Tumour vaccines expressing IL-2, CD80, and IL-2 plus CD80 gene.

Experiments were designed to investigate immunogenicity and therapeutic efficacy of tumour vaccines constructed by transfection of poorly immunogenic murine sarcoma Mc12 with synergistic CD80 and IL-2 genes. Immunization/challenge experiments demonstrated that both, IL-2(+) and IL-2(+) plus CD80(+) live cell vaccines can exert an immunizing stimulus, the IL-2(+) plus CD80(+) vaccine being superior to the IL-2(+) vaccine. Live CD80(+) Mc12 cell vaccine could not be tested, since the vaccine was highly tumorigenic in the doses required for immunization. Preimmunization with IL-2(+) and IL-2(+) plus CD80(+) vaccines induced regressions of a proportion of the parental Mc12 challenge inocula after their temporary growth. Areas of necrosis and extensive infiltration with Mac1(+) and CD4(+) leukocytes have been observed in the regressing sarcomas. When the therapeutic efficacy of the irradiated CD80(+), IL-2(+), and mixed CD80(+) plus IL-2(+) vaccines was compared, it was found that the insertion of the IL-2, but not CD80 gene alone was efficient. The mixed IL-2(+) plus CD80(+) tumour vaccine was able to protect and prolong survival of a higher proportion of mice than the IL-2(+) tumour vaccine.

Tumour inhibitory effects of plasmid DNA.

The experiments were designed to examine whether direct in vivo transfer of plasmid DNA not carrying any 'therapeutic' genes ('empty' plasmid DNA) can influence tumour growth. Murine MC-induced sarcoma Mc12 was transplanted i.m. in syngeneic recipients and the tumour-inoculated mice were treated either with repeated peritumoral (i.m.) injections of the naked plasmid DNA or with a single peritumoral (i.m.) injection of plasmid DNA incorporated in liposomes. Two plasmid DNA preparations, BCMGNeo and pON1 were utilized. The direct in vivo transfer of both 'empty' plasmid DNA preparations inhibited tumour growth and significantly prolonged survival of tumour-bearing mice. Our results emphasize the importance of plasmid DNA controls for evaluation of gene therapy of cancer based on the transfer of 'therapeutic' genes in tumour-bearing individuals.

Effects of magnetic field exposure on the development of lung fibrosis elicited by industrial pollutants.

Lung injury elicited by a single intratracheal instillation of fibrogenic (quartz) and nuisance (anatase) dusts and/or weekly repeated instillation of CdCl2 solution combined with sinusoidal (50 Hz, 10 mT) magnetic field (MF) exposure was studied in male rats. Combined effects in bronchoalveolar lavage (BAL), rat lungs and regional lymph nodes after 4 months of MF exposure (1 h/5 days per week) were evaluated biochemically and by cytological and histopathological examination. Damage of cell membranes in the cell part of BAL due to MF exposure was not observed in the examined animal groups. Following MF exposure, decreased synthesis of collagen proteins (incorporation of [14C]proline) was demonstrated in lungs with quartz dust burden. Histological examination revealed differences in the lung tissue reaction suggesting the modification of the repair process due to MF exposure following experimental injury in both quartz and cadmium groups.

Interleukin-2 gene therapy of residual EL-4 leukaemia potentiates the effect of cyclophosphamide pretreatment.

Experiments were designed to investigate a possible therapeutic role of interleukin-2 (IL-2) gene transfer in the model of murine (EL-4) leukaemia pretreated with cyclophosphamide. It has been found that i.p. pretreatment of the leukaemic mice with cyclophosphamide, followed by i.v. administration of irradiated cells, genetically engineered to produce IL-2 and used as a source of the cytokine (IR-IL-2 cells), cured a substantial percentage of the leukaemic mice. Neither treatment with cyclophosphamide nor administration of the IR-IL-2 cells alone had any significant therapeutic effect. Labelling of the EL-4 and IR-IL-2 cells with different fluorescent cell linkers followed by i.v. injection and detection of the labelled cells in cryostat sections of various organs has shown that both cell populations can be detected almost exclusively in the red pulp of the spleen, close to the white pulp nodules, thus providing the possibility of short-range local interactions among the IL-2-producing cells, IL-2-responsive defence effector cells and EL-4 leukaemia targets.

Gene therapy of plasmacytoma: comparison of the therapeutic efficacy of tumour cells transduced with the interleukin-2, interleukin-4, or interleukin-6 genes.

