PubMed Health⌕ Search

Biomedical subjects

B Gansbacher

Publications and source records attributed to B Gansbacher.

At least 37 records · Page 2Linked to original sources
PubMed1995

Imaging the expression of transfected genes in vivo.

Imaging the expression of successful gene transduction has been demonstrated in vivo for the first time by using an appropriate combination of "marker gene" and "marker substrate" in an experimental animal model. The herpes simplex virus 1 thymidine kinase (HSV1-tk) gene was selected as an example of a marker gene, and the recombinant STK retrovirus containing HSV1-tk was used to transduce RG2 glioma cells in vitro and in vivo. RG2TK+ cell lines expressing the HSV1-tk gene and three potential marker substrates for the HSV1-TK enzyme were evaluated. Radiolabeled 5-iodo-2'-fluoro-2'deoxy-1-beta-D-arabinofuranosyluracil (FIAU) was shown to be a substantially better marker substrate for the HSV1-TK enzyme than 5-iodo-2'-deoxyuridine or ganciclovir. The magnitude of FIAU accumulation in different RG2TK+ clones corresponded to their sensitivity to ganciclovir and to the level of HSV1-tk mRNA expression. Imaging the expression of HSV1-tk in transduced RG2 tumor cells was demonstrated in animals using quantitative autoradiography; 2-[14C]FIAU accumulation was shown to be high in RG2TK+ brain tumors growing in one hemisphere and very low in nontransduced RG2 tumors in the contralateral hemisphere. Transduction of RG2 tumor cells with the HSV-tk gene in vivo resulted in tumors which accumulated FIAU to high levels and produced clearly defined images. Given the level of FIAU accumulation in the transduced tumors, it is likely that a clinically applicable method for imaging HSV1-tk gene expression can be implemented using existing clinical imaging techniques.

Animals↗
PubMed1995

RG-2 glioma growth attenuation and severe brain edema caused by local production of interleukin-2 and interferon-gamma.

Two aspects of cytokine therapy of intracerebral tumors are considered in this study: modulation of tumor growth in vivo and central nervous system toxicity. Coimplantation of RG-2 glioma cells and retroviral vector producer cell lines was performed to provide a local source of interleukin-2 (IL-2) or IFN-gamma within the tumor and coinitiate an antitumor immune response. We demonstrated that local intratumoral production of IL-2 and IFN-gamma generates a cell-mediated antitumor response in vivo. This response was manifest as a diffuse infiltration of monocytes/macrophages, CD4+ and CD8+ T cells, and activation of microglial OX42+ cells in intracerebral RG2 tumors. The cell-mediated antitumor immune response resulted in the early suppression of intracranial and subcutaneous tumor growth, but the effect was not sustained and there were no tumor regressions. The absence of increased survival of animals with intracranial tumors is explained in part by the severe central nervous system toxicity caused by local production of IL-2 and IFN-gamma. Central nervous system toxicity induced blood-brain barrier disruption, vasogenic brain edema, and dislocation of the brain midline structures, as observed by dynamic magnetic resonance imaging and direct measurements of tissue water content. The clinical application of IL-2 and IFN-gamma gene transfer therapy for intracerebral tumors must consider the potential for severe vasogenic brain edema associated with intracerebral production of these cytokines.

3T3 Cells↗
PubMed1995

Rejection of mouse melanoma elicited by local secretion of interleukin-2: implicating macrophages without T cells or natural killer cells in tumor rejection.

Tumor cells transduced with cytokine genes provide a model to study host-effector mechanisms involved in tumor rejection. Local IL-2 production within a tumor site mimics a specific helper-T-cell response, bypassing an immunization phase. Growth of mouse B16F10 melanomas transduced with interleukin-2 (IL-2) in syngeneic hosts were significantly delayed. IL-2-producing B16F10 cells were super-transduced with interferon-gamma to up-regulate expression of major-histocompatibility-complex (MHC) antigens. Expression of class-I- or class-II-MHC molecules did not augment tumor rejection of IL-2-secreting tumor cells. Rejection of IL-2-transduced B16F10 cells in syngeneic mice was unaffected by depletion of CD8+ T-cell and NK1.1+ natural-killer (NK) cell populations. Tumor rejection occurred in SCID mice even after depletion of NK1.1+ cells, confirming that T cells and NK cells were not required for tumor rejection. Histologic examination of sites of tumor rejection showed inflammation, characterized by infiltrates of macrophages, occasional neutrophils, and areas of necrosis. When mice were treated systemically with macrophage-colony-stimulating factor to expand monocyte pools, tumor rejection was significantly augmented further. This study shows that in situ IL-2 production can result in tumor rejection mediated by inflammatory events, possibly involving macrophages, and mimicking a delayed-type hypersensitivity (DTH) response even in the absence of T cells and NK cells. Furthermore, tumor rejection can be enhanced by systemic administration of a cytokine to expand potential inflammatory cell populations.

