[Application of gene therapy to antitumoral vaccination].
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Biomedical subjects
H Haddada
Publications and source records attributed to H Haddada.
Complete recovery of mice from a pre-established tumor by direct intratumoral delivery of an adenovirus vector harboring the murine IL-2 gene.
Direct introduction of exogenous genes into pre-existent tumors could provide an effective therapeutic approach for treatment of localized tumors. In this report we show that direct intratumoral delivery in animals of a replication-deficient adenovirus vector harboring the murine interleukin (IL)-2 gene (AD-mIL2) causes complete disappearance of P815 murine mastocytoma tumors in up to 75% of cases. Histological analysis of treated tumors revealed the presence of several zones of necrosis and the infiltration of macrophages and T cells. Moreover, the successfully treated animals develop a long lasting state of immunity during which further challenges with the tumor cells are rejected. To our knowledge this is the first successful in vivo treatment of an established tumor using adenoviral gene therapy methods.
Adenoviruses, Human↗
Efficient adenovirus-mediated gene transfer into human blood monocyte-derived macrophages.
The efficiency of gene transfer into human blood monocyte-derived macrophages has been evaluated using a replication-defective adenovirus vector harboring a lac Z gene of E. coli as a reporter gene. Whereas, no beta-galactosidase activity was found in freshly infected purified monocytes, 40% to 80% of infected macrophages which derived from these monocytes showed a beta-galactosidase activity, 2 to 4 days after infection and lasted for at least 3 weeks. Moreover, beta-galactosidase activity was found in infected monocyte/macrophages 7 days after their injection into a human tumor preestablished in nude mice. These data indicate that it is possible to transfer and stably express a gene of potential therapeutical function into human monocyte-derived macrophages using an adenovirus vector.
Adenoviridae↗
Adenoviral interleukin-2 gene transfer into P815 tumor cells abrogates tumorigenicity and induces antitumoral immunity in mice.
The murine mastocytoma cell line P815 was used as a model to evaluate the effect on its tumorigenic capacity following interleukin-2 (IL-2) gene transfer into the tumor cells using a replication-defective adenovirus vector. The data show that P815 cells infected in vitro with this recombinant adenovirus secreted significant amounts of functional IL-2 as tested on CTL-L2 cells. Furthermore, when injected into syngeneic DBA/2 mice, the tumorigenic phenotype is lost in up to 80% of the animals. The rejection of the infected cells was host dependent, because co-injection at the same site or concomitant injection at the opposite side of the animal with a tumorigenic dose of noninfected P815 cells did not lead to tumor development in 50-70% of the mice. Moreover, protected animals developed a long-lasting state of immunization against the P815 tumor cells and their splenocytes were able to transfer the immunity to syngeneic naive recipients.
Adenoviridae↗
TGF-alpha production correlates with tumorigenicity in clones of the SW613-S human colon carcinoma cell line.
The c-myc gene is amplified and the c-Ki-ras gene is mutated in the SW613-S human colon carcinoma cell line. Two cell types with different levels of c-myc amplification are present in the SW613-S cell population and representative cell clones can be isolated. The clones with a high level of amplification and expression of the c-myc gene are tumorigenic in nude mice whereas those with a low level are not. The tumorigenic clones secrete transforming growth factor alpha (TGF-alpha) in the culture medium whereas the non-tumorigenic clones do not produce any detectable amount. Accordingly the level of TGF-alpha mRNA is higher and the transcription rate of the gene is increased in the tumorigenic clones. The acquisition of the tumorigenic phenotype by cells of non-tumorigenic clones, following introduction of c-myc gene copies by transfection, is accompanied by an increase in the steady-state level of TGF-alpha mRNA. These findings suggest a role for an elevated level of TGF-alpha production in the tumorigenic phenotype of SW613-S cells. The possibility that this role is indirect is discussed.
Animals↗
Induction of simian virus 40 transplantation immunity in hamsters by gel-purified SV40 large T antigen.