The present study was designed to compare the tumour inhibitory effects of local therapy based on insertion of cloned IL-2, IL-4, or IL-6 genes into the genome of murine plasmacytoma X63-Ag8.653, followed by injection of the genetically modified cells to the vicinity of the parental plasmacytoma growing in syngeneic mice. It was also designed to investigate the effects of combined gene therapy with IL-2- and IL-6-producing plasmacytoma cells. It has been found that insertion of the IL-2 gene into the genome of X63-Ag8.653 cells can abrogate tumorigenicity of the transduced cells more effectively than insertion of the IL-4 or IL-6 gene. Peritumoral administration of the genetically modified plasmacytoma cells producing IL-6 (X63-h-IL-6) resulted in a therapeutic effect comparable with that of the IL-2-producing cells (X63-m-IL-2), whereas no substantial therapeutic effect of IL-4-producing cells (X63-m-IL4) was observed. The combined gene therapy with IL-2- and IL-6-producing cells did not give any additive or potentiated therapeutic effect.

Kinetics and function of peritoneal-exudate cells during local IL-2 gene-therapy of cancer.

The present study was designed to examine the kinetics and function of peritoneal exudate cells (PEC) during local interleukin 2 (IL-2) gene therapy of the X63-Ag8.653 plasmacytoma growing in the peritoneal cavity. BALB/c mice were inoculated i.p. on day 0 with a tumorigenic dose of the syngeneic plasmacytoma and on day 1 with non-tumorigenic plasmacytoma cells carrying an inserted IL-2 gene and producing constitutively IL-2. At regular time intervals the experimental mice were sacrificed and their peritoneal exudate cells were used for phenotypic analysis and Cr-51 microcytotoxicity assay. On the first day after i.p. inoculation of the genetically modified plasmacytoma cells the percentage of Thy 1.2+, CD3+, TCR alphabeta+ T lymphocytes and NK+ cells in the peritoneal fluid dramatically increased. The levels of the positive cells continually decreased until day 11, when the values of normal, healthy mice were obtained. The percentage of Thy 1.2+ and CD3+ cells remained at these, or slightly lower values, until the end of the observation period. A similar, though more slowly descending kinetics was seen in the CD5+ cell population, whereas the CD8+ cells, compared to the controls, exhibited only a short-term peak between days 3 and 5, and the values of TCR alphabeta+ and NK+ cells exhibited a second peak between days 25 and 48. The percentage of TCR gammadelta+ cells showed a permanent, moderately elevated plateau from day 1 till the end of the observation period. In control, untreated mice, inoculated i.p. with the X63-Ag8.653 plasmacytoma, the kinetics of peritoneal exudate cells was different. A moderate, permanent elevation of all of the T and NK cell subsets examined occured during the observation period. In addition, the percentage of TCR alphabeta+, TCR gammadelta+ and NK+ cells further increased continually from day 11 till the end of the observation period. The cytolytic activity of the peritoneal exudate cells was examined in vitro concurrently with FACS phenotyping. Free tumour-specific killer cells generated in the peritoneal fluid due to the local IL-2 gene therapy were found only on day 6, and these cells were cytolytic for both, the parental X63-Ag8.653 and the genetically modified X63-m-IL-2 plasmacytoma cells.

Gene therapy of cancer: use of IL-2 gene transfer and kinetics of local T and NK cell subsets.

Experiments were designed to compare the efficacy of recombinant IL-2 immunotherapy and IL-2 gene therapy of i.p. growing murine plasmacytoma X63-Ag8.653. The kinetics of peritoneal exudate mononuclear cells were monitored during the progression and gene therapy of the plasmacytoma, using cytofluorometric analysis and monoclonal antibodies against T and NK cell subsets. It has been found that the percentage of mice protected against plasmacytoma transplants was higher in mice treated by transfer of genetically manipulated IL-2-producing plasmacytoma cells as compared to the mice repeatedly injected with recombinant IL-2. Intraperitoneal inoculation of the X63-Ag8.653 plasmacytoma led in most of the inoculated mice to an increased percentage of NK+, ASGM1+, Thy 1.2+, CD3+ and TCR alpha beta+ cells in the peritoneal fluid. The presence of macroscopically detectable i.p. tumours was accompanied by a higher increase in the percentage of NK+ and TCR gamma delta+ cells. Local IL-2 gene therapy of the plasmacytoma either prevented or diminished an increase in the percentage of CD3+, Thy 1.2+ and TCR alpha beta+ lymphocytes.

Utilization of interleukin-2 gene transfer in local immunotherapy of cancer.

