In vitro and in vivo effects of reovirus on HPV16-transformed mice cells.

UNLABELLED: Oncolytic viruses are examined to serve as anticancer therapeutics. It is expected that in addition to direct oncolytic effect their action will also help eliciting a solid antitumor immunity. In presented series of experiments we have employed two HPV16-transformed mouse (strain C57/B6) cell lines, TC-1 and MK16/III/ABC (MK16), and reovirus type 3, strain Dearing (RV). Both cell lines are highly susceptible to RV and produce large amounts of infectious virus in vitro while normal human are not susceptible to RV. Still, some differences were encountered. TC-1 cells produced moderately lesser amounts of infectious virus, but, paradoxically, were more efficient producers of delta1 antigen of RV and as a consequence of virus infection died more rapidly than simultaneously infected MK16 cells. Minor differences between the cell lines were observed in the percentage of cells arrested in theG2/M phase of the cell cycle and in some markers of apoptosis. When inoculating high doses (5x106) of infected cells (MOI 10 PFU/cell) into syngeneic animals their oncogenic activity was strongly suppressed, nearly completely in the case of MK16 cells and somewhat less efficiently in the case of more oncogenic TC-1 cells. Immunizing experiments in which non-oncogenic doses (106) of RV infected TC-1 cells were tested in parallel with the same doses of irradiated cells brought surprising results. When immunized animals were challenged with TC-1 cells, the irradiated cells proved to be a much better immunogen that the infected cells. However, when challenged with MK16 cells the opposite was true. It is believed that this difference was associated with the different biological properties of the cell lines tested. KEYWORDS: reovirus type 3, HPV16-transformed mouse cell lines, apoptosis, cell cycle, immunization/challenge experiments.

Isolation and properties of gene-modified mouse bcr-abl-transformed cells expressing various immunostimulatory factors.

UNLABELLED: B210 cells are murine (BALB/c) cells transformed by bcr-abl fusion gene. After intravenous administration they are capable of inducing leukaemia-like disease in syngeneic mice. From these cells a thymidine-kinase less subline was derived. It was significantly less pathogenic than the parental cells. However, a highly pathogenic clone denoted B210cTK-/cl-2 was isolated from its population. As determined by Western blotting, these cells produced more p210 protein than the parental B210 cells. To successfully transfect these cells a modified electroporation method was introduced. Bicistronic plasmids carrying gene for herpes simplex thymidine kinase (HSV TK) and the gene for either granulocyte-monocyte colony stimulation factor (GM-CSF), interleukin-2 (IL-2) or interleukin 12 (IL-12) were used for the transfection experiments. Gradually, cell lines producing these cytokines were isolated in media supplemented with hypoxantin, aminopterin and thymidine (HAT). All of them were highly sensitive to ganciclovir in vitro confirming that the cells produced HSV TK. The genetic modification of B210cTK-/cl-2 was associated neither with the alteration of p210 bcr-abl production nor with any changes in expression of MHC class I molecules. From populations of each of the three lines several cell clones were isolated and tested for the production of the respective cytokines. The original uncloned population and several clones differing in the cytokine production were administered intravenously into mice. All animals survived without symptoms of the disease suggesting that the gene-modification was associated with the loss of pathogenicity. KEYWORDS: CML, Bcr-Abl, HSV TK, cytokines, gene-modified tumour cells, pathogenicity.

Immunological profiles of patients with chronic myeloid leukaemia. I. State before the start of treatment.

In view of the increasing interest in the immunotherapy of CML it seems highly desirable to broaden the present knowledge on the immune reactivity of CML patients. A group of 24 patients and 24 healthy controls were studied for the total of 15 immunological parameters, including the prevalence of antibodies against human herpesviruses and papillomaviruses. To clearly discriminate between changes associated with the disease and those induced by the therapy, all patients were enrolled prior to the start of any anti-leukaemic therapy. Statistically significant differences between patients and controls were found in the levels of IgA, C4 component of complement, CRP and IL-6, the production of Th1 cytokines in stimulated CD3 cells and the E. coli stimulatory index. The analysis of the interrelationship between the results obtained in the individual patients presented some unexpected findings, such as the lack of correlation between the CRP and IL-6 levels. It will be the purpose of a follow-up to determine whether and how the immune status of the patients prior to the treatment correlates with their response to therapy and how the individual immunological profiles change in the course of the disease. These observations will be utilized in the future immunotherapeutic studies to constitute the vaccine- and placebo-treated groups.