Animals↗
PubMed1995

The impact of gene therapy on T cell function in cancer.

Studies of signal transduction by T cells are slowly identifying the intracellular messengers that must be generated for full T cell activation to take place. The recent, convincing identification of several tumor-associated antigens (TAA) has transformed our task into trying to define the mechanisms that underlie the failure of T cells to destroy antigenic tumor cells. Although there are a variety of hypotheses that explain why tumors grow progressively, even if they are antigenic, recent evidence suggests that T cells from tumor-bearing patients exhibit abnormalities in signal transduction that render them unable to respond to appropriate activation signals, even following proper stimulation. Gene therapy with interleukin-2 (IL-2)-secreting tumor cells in an animal model has been effective in preventing the onset of these signaling defects. Discovery of the molecular mechanisms by which such cytokine-secreting tumor cells induce immune responses and how they may best be applied clinically may provide clearer indications of the directions to pursue to alter the balance between the T cell and the tumor cell in the patient's favor.

Animals↗
PubMed1995

B7-1 amplifies the response to interleukin-2-secreting tumor vaccines in vivo, but fails to induce a response by naive cells in vitro.

Parental and interleukin-2 (IL-2)-secreting CMS5 tumor cells were transfected with the B7-1 costimulatory molecule to amplify anti-tumor responses. CMS5 cells transfected with B7-1 grew more slowly in vivo than did parental CMS5 cells. Moreover, tumor cells secreting levels of IL-2 too low to cause rejection alone were rejected following transfection with B7-1. To determine whether the expression of B7-1 enabled the tumor cells to activate T cells directly, their ability to stimulate in vitro functional responses by T cells was examined. We found that neither B7-1+ nor IL-2-secreting, B7-1+ CMS5 cells stimulated naive spleen cells to proliferate or to become cytotoxic. In contrast, restimulation of primed T cells by B7-1+ CMS5 cells resulted in stronger cytotoxicity responses than seen following restimulation by parental CMS5 cells. Lysis was even higher if the B7-1+ tumor cells also secreted IL-2. Our results suggest that the expression of costimulatory molecules can augment responses generated by vaccinating with IL-2-secreting tumor cells. Furthermore, they are consistent with the hypothesis that the initiation of an anti-tumor response by naive T cells may depend upon initial antigen presentation by another unidentified cell and that the major action of IL-2-secreting and/or B7-1+ tumor cell vaccines might be to potentiate the response of already primed cells.

Animals↗
PubMed1995

IL-2 gene-transduced human HLA-A2 melanoma cells can generate a specific antitumor cytotoxic T-lymphocyte response.

This study was designed to assess whether transfer of the interleukin-2 (IL-2) gene into human tumor cells could generate cytotoxic T lymphocytes (CTL) directed specifically against the autologous tumor. Two HLA class I+ melanoma cell lines, one (TOM) A2+ and the other (CLB-M) A2-, obtained from living patients were transduced with the IL-2 gene. The patients' peripheral blood lymphocytes (PBL) were incubated with irradiated IL-2 gene-transduced autologous tumor cells for up to four weeks. After seven days, PBL from both patients showed non-specific cytotoxic activity against the K562 cell line. When the co-culture incubation time was prolonged to 28 days, PBL from patient TOM (A2+) developed a lytic activity directed specifically against the autologous tumor cells. In contrast, after 28 days of incubation, PBL from CLB-M (A2-) displayed only non-specific cytotoxic activity. Inhibition experiments demonstrated that the specific lytic function observed with TOM cells transduced with the IL-2 gene could be reversed following incubation with monoclonal antibodies directed against HLA class I and CD8. The evidence that K562 cells were incapable of blocking the PBL killing capacity in a cold-target inhibition assay further confirmed that engineered TOM cells induced the generation of specific CTL. This study indicates that retroviral vector mediated transfer of the IL-2 gene into human melanoma cell lines can lead to the amplification of the autologous cytotoxic compartment and to the generation of specific antitumor CTL, and that the A2 allele may play an important role in the process of tumor recognition.