Simian virus 40 (SV40) large T antigen and p53 cellular protein were isolated from an SV40-transformed hamster cell line by immunoprecipitation with anti-T sera and purified by sodium dodecyl sulfate-gel electrophoresis. These two protein were tested in hamsters for the presence of SV40 transplantation rejection antigenic sites by in vivo transplantation rejection assay. The large T antigen immunized the hamsters against a challenge of SV40 tumor cells and the protected animals generated cytotoxic spleen cells. Hamsters immunized with the p53 cellular protein were not protected against SV40-induced tumor but there was some delay in the appearance of tumor.
Animals↗
Viral gene inhibition of class I major histocompatibility antigen expression: not a general mechanism governing the tumorigenicity of adenovirus type 2-, adenovirus type 12-, and simian virus 40-transformed Syrian hamster cells.
The association between the level of class I major histocompatibility (MHC) antigen expression and the tumorigenic phenotype was determined for cells from a series of 15 lines of adenovirus type 2 (Ad2)-, Ad12-, and simian virus 40 (SV40)-transformed hamster cells and 16 lines of cells established from hamster tumors induced by SV40 mutants. These cells range from nontumorigenic to highly tumorigenic in both syngeneic and allogeneic adult hamsters. The Ad2-transformed cells--cells that were nontumorigenic in syngeneic adult hamsters--expressed either high levels or low levels of class I MHC antigens. The SV40-transformed cells--cells transformed in vitro that produced tumors with equal efficiency in both syngeneic and allogeneic adult hamsters--or cells derived from SV40-induced tumors expressed very high levels of class I MHC antigens. The Ad12-transformed cells uniformly expressed low levels of class I MHC antigens; these cells produced tumors 200- to 1,000-fold less efficiently in allogeneic adult hamsters than in syngeneic adult hamsters and produced tumors with about the same efficiency in immunoimmature newborns and immunocompetent syngeneic adult hamsters. We conclude that the expression of either high levels or low levels of class I MHC antigens is, at most, a minor factor in the differences observed among these adenovirus- and SV40-transformed cells in their tumor-inducing capacity in naive, immunocompetent hamsters.
Adenoviridae↗
Mitogenic and antimitogenic transforming growth factors secreted by adenovirus 2- and simian virus 40-transformed hamster cells: possible roles in promoting tumorigenesis.
Adenovirus 2 (Ad2)- and simian virus 40 (SV40)-transformed hamster embryo cells differ markedly in a number of phenotypic properties including their potential for inducing tumors in hamsters. Both Ad2- and SV40-transformed cells are immortalized and readily induce tumors in immunoincompetent newborn syngeneic hamsters, but only SV40-transformed cells are highly oncogenic in both adult syngeneic and allogeneic immunocompetent hamsters. The reasons for the difference in the oncogenic potential of the Ad2- and SV40-transformed phenotypes remain elusive. However, recent studies with transforming growth factors (TGFs) indicate that these factors play an important role in determining many phenotypic characteristics of transformed cells. To determine whether TGFs secreted by Ad2- and SV40-transformed hamster embryo cells differ, we have examined the ability of media conditioned by these two transformed cell phenotypes to modulate thymidine uptake in quiescent, untransformed cells. We found that both transformed phenotypes secrete very similar TGF alpha-like mitogenic factors which inhibit binding of 125I-labeled epidermal growth factor to its receptor. Our results also show that SV40-transformed cells, but not Ad2-transformed cells, secrete a powerful mitogenic inhibitor (MI). The MI secreted by SV40-transformed cells is inhibitory for several transformed and untransformed cell types and exerts a cytostatic, not cytolytic, action on untransformed primary hamster embryo cells. MI elutes from size exclusion high-performance liquid chromatography columns with a molecular weight of 24,000. Although MI has about the same molecular weight as TGF beta, it differs from TGF beta in two important respects: it is heat labile and it has a different target specificity for antimitogenic activity. The MI secreted by SV40-transformed cells also inhibits thymidine uptake by concanavalin A-stimulated spleen lymphocytes. This finding suggests that MI might contribute to the extreme oncogenicity of SV40-transformed cells by inhibiting mobilization of immune effector cells at the site of tumor cell proliferation.