Use of polyclonal rabbit antibodies for detection of the bcr-abl fusion zone in cells transfected with experimental bcr-abl DNA vaccines.

Rabbits were immunized with peptides covering the fusion zone of the chimeric bcr-abl protein in order to prepare antibodies capable of detecting the expression of a selected portion of this fusion zone, by a variety of experimental genetic vaccines. Three peptides of different size covering the b3a2 fusion zone, either unmodified or modified by the omission of alanine at the N-terminal of the a2 section of the fusion zone, and one peptide covering the unmodified b2a2 fusion zone were used. All were capable of eliciting antibodies reactive with the respective immunizing peptides. Their cross-reactivities, especially the results of cross-absorption experiments, strongly suggested that the serum of the rabbit immunized with an octadekapeptide mimicking the b3a2 fusion zone contained antibodies against a novel antigenic determinant created by the chimeric protein, and also against an epitope present in the adjacent a2 section but no antibody reactive with the adjacent b3 region. In Western blotting, these antibodies were capable of detecting the p210bcr-abl or a portion of it (a 25 amino acid-long sequence covering the b3a2 fusion zone) in lysates of 293T cells transfected with plasmids that carried either the full cDNA of the bcr-abl gene or a fragment thereof fused with either the HSP70 gene or certain other genes.

Characteristics of two mouse bcr-abl-transformed cell lines. II. Pathological lesions induced in mice.

Groups of six BALB/c mice each were intravenously inoculated with lethal doses of Ba-P210 (B210) or 12B1 cells and examined by autopsy, histology, special staining methods, enzyme histochemistry and immunohistochemistry. Clinical symptoms related to neoplasia consisted of a poor nutritional state, anaemia, mild to moderate dehydration and apathy. Paresis was apparent in three mice inoculated with 12B1 cells. Necropsy revealed splenomegaly in all animals. Sporadic haemorrhages in the lungs and enlargement of some lymph nodes were seen in some of the animals. Histological examination showed neoplastic cells in the spleen, in the bone marrow of the sternum, in the lung interstitium and in sinusoids of the liver in all mice. In six of nine brains examined, mild to moderate infiltration by neoplastic cells was observed. In all but two mice mild infiltration of the kidneys was found. The enlargement of lymph nodes was caused by an accumulation of neoplastic cells. The paresis was due to neoplastic infiltration of the vertebra, epidural space and spinal roots. Staining with Sudan black revealed cytoplasmic granules in neoplastic cells; however, the peroxidase reaction was negative. Numerous neoplastic cells disseminated in the red pulp of the spleen were reactive with CD3, CD79beta, CD11b and with neutrophil antibodies. We classified the disease induced by both of the cell lines as acute myeloid undifferentiated leukaemia (AML MO).

Characteristic of two mouse bcr-abl-transformed cell lines: I. General properties of the cells.

In an effort to develop an experimental system suitable for immunological studies in which Bcr-Abl-positive cells are to be used as antigens, we examined the properties of two mouse (Balb/c) established cell lines that express the Bcr-Abl protein and are oncogenic for syngeneic animals. Under standard conditions the two cell lines, viz. Ba-p210 (B210) and 12B1, expressed comparable amounts of the Bcr-Abl protein. However, they differed in a number of characteristics. From the morphological point of view, B210 cells were the more homogeneous, being mainly represented by leukaemic blastic cells with a large number of AgNORs as markers indicating a high proliferative activity. 12B1 cells were more polymorphic and giant cells were detected within their populations. Many 12B1 cells exhibited nuclear segmentation and "band-like" structures. Markers of proliferation were less frequent in 12B1 and the tendency for aging was more pronounced in these cells. The 12B1 cells were slightly more sensitive to imatinib mesylate than B210 cells. In B210 cells, the expression of MHC class I was downregulated, which was not the case with 12B1 cells. Both cell lines induced leukaemia-like disease in mice after intravenous application but, as compared with B210, 12B1 cells were about 100 times more oncogenic and the disease they induced was more aggressive. Moreover, 12B1, but not B210, induced tumours after subcutaneous or intraperitoneal inoculation.