Animals↗
PubMed1994

Abnormal signal transduction by T cells of mice with parental tumors is not seen in mice bearing IL-2-secreting tumors.

There is considerable evidence to demonstrate that immune function is abnormal in tumor-bearing mice, perhaps accounting, at least in part, for progressive tumor growth. In an attempt to generate an antitumor response, we used retroviral vectors to express IL-2 cDNA in CMS5, a murine fibrosarcoma. Mice inoculated with unmodified tumor cells suffered progressive tumor growth, whereas tumors secreting IL-2 were rejected or grew slowly. Animals bearing unmodified but not IL-2-secreting tumors also were immunosuppressed. On the basis of these observations, we were interested in how IL-2 secretion by the tumor cells prevented the onset of hyporesponsiveness. To identify biochemical differences between T cells of mice with parental vs slowly growing IL-2-secreting tumors, we examined signal transduction after activation through the CD3/TCR complex. Protein tyrosine phosphorylation was altered and calcium flux was reduced in cells of mice with parental tumors compared with animals with slowly growing IL-2-secreting tumors. In addition, levels of protein for the tyrosine kinases p56lck and p59fyn, as well as the TCR-zeta-chain, were reduced. These differences in signal transduction were observed for T cells of mice with parental and IL-2-secreting tumors of the same size, demonstrating that differences in tumor size alone could not explain our findings. Thus, IL-2 secretion by tumors seems to be able to prevent immunosuppression by maintaining normal signal transduction in T cells, facilitating the generation of antitumor responses.

Animals↗
PubMed1994

Cytokine therapy with gene-transfected cells: single injection of irradiated granulocyte-macrophage colony-stimulating factor-transduced cells accelerates hematopoietic recovery after cytotoxic chemotherapy in mice.

Development of cell-based delivery systems that can release therapeutic levels of hematopoietic growth factors into the systemic circulation would facilitate treatment of patients requiring cytokine therapy. In this study, we have investigated the potential of granulocyte-macrophage colony-stimulating factor (GM-CSF)-secreting, irradiated syngeneic murine cells to accelerate hematopoietic recovery after cytotoxic chemotherapy. As a model, CMS-5 fibrosarcoma cells, were transduced with a retroviral vector containing the murine GM-CSF cDNA. Transduced cells secreted 38 ng GM-CSF/10(6) cells in 24 hours. After irradiation, in vitro GM-CSF production initially increased up to fivefold and was measurable for about 2 weeks. One and 2 days after injection of irradiated, GM-CSF-secreting CMS-5 cells (N2/CMVGM-CSF/CMS5 # 6 cells) into mice, GM-CSF serum levels of 405 +/- 58 pg/mL and 183 +/- 36 pg/mL were measured, respectively. Serum levels were comparable with levels detected 3 hours after injection of 100 ng recombinant murine GM-CSF (rmGM-CSF) subcutaneously (90 pg/mL). Injection of N2/CMVGM-CSF/CMS5 # 6 cells in cyclophosphamide-treated mice was as effective in accelerating neutrophil recovery as twice daily subcutaneous injections of rmGM-CSF. These data suggest that irradiated hematopoietic growth factor-secreting cells might offer an alternative to parenteral injections of recombinant cytokines in the treatment of neutropenic patients.

Animals↗
PubMed1994

Immunotherapy of bladder cancer with cytokine gene-modified tumor vaccines.

This study explored the use of cytokine gene-modified tumor cells as cellular vaccines for the treatment of bladder cancer. The mouse MBT-2 tumor is an excellent model for human bladder cancer. This carcinogen-induced tumor of bladder origin resembles human bladder cancer in its etiology and histology and responds to treatment in a manner similar to that of its human counterpart. In a previous study we have shown that interleukin 2 (IL-2)-secreting, irradiated, MBT-2 cell preparations were capable of curing animals from orthotopically established tumors and engendered protective immunological memory in the cured animals. In this study we have compared the effectiveness of several cytokines and found that while IL-1 alpha, IL-1 beta, and gamma-interferon were only weakly effective in the therapeutic vaccination protocol, granulocyte-macrophage colony-stimulating factor was almost as effective as but not superior to IL-2, as reported previously for another tumor model system. Induction of cytotoxic T-lymphocyte correlated only poorly with the therapeutic benefit of the cytokine gene-modified tumor cell preparations, questioning its prognostic value for the development of improved genetically modified tumor vaccines.