It has been previously found that local administration of Balb/c plasmacytoma cells transformed and made non-tumorigenic by insertion of the cloned murine interleukin-2 (IL-2) gene induced regressions of a variety of murine tumours including the original Balb/c plasmacytoma X63-Ag8.653 growing in syngeneic mice. The tumour-inhibitory effect of the plasmacytoma cells transformed by IL-2 cDNA and designated as X63-m-IL-2 was due to their high constitutive production of IL-2. Here we show that admixture of syngeneic spleen cells to the X63-m-IL-2 transformants substantially (P < 0.025) increased the antitumour efficacy of the transformants. Balb/c spleen cells co-cultivated with X63-m-IL-2 cells in vitro yielded predominantly Thy 1.2+, CD3+, LFA-1+ lymphocytes, cytolytic for the X63-Ag8.653 plasmacytoma as well as for other murine tumours, including the X63-m-IL-2 target cells.

Therapeutic efficacy of repeated cycles of local IL-2 injections in mice carrying slowly growing tumour grafts.

Local IL-2 administration prior to transplantation of murine sarcoma virus (MSV Harvey)-induced tumour MSVT2 provided a model of slowly growing tumours suitable for long-term investigation of the therapeutic efficacy of repeated IL-2 injection cycles. Challenge of mice with the dose of sarcoma cells, which was lethal for 20/20 untreated control recipients, revealed that 8/20 IL-2-pretreated mice were protected by the local IL-2 treatment and survival indefinitely. Nine out of twenty IL-2-pretreated mice died during the same time period as the control mice, i.e., during 36 days, and 3/20 IL-2 pretreated mice were tumour-negative until day 60, when incipient tumours arose. The three late tumours were used as a model to investigate the therapeutic efficacy of the new cycles of repeated local IL-2 administration. It was found that no resistance to IL-2 immunotherapy was induced by pretreatment of the late tumours and that the tumours were repeatedly susceptible to local IL-2 treatment. Spleen cells of the tumour-bearing mice, which were not cytotoxic for MSVT2 tumour cells in vitro, could be made cytotoxic by addition of exogenous IL-2.

Activation and phenotyping of LAK generated by exposure to product of cells transformed by interleukin 2 cDNA.

It has been previously found that local administration of X63-m-IL-2 cells transformed by interleukin 2 (IL-2) cDNA and constitutively producing large quantities of IL-2 mediated regressions of murine plasmacytomas and 3-methyl-cholanthrene-induced sarcomas transplanted in syngeneic mice. Here we show that killer cells generated by cultivation of spleen cells in supernatants from X63-m-IL-2 cultures (LAK) or by co-cultivation of murine splenocytes with X63-m-IL-2 cells were cytolytic for natural killer (NK)-sensitive as well as NK-resistant target cells, including the IL-2-producing X63-m-IL-2 cells. Spleen cells cultured in X63-m-IL-2 supernatants or co-cultivated with X63-m-IL-2 cells yielded predominantly Thy 1.2+, CD3+, LFA-1+ lymphocytes. The in vitro results suggest that the LAK cells generated due to the IL-2 production by genetically engineered cells probably help to terminate the treatment by killing the IL-2 producers.

Use of IL-2 gene transfer in local immunotherapy of cancer.

Insertion of functional interleukin-2 (IL-2) gene into a plasmacytoma cell line X63-Ag8.653 substantially reduced tumorigenicity of the resulting cloned cells, designated as X63-m-IL-2. Peritumoral administration of the X63-m-IL-2 cells, producing constitutively large quantities of IL-2, resulted in regressions of established X63-Ag8.653 plasmacytomas growing in the peritoneal cavity of syngeneic mice. In vitro activation of BALB/c spleen cells by co-culture with X63-m-IL-2 cells or their supernatants gave rise to cytotoxic lymphocytes with lymphokine-activated killer (LAK) activity against syngeneic X63-Ag8.653 plasmacytoma and other tumor targets. In contrast, peritumoral administration of X63-Ag8.653 cells carrying an inserted interleukin-4 (IL-4) gene (designated X63-m-IL-4 cells) and producing constitutively large quantities of IL-4 did not result in a therapeutic effect. Moreover, the admixture of the X63-m-IL-4 and X63-m-IL-2 cells substantially diminished the X63-m-IL-2 cell-mediated therapeutic effect. Similarly, IL-4-containing supernatants generated from X63-m-IL-4 cell cultures substantially diminished LAK activation by X63-m-IL-2 cell produced supernatants.

Hemoblastoses in mice contaminated with low activities of 239Pu.