Immunomodulatory properties of monoclonal anti-Thy-1.2 antibody in vivo.

Monoclonal mouse anti-Thy-1.2 antibody of IgG3 isotype (MTA) inhibits in a significant and long-term manner the regional graft-versus-host reaction (GVHR) when administered to donors as well as to recipients. The process of sensitization in the reaction of delayed type hypersensitivity to SRBC (DTH) is, on the other hand, suppressed only if MTA is administered immediately prior to sensitization. If the time interval between the injection of MTA and of SRBC is extended, the resulting DTH is stimulated. Similarly, the titers of total Ig against SRBC are decreased only if MTA is administered shortly before immunization. Contrasting effects of MTA on the production of IgM and IgG were observed. While the production of IgG was inhibited even after the time interval between the administration of MTA and immunization had been increased, the production of IgM antibodies was stimulated. In recipients of allogeneic Sarcoma I treated with MTA, the primary growth of the tumor is enhanced and the percentage of permanent progressors is increased. Mechanisms of MTA activity are discussed.

Interleukin 2 gene therapy of residual disease in mice carrying tumours induced by HPV 16.

Experiments were designed to examine the efficacy of IL-2 gene therapy in a surgical minimal residual tumour disease, using moderately immunogenic MK16/1/IIIABC murine cells transformed by activated ras and HPV 16 E6/E7 oncogenes (MK16 cells). Previously we demonstrated that surgical minimal residual tumour disease (SMRTD) could be effectively cured when murine Mc12 sarcoma had been resected and the operated mice were treated with irradiated Mc12 sarcoma cells engineered to secrete IL-2. In this study we performed IL-2 gene therapy of MK16 carcinoma with two types of irradiated MK16-unrelated tumour cell vaccines. One type of vaccine was derived from MHC class I-matched Mc12 sarcoma cells engineered to secrete IL-2 and the other from MHC class I-discordant IL-2 producing plasmacytoma X63-m-IL-2. The vaccines did not share any tumour rejection antigen with the MK16 cells and served exclusively as a local source of IL-2 production. Both vaccines were capable of inhibiting MK16 tumours when administered peritumorally up to 15 days after MK16 tumour challenge. The irradiated MHC class I-matched and IL-2-producing Mc12 sarcoma vaccine was then selected for therapy of MK16 SMRTD. Whereas the recurrence rate in the operated MK16 carcinoma bearers was 80%, so that only 20% of mice were cured by surgery, approximately 65% of the MK16 carcinoma bearers were permanently protected when the surgery was followed by local administration of the IL-2-producing Mc12 sarcoma vaccine.

DNA vaccine against oncogenic hamster cells transformed by HPV16 E6/E7 oncogenes and the activated ras oncogene.

The capability of DNA to elicit anti-tumour immunity was studied using human papillomavirus type 16 (HPV16)-transformed Syrian hamster cells denoted K3/II. These cells had been derived after cotransfection of primary kidney cell cultures with p16HHMo plasmid containing E6/E7 oncogenes of HPV16 and pEJ6.6 plasmid containing the activated human H-ras oncogene; they express both the HPV16 and activated H-ras genes. As a DNA vaccine, the p16HHMo plasmid was used. Three doses of the plasmid (either 100 microg or 10-15 microg per dose) were administered intramuscularly at 3-week intervals. The animals were challenged with four different doses (10(3)-10(6) per animal) of K3/II cells 10 days after the last plasmid injection. In one experiment the lower dose of plasmid DNA was also given in a mixture with the cationic lipid DOTAP. In another experiment, the pEJ6.6 plasmid (100 microg per dose) was used either alone or in combination with p16HHMo. In all experiments animals inoculated with the same doses of pBR322 plasmid served as controls. A moderate protective effect was observed in animals inoculated with the 100-microg doses of p16HHMo, but not in those inoculated with 10-15 microg of the same plasmid, whether given with or without DOTAP. A protective effect was also observed after administration of the pEJ6. 6 plasmid. At the time of challenge a portion of the p16HHMo-immunized, but not the pBR322-treated, animals possessed antibodies reactive in ELISA with peptides derived from the N-terminal portion of HPV16 E7 protein and with one peptide derived from E6 protein, while two other E6 peptides exhibited non-specific reactivity.

