syngeneic mice.

In previous communications we have demonstrated that crude rat interleukin 2 and partially purified mouse interleukin 2 were capable of inhibiting growth of transplantable, MC-induced mouse sarcomas in syngeneic recipients. Here we report that repeated peritumoral injections of highly purified human recombinant interleukin 2 can inhibit growth of these mouse sarcomas and prolong survival of tumour-bearing mice. These findings taken together and the ready availability of high doses of recombinant human interleukin 2 substantiate our proposal for initiation of clinical trials using local administration of the interleukin 2 (Bubeník et al. 1983) in selected cancer patients.

Animals↗
PubMed1986

Immunotherapy of murine sarcomas with interleukin 2. I. Local administration of human recombinant IL-2 preparations.

The immunotherapeutic effect of human recombinant interleukin 2 was examined with a panel of MC-induced murine sarcomas carrying individual tumour-specific transplantation antigens. Repeated peritumoral injections of RIL-2 inhibited growth of five (MC11, MC13, MC14, MC15, MC16) out of six sarcomas in syngeneic mice. The sixth murine sarcoma (MC12) was resistant to the tumour-inhibitory effect of human recombinant IL-2 as well as to the tumour-inhibitory effect of murine and rat lymphoid IL-2 preparations. Since the IL-2-sensitive and IL-2-resistant sarcomas were induced with MC in mice of identical genotype and share most of their characteristics, they represent a useful model for investigation of structural target cell determinants and functional target cell properties responsible for the sensitivity of tumours to the immunotherapeutic effects of IL-2.

Administration, Topical↗
PubMed1986

Immunotherapy of murine sarcomas with interleukin 2. II. Activation of killer cells by human recombinant IL-2.

Highly purified human recombinant interleukin 2 induced cytotoxicity in mouse spleen cells against mouse sarcoma cells when added during the 51Cr microcytotoxicity assay. It elicited similar levels of killer cell activation as did human lymphoid (Jurkat leukaemia-derived) or mouse lymphoid (EL-4 leukaemia-derived) IL-2 preparations. The susceptibility of six MC-induced mouse sarcomas to the cytolytic effect of lymphokine-activated killer cells was compared. Five (MC11, MC13, MC14, MC15, MC16) of six mouse sarcoma cell lines examined were sensitive in vitro to the LAK cell effect, whereas one cell line (MC12) was resistant. Since the sensitive and resistant target cell lines had been induced with the same carcinogen and in mice of the same genotype, they represent a very useful model for investigation of target cell structures responsible for the sensitivity to the LAK cell effect.

Animals↗
PubMed1985

Tumour inhibitory effects of TCGF/IL-2/-containing preparations.

Supernatants from ConA-stimulated rat spleen cell cultures and from cultures of PMA-stimulated murine lymphoma subline EL-4TF were found to contain TCGF and to inhibit growth of a transplantable, MC-induced sarcoma MC11 in syngeneic mice. Tumour-inhibitory effects of the supernatants were dependent on local and repeated administration. Prior to use of the supernatants obtained from PMA-stimulated EL-4TF cell cultures, the dialysable PMA had to be removed; contamination with PMA was found to abolish the tumour-inhibitory effect of the supernatants and to produce enhancement of tumour growth. A significant tumour-inhibitory effect has also been obtained with partially purified TCGF prepared from culture supernatants of cloned EL-4TF cells by ammonium sulphate precipitation, ion-exchange (FPLC) chromatography, and AcA 44 Ultrogel filtration.

Animals↗
PubMed1985

The in vitro cytotoxic effect of bilirubin on human lymphocytes and granulocytes.

Using an in vitro cytotoxicity assay (51Cr-release), it was shown that bilirubin exerts a cytotoxic effect on both adult and newborn human lymphocytes after a 1-d incubation of cells with bilirubin. The effect of bilirubin on human granulocytes was less pronounced; the association of the cytotoxic effect with a functional immunological perturbation of these cells is discussed.

Adult↗
PubMed1985

Tumour localization of radiolabelled monoclonal antibody in mice bearing human urinary bladder (T24) carcinoma xenografts.

Selectivity of mouse monoclonal antibody 7E9 (IGG3) directed against human urinary bladder carcinoma cells has been examined by indirect membrane immunofluorescence, using a panel of 31 human cell lines. The 7E9 monoclonal antibody discriminated between urinary bladder carcinoma cells and normal urothelium or cells of non-urothelial origin, although occasional reactions with bladder carcinoma-unrelated cell types were observed. The 7E9 antibody was purified by protein A affinity chromatography, labeled with 131I and used for gamma scintigraphy in nude mice xenografted with human urinary bladder carcinoma T24. The 7E9 antibody was capable of locating the T24 xenografts in nude mice; it localized preferentially in the T24 tissue compared to normal mouse tissues. The T24 xenografts could not be detected by gamma scintigraphy with 131I-labelled monoclonal antibody against human mammary carcinoma cells and two other control antibodies. Likewise, the 131I-labelled 7E9 antibody was not capable of locating human mammary carcinoma xenografts in nude mice.