Female ICR mice were injected intravenously with low activities of 239Pu (3.0 kBq, 6.0 kBq, 12.3 kBq/kg). In these mice with high spontaneous incidence of hemoblastoses the occurrence of myeloid leukemia, lymphocytic leukemia, lymphosarcoma, reticulum-cell sarcomas and osteosarcoma was studied. Hemoblastoses, on the whole, remained in their numbers radiation-independent, nevertheless, the distribution into specific types changed, with moderate prevalence of myeloid and lymphocytic leukemia and lymphosarcoma. After plutonium injection the mean survival time of mice bearing myeloid and lymphocytic neoplasias was significantly shorter than the survival of mice that died of retothelosarcoma and from other causes. These contamination-dependent differences could not be observed in matched controls. As expected, 239Pu activities used in this experiment induced osteosarcomas. Whereas in leukemogenesis alpha-radiation appeared as a factor promoting and modifying the leukemogenic process, in osteosarcoma the alpha-particles acted rather as an initiator, the effect of which was dependent on the dose to the endosteal progenitor cells.

Local administration of cells containing an inserted IL-2 gene and producing IL-2 inhibits growth of human tumours in nu/nu mice.

We have prepared a retroviral expression construct, pPS-IL-2, in which human IL-2 cDNA has been inserted into the polylinker region, and have used the retroviral vector to introduce the functional IL-2 gene into a fibroblast cell line, RAT-1. Peritumoral administration of IL-2-producing RAT-1 cells into congenitally athymic (nu/nu) mice carrying subcutaneous transplants of human carcinoma cells inhibited the growth of the human tumour xenografts.

Effect of plutonium-239 on the mitotic activity of mouse bone marrow cells.

Mitotic index of the bone marrow cells was studied in femoral bone marrow of mice given 313 kBq 239Pu kg-1. The attention was turned to the femoral midshaft and the mitosis concentration, intensified by Colcemid stathmokinetic effect, was evaluated in a sampling field from endosteal surface to the central venous canal, throughout 68 weeks. It has been found that the plutonium effect in the sampling band is rather uniform except the points in subendosteal zone early after plutonium injection, where the mitotic index was reduced in such a way that the mitotic gradient, observed in controls, was affected. The mitotic activity in femoral diaphysis of plutonium injected mice was mobilized approximately till the 30th week of contamination. Later it deteriorated progressively. The results are discussed and should not be regarded as representative for the entire bone marrow hemopoiesis.

Long-term effects of low-level 239Pu contamination on murine bone-marrow stem cells and their progeny.

The effects of long-term internal contamination with 13.3 kBq kg-1 239Pu injected intravenously were studied in 10-week-old ICR (SPF) female mice. Radiosensitivity of spleen colony-forming units (CFU-S) and 125IUdR incorporating into proliferating cells of vertebral bone marrow and spleens were determined in plutonium-treated and control animals one year after nuclide injection. The CFU-S in 239Pu-treated mice were more sensitive to X-rays (D0 = 0.52 +/- 0.01 Gy) than in controls (D0 = 0.84 +/- 0.02 Gy). 125IUdR incorporation into bone marrow and spleen cells was reduced after plutonium contamination. At one year following plutonium injection, the occurrence of chromosome aberrations was evaluated in metaphase figures of femoral bone marrow cells. The frequency of aberrations increased early after plutonium treatment, at later intervals it tended to decrease but not below the control level. While the relative numbers of vertebral marrow CFU-S decreased significantly, but only to 86 per cent of normal, cellularity of vertebral bone marrow, peripheral blood counts and survival of 239Pu-treated mice did not differ from the control data.

Self-renewal capacity of murine hemopoietic stem cells under internal contamination with 239Pu and 241Am.

The self-renewal capacity of murine pluripotent hemopoietic stem cells (CFU-S) of vertebral bone marrow was studied under conditions of short-term and long-term internal contamination with 239Pu or 241Am in female mice. Measurement of the CFU-S self-renewal capacity was carried out using double transplantation assay. To evaluate the production of differentiated progeny of stem cells average erythroblast numbers/visible spleen colony and 59Fe-uptake/colony were computed. The marrow cellularity/vertebra and the number of CFU-S/vertebra were decreased and affected more by 239Pu than by 241Am. The production of erythroblasts per a single CFU-S and the 59Fe-uptake/colony were reduced, similarly the numbers of secondary spleen colonies and of secondary CFU-S in primary colonies. The above changes resulting from impaired functions of surviving CFU-S were more serious with 241Am than with 239Pu. The biological effects of plutonium and americium appeared independent of the phase of contamination.

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.

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.