Regulatory role of T lymphocytes and NK cells in tumor allograft development.

In the present study, the respective roles of T cells and their subpopulations as well as of NK (natural killer) cells in antitumor immune responses were followed using the SaI (H-2a) allograft model. The development of this tumor in B10 (H-2b) mice was evaluated after pretreatment of the recipients with xenogeneic antithymocyte serum (ATS). Anti-Thy 1.2, anti-Lyt 2.2 and anti-L3T4 monoclonal antibodies were used in order to determine T lymphocyte phenotypes and to assess the frequency of TC/S and TH subpopulations at various periods of tumor development. Rabbit polyclonal anti-asialo GM1 antiserum was used for the identification of NK cells. In a previous work it was suggested that the first week following transplantation, the cells predominantly involved in the growth regulation of SaI belong to the TS subclass. Our results based on the use of anti-Lyt 2.2 monoclonal antibodies have further supported this finding. The application of anti-Thy 1.2 on the 3rd and 5th day has hampered a secondary tumor growth while anti-Lyt 2.2 was effective when given on day 5. The depletion of Lyt. 2.2+ cells on day 3 resulted in the inhibition of both primary and secondary tumor development. On the other hand, when anti-Thy 1.2 was applied on day 7 after transplantation, the primary and secondary tumor growth was strikingly enhanced. It appears that Thy 1.2+ lymphocytes display at this period effector functions and contribute, in conjunction with macrophages, to subsequent tumor regression. The depletion of L3T4 cells on days 3 and 5 after tumor inoculation has resulted in primary tumor growth enhancement. This suggests that cells of the L3T4+ phenotype display at this time helper functions contributing to CTL proliferation and maturation. A further indication, supporting the possible suppressor effect of L3T4+ cells, counts from the finding that anti-L3T4 treatment results in an inhibition of secondary tumor growth. The anti-asialo GM1 treatment has not enhanced, at least significantly, primary tumor development but has partially or totally inhibited the growth of secondary tumors. It appears that cells of the GM1+ (NK cells) phenotype do not participate in any substantial way in the early phases of SaI tumor development in ATS treated allogeneic recipients.

Comparison of the effects of anti-Thy-1.2 monoclonal antibody and xenogeneic antithymocyte serum on the development of SaI tumor allograft in mice.

Using the model of Sarcoma I (SaI) (H-2a) tumor allograft transplanted to normal B10 (H-2b) mice and to B10 mice pretreated with ATS, we studied the effects of anti-Thy-1,2 monoclonal antibody (MTA) and compared them with those of rabbit polyvalent antiserum against mouse thymocytes (ATS). When administered shortly before tumor transplantation, MTA showed immunosuppressive effects, at one dose identical to those of ATS, at two doses exceeding ATS effects. In the period following the transplantation of SaI to normal mice, single doses of MTA and ATS showed weak antitumor effects. In mice immunosuppressed with ATS before the tumor transplantation the effects of each agent differed: MTA showed opposite immunomodulatory effects depending on the period of its administration, while ATS supported the primary growth of tumors independent of the time of administration. The results indicate that the immunomodulatory influence of MTA on the development of the tumor allograft is more selective than that of ATS.

Functional heterogeneity of Sarcoma I cells in transplantation experiments.

Cell suspensions prepared from Sarcoma I (SaI) allografts at various stages of development in C57BL10/ScSn (B10) mice immunosuppressed with xenogeneic antithymocyte serum (ATS) were adoptively transferred into secondary syngeneic (A/Ph) and allogeneic (B10) recipients. In the syngeneic recipients, a gradual decrease in the tumorigenic capacity of transferred suspensions was proved, while in the allogeneic recipients the tumorigenic activity was proved in early and late periods. The suspensions from the period of both permanently and temporarily regressing tumors showed a suppressed growth capacity in syngeneic and immunosuppressed allogeneic recipients. On the other hand, the suspensions from growing tumors produced in both types of secondary recipients a sharp and permanent growth. The data obtained suggest changes in functional properties of SaI cells during the course of their development in allogeneic recipients.