Adenoviridae↗
Tumorigenicity of hamster and mouse cells transformed by adenovirus types 2 and 5 is not influenced by the level of class I major histocompatibility antigens expressed on the cells.
Inbred hamster and mouse cells transformed by the nononcogenic adenovirus (Ad) serotypes, Ad2 and Ad5, are nontumorigenic in syngeneic adult animals, while cells from these species transformed by the highly oncogenic Ad12 are tumorigenic in such rodents. By immunoprecipitation and flow cytometry, cells from four of six Ad2- and Ad5-transformed hamster and mouse lines expressed high levels of cell-surface class I major histocompatibility complex (MHC) antigens, while cells from two of these six lines expressed low levels of cell-surface class I MHC antigens. The levels of class I MHC proteins expressed by cells from these latter two lines were comparable to the levels of cell-surface class I MHC proteins expressed by cells from Ad12-transformed hamster and mouse lines. Moreover, an Ad2-transformed line that had become highly oncogenic after in vivo adaptation showed the same high level of MHC expression as the nononcogenic parent. The amounts of class I mRNA, analyzed by RNA blotting, were, in general, consistent with the levels of class I antigens expressed on the surfaces of these cells. These results indicate that there is no correlation between the tumorigenicity in immunocompetent syngeneic adult rodents of Ad2- and Ad5-transformed hamster and mouse cells and the level of class I MHC antigens expressed on the surfaces of these cells. Thus, the expression of different levels of class I MHC proteins does not seem to explain the differences in the oncogenicity between nononcogenic and highly oncogenic human Ad serotypes.
Adenoviruses, Human↗
Spleen cell populations in normal and tumor-bearing hamsters.
Spleen cell subpopulations from normal and tumor-bearing hamsters (TBH) were quantified using Petri dishes coated with specific antibodies, and by flow cytometric immunofluorescence analysis. The relative numbers of T cells (Thy + cells) decreased, by both methods, as a function of tumor growth, while the number of B cells (bearing surface Ig) increased. Cells without T or B markers (null cells) were more numerous in the spleen of TBH and a large number of them expressed the receptor for the Fc fragment of IgG. Splenic cells were also sorted according to their light-scattering properties, and electron microscopic analysis was performed on the sorted fractions. It showed the presence of secreting plasmocytes and activated macrophages in the spleen of TBH.
Animals↗
In vivo studies of spleen lymphoid cells implicated in antitumor immunity in hamsters.
Hamsters were immunized with repeated and progressive doses of strongly antigenic SV40-transformed cells (ZDCl25) and these animals rejected a tumor graft. According to the in vivo Winn test, spleen cells from such immunized animals could be used to transfer the antitumor immunity and protect naive recipients. To characterize the immune effector cells, different techniques of fractionation were used. In all cases the best protection was obtained by whole cell populations, even after reconstitution. The "T-depleted' fraction was more active than the T cell fraction. As shown by the treatment with specific antisera, part of the active population bore surface IgG. The protective activity could be the result of cooperation between different cell populations, one of which, unknown at present, could be the promotor of the transferred immunity and of the tumor neutralization effect.
Animals↗
Immunologic and enzymatic studies of two breeding lines of NZB mice that differ in chromosome breakage.