Animals↗
PubMed1984

Inhibition of tumor growth in mice treated with synthetic muramyl dipeptide.

Treatment with synthetic MDP inhibited growth of transplantable, chemically induced tumors in syngeneic mice. The tumor-inhibitory effect was dependent on the schedule of MDP administration. Growth of SC transplants of a nonmetastasizing, MC-induced fibrosarcoma, MC11, was inhibited by local treatment with 200 micrograms and 1,000 micrograms MDP given SC 5-7 weeks before challenge. Treatment with lower (10 micrograms and 100 micrograms) doses of MDP and shorter (1-4 weeks) time intervals was not effective. Single doses of MDP (10-1,000 micrograms) 1-3 weeks after challenge had no effect. Growth of IV-inoculated, metastasizing AAT-induced hepatoma A was inhibited by IV injections of 20 micrograms MDP given 1 and 2 days prior to the challenge. Significant increases in the survival of hepatoma-bearing mice were observed only after injections of MDP incorporated in multilamellar liposomes.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed1984

Phorbol myristate acetate-stimulated production of interleukin 2 by T-cell lymphoma and constitutive production by derived hybridomas.

To isolate a stable tumor cell line source of IL-2 (TCGF), 19 murine T-cell lines and their derivatives were screened for both constitutive and mitogen-stimulated IL-2 production. The cloned subline of a mouse thymic lymphoma EL-4 designated as EL-4TF could be stimulated with PMA to produce 80 U/ml of IL-2. A TK- EL-4TFR mutant line has been selected from the EL-4TF cell population by treatment with 5-BrdUrd. The EL-4-TFR cells were stimulated with PMA and fused with the cells of thymic lymphoma BW5147. The resulting BH3 hybrid cell population was repeatedly cloned and tested for constitutive IL-2 production: two of the BH3 hybridoma clones were found to produce IL-2 constitutively. The IL-2 of EL-4TF origin was found to support permanent in vitro growth of IL-2 dependent, tumor-specific T killer cell line CTLL when present in culture medium at a concentration of at least 0.1 U/ml. Since the EL-4TF-derived IL-2 preparations were contaminated with PMA, it was of interest whether PMA alone has a growth-promoting activity in CTLL cell cultures. Permanent cultivation of CTLL cells in an IL-2-free medium containing PMA was not possible. However, both mitogenic and co-mitogenic effects of PMA on CTLL cells were observed.

Animals↗
PubMed1984

BW 5147 supernatants suppress Con A stimulation of mouse spleen cells.

Conditioned medium from mouse T lymphoma BW 5147 cell cultures suppressed Con A-stimulated DNA synthesis in cultures of mouse spleen cells. Supernatants from control mouse T cell lymphoma EL-4 were not effective. Conditioned medium from BW 5147 cells did not inhibit DNA synthesis in control cultures of IL-2-dependent mouse T cells or mouse leukaemias of T cell or erythroblastic origin. It is suggested that constitutive production of a soluble suppressor factor by BW 5147 lymphoma cells is responsible for the effects observed.

Animals↗
PubMed1984

Increase in sensitivity of the microcytotoxicity assay by T cell growth factor (IL-2)-containing preparations.

Supernatants from Con A-stimulated rat spleen cell cultures containing TCGF were able to increase the sensitivity of the microcytotoxicity assay. Spleen cells from mice immunized with irradiated syngeneic tumour were precultivated for 24 h in a TCGF-containing medium. The precultivated immune spleen cells, when allowed to react with syngeneic tumour target cells, displayed a higher cytotoxic effect than the same immune spleen cells precultivated in a medium without TCGF. Similarly, the addition of TCGF-containing medium to the effector cells during the microcytotoxicity assay increased the cytotoxic effect. Combination of both schedules, i.e., precultivation of the immune effector cells in a TCGF-containing medium and addition of TCGF-containing medium to the effector cells during the microcytotoxicity assay produced the highest increase in sensitivity of the microcytotoxicity assay.

Animals↗
PubMed1983

Thy-1 epitopes are expressed on murine myeloid leukemia and reticulum sarcoma cells.

Expression of Thy-1.2 specificities in cells from 29 primary spontaneous leukemias of random-bred ICR Swiss mice was examined by cell membrane and cytoplasmic immunofluorescence with monoclonal HO-13-4 antibody [1]. The Thy-1.2 epitopes were detected in all thymic lymphomas and were absent in the lymphomas of non-thymic origin. Unexpectedly, the Thy-1.2 epitopes were also detected in 71% (5/7) of myeloid leukemias and 40% (4/10) of reticulum cell sarcomas examined.

Animals↗
PubMed1983

T-cell hybrids. IV. One of parental cell lines is dominant in determining membrane characteristics of hybrid cells.

Membrane characteristics of parental thymic lymphoma cells (BW5147, EL-4R) and their hybrids (BH2) derived by PEG-promoted cell fusion were compared. Isoelectric focusing of the cell populations, titration of membrane proton binding groups and quantitative examination of cell-substrate adhesiveness indicated that the BW5147 cells behaved as dominant in determining cell membrane characteristics of the BH2 hybrids.

Animals↗