Immunomodulatory action of double-stranded RNA in unirradiated and irradiated mice.

Double-stranded RNA (ds RNA) stimulates the regional graft-versus-host reaction (GVHR) in mice irradiated with 3 Gy or 5 Gy (but not 7 Gy) of gamma-rays; in control animals GVHR is not influenced. Sensitization of animals to delayed-type hypersensitivity (DTH) reaction is suppressed by ds RNA in both irradiated and unirradiated mice. In the antibody formation to sheep and blood cells (SRBC) ds RNA given before immunization inhibits the switch of IgM to IgG. When injected simultaneously with or after immunization, ds RNA markedly stimulates antibody formation. The differences in the influence of ds RNA on individual immune reactions can be explained by its different action on the regulatory mechanisms.

D-Glucosamine as inhibitor of early processes of transformation in Bacillus subtilis 168 trp2.

Glucosamine added to a transformation medium (TM2) after a 30-min cultivation of cells exhibited the highest inhibitory effect on the transformation process in Bacillus subtilis 168 trp2. The recipient culture was least sensitive to glucosamine added after 50 min. Glucosamine had no inhibitory effect when added 10 min after the transformation DNA.

Properties of Syrian hamster cells transformed by human papillomavirus type 16.

Adult Syrian hamster kidney cells were transfected with a mixture of plasmids containing human papillomavirus type 16 (HPV16) E6/E7 open reading frames (ORFs), activated Ha-ras gene and neomycin resistance gene. From these cultures two lines were isolated which were oncogenic for newborn and 5-day-old but not for 3-week-old hamsters. Sublines oncogenic for 3-7-week-old hamsters were derived from tumours formed in animals inoculated within 5 days of birth. The cells contained HPV 16 DNA in an integrated form and HPV16 transcripts. The transcript patterns in low and high oncogenicity sublines were different. Very few tumour-bearing animals possessed antibodies reactive with E6- and E7-derived synthetic peptides. On the other hand a majority of these animals gave a positive reaction in the lymphoproliferation assay with either E6 and E7 peptides or extracts from the transformed cells.

[Comparison of the immunomodulating properties of the cyclosporin Sandimmune (Sandoz) with the newly developed Consupren (Galena)]. / Srovnání imunomodulacních vlastností cyklosporinu Sandimmune (Sandoz) s nove vyvinutým Consuprenem (Galena).

An experimental comparative investigation provided evidence of identical immunomodulating properties of the Swiss cyclosporin A of Sandoz Company (Sandimmune) and the Czechoslovak cyclosporin of Galena Company (Consupren). Both preparations administered by the oral route to mice in low doses stimulated the reaction of the late hypersensitivity and in higher doses they inhibited markedly this reaction. Immunostimulation was also observed when influencing the host's reaction against the graft by low doses of the substance. The systemic reaction of the graft against the host was markedly suppressed by repeated doses. The development of antibody-producing cells in the spleen was markedly inhibited by high as well as small doses of the preparations. Inhibition of the formation of total antibodies is caused by inhibition of IgG formation, while IgM formation is enhanced and persists longer. This phenomenon is due to a block of the shift from IgM to IgG formation. In the allogenic tumour model both preparations caused a dose-dependent temporary immunosuppression.

[Papillomaviruses and human tumors]. / Vztah papillomaviru k lidským nádorum.

The report summarizes the main results obtained in the course of our research project. The results of immunological and epidemiological studies provide further proofs that human papillomaviruses (HPV) are the etiological agents in cervical neoplasia. In addition, they raise hopes that immunological methods may be utilized in diagnostics of cervical cancer and for monitoring the clinical course of this disease in the near future. Since the etiological relationship between HPV and cervical carcinoma seems to be proven beyond reasonable doubt, the development of prophylactic and therapeutic vaccines has become the dominant of the contemporary HPV reseach. For studying immune reactions against HPV-induced tumours we developed a model of HPV16-transformed rodent cells.