Two sublines of NZB/BI mice were developed by selective matings according to chromosome breakage frequencies. These sublines--HB, a line with high chromosome breakages, and LB, a line with low or normal breakage rates--were studied in regard to the age of the animals as it related to two different aspects. The first aspect was immunologic: A decreased response to T-cell mitogens was found in old NZB mice, but this response was more pronounced in HB mice. The response to the B-cell mitogen (lipopolysaccharide) was increased in both sublines as compared to that in BALB/c mice. The percentages of IgG-positive and theta-positive spleen cells were evaluated in both sublines: Some increase in IgG-positive cells was observed in the spleens of 2- to 8-month-old NZB mice and a slight decrease was seen after age 8 months. The percentage of theta-positive cells diminished according to the age of the mice, and the decrease occurred earlier in HB than in LB mice. The second aspect studied was enzymatic and concerned the levels of DNA alpha- and beta-polymerases and terminal DNA nucleotidyltransferase in the thymuses and spleens of these animals. The major finding noted was an augmentation of 100-200% in the terminal DNA nucleotidyltransferase levels in HB thymuses by comparison with LB thymuses. The levels of both polymerases were increased in spleen cells of HB mice as compared to those of LB mice.
Age Factors↗
Isolation of two immunoglobulin G subclasses, IgG2 and IgG1, from hamster serum using protein A-sepharose.
The isolation of hamster immunoglobulin classes and subclasses by affinity chromatography on protein A and selective elution was studied using 0.1 M phosphate buffer, pH 8. The IgG fraction was completely absorbed, while IgM did not bind. Sequential application of buffers of decreasing pH allowed the elution of pure IgG2 (eluted at pH 6) and IgG1 (eluted at pH 5). Both subclasses were fully recovered. IgG2 could be subfractionated into 2 peaks eluted respectively at pH 6.5 and 6. Immunodiffusion of the whole IgG2 fraction against anti-hamster immunoglobulin serum gave 2 precipitation lines. One of these lines was missing in the pH 6.5 fraction. Until now only 2 IgG subclasses have been described and these results suggest heterogeneity of hamster IgG2.
Animals↗
Nude Syrian hamsters: some immunological and histological characteristics.
Nude mutants appeared in our colony of Syrian hamsters. They were hairless and just had rudiments of thymus. Only 3.5% of splenic cells were killed by rabbit anti-hamster thymocytes serum + C' whereas 55.5% of these cells were lyzed by an anti-hamster IgG + C' and 60-70% fixed the fluorescent protein A, but could not respond either to B-cell mitogens or to rat cell mitogens. The natural cytotoxic activity of the spleen cells from nude hamsters was evaluated in comparison with the same activity expressed by spleen cells of golden Syrian hamsters.
Animals↗
Evolution of DNA polymerase alpha, beta and terminal deoxynucleotidyl transferase in hamster lymphoid populations during the development of different types of tumors.
We followed the evolution of DNA polymerase alpha, beta and terminal deoxynucleotidyl transferase (TdT) in the thymus, spleen and bone marrow of tumor bearing hamsters. Tumors were induced by spontaneously transformed fibroblasts (EHB), by SV40 (ZD) or by 20-methylcholanthrene (MCH2) transformed fibroblasts. We have shown that, in the thymus, the TdT activity of the various tumor bearing hamsters is generally inferior to the TdT activity of the control. In the spleen of animals bearing viral or spontaneously induced (ZD or EHB) tumors, the TdT activities were higher than in the controls. Moreover, DNA polymerase alpha and DNA polymerase beta activities in the spleen of the EHB tumor bearing hamsters were higher than in the controls. This fact, however, was not observed in the two other kinds of tumor, possibly because EHB tumors were growing much faster and led to earlier changes in DNA polymerases activities, as well as in spleen size and cellular populations. Finally, in the bone marrow, TdT, polymerase alpha and beta of ZD and EHB tumor bearers reached much higher activities than in the controls; for the MCH2 tumor bearing hamsters, no difference with the controls could be observed.
Animals↗
Modifications of the lymphoid B and T cell populations in spleen and thymus of tumor-bearing hamsters.
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Suppressive and cytostatic activities in the spleen of tumor-bearing hamsters.
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Animals↗
Protein A : a B cell mitogen for hamster splenic lymphoid cells.
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