Animals↗
PubMed1994

Transduction of the IL2 gene into human acute leukemia cells: induction of tumor rejection without modifying cell proliferation and IL2 receptor expression.

BACKGROUND: Previous studies have suggested that some of the limitations associated with the administration of high-dose exogenous interleukin 2 (IL2) may be overcome, at least partly, by cytokine gene transfer modalities. These findings have prompted investigations into whether human tumor cells may be transduced with the IL2 gene and whether tumor cell lines could be engineered to release IL2. PURPOSE: The purpose of this study was to evaluate the possibility of inducing a productive transfer of the IL2 gene into human acute leukemia cells and to assess the phenotypic and proliferative changes generated in the engineered cells, as well as their tumorigenic potential in nude mice. METHODS: Three retroviral vectors (DC/TK/IL2, DC/AD/R/IL2, and N2/CMV/IL2) carrying the IL2 gene were used to transduce three human leukemic cell lines: K562 and U937 (myeloid) and ST4 (lymphoid). Messenger RNA expression of the IL2 gene was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) and productive IL2 release using a human IL2 assay and an enzyme-linked immunosorbent assay kit. The expression of the p55 (alpha) and p75 (beta) chains of the IL2 receptor were determined by RT-PCR and indirect immunofluorescence. The kinetics of in vitro growth and proliferation of parental and engineered cells were also measured. Parental and IL2 gene-transduced ST4 lymphoblasts were injected into immunosuppressed nude mice that had their tumors measured twice weekly. RESULTS: The productive insertion of the IL2 gene was achieved in all three cell lines studied. The amounts of IL2 constitutively released by the engineered neoplastic cells ranged between 1 and 11 U/mL of IL2 produced from 10(6) cells in 72 hours. A fivefold increase in IL2 production was obtained in ST4 cells by further limiting dilution cloning of the bulk-infected cells. The stable integration of the IL2 gene did not modify the phenotype of the leukemic cells, the expression of the IL2 receptor alpha and beta chains and of several cytokine genes, or the kinetics of in vitro growth and proliferation. In nude mice injected with various IL2-producing ST4 clones, tumor growth associated inversely with the amounts of IL2 secreted by the leukemic cells. CONCLUSIONS: The results of this study demonstrate that the IL2 gene can be productively transduced into human myeloid and lymphoid leukemic cells without modifying their phenotypic and proliferative properties and that this transduction leads to a reduced or abrogated in vivo tumorigenic potential.

Acute Disease↗
PubMed1994

Immunotherapy of prostate cancer in the Dunning rat model: use of cytokine gene modified tumor vaccines.

Adenocarcinoma of the prostate is the most common cancer in men. The majority of cancers are discovered once they have already metastasized, and there is no effective therapy for prostatic cancer at this stage. The use of cytokine-secreting tumor cell preparations as therapeutic vaccines for the treatment of advanced prostate cancer was investigated in the Dunning rat R3327-MatLyLu prostatic tumor model. IL-2 secreting, irradiated, tumor cell preparations were capable of curing animals with s.c. established tumors, and induced immunological memory that protected animals from subsequent tumor challenge. Immunotherapy was less effective when tumors were induced orthotopically, but nevertheless led to improved outcome, significantly delaying, and occasionally preventing, recurrence of tumors after resection of the cancerous prostate. Granulocyte-macrophage colony stimulating factor secreting tumor cell preparations were less effective, and interferon-gamma secreting cells had only a marginal effect. Induction of a potent immune response in tumor bearing animals against the nonimmunogenic MatLyLu tumor supports the view that active immunotherapy warrants further investigation as a potential therapeutic approach to prostate cancer.

Adenocarcinoma↗
PubMed1994

Augmentation of antitumor immunity by tumor cells transduced with a retroviral vector carrying the interleukin-2 and interferon-gamma cDNAs.

Therapeutic models using gene transfer into tumor cells have pursued three objectives: (1) to induce rejection of the tumor transduced with therapeutic genes, (2) to induce immune-mediated regression of metastatic disease, and (3) to induce long-lasting immunity to protect against challenge with tumor cells or clinical regrowth of micrometastatic disease. Because in vivo therapy for patients with cancer using gene transfer would, as a first step, attempt to eliminate the existing tumor, we have investigated whether antitumor immunity induced by tumor cells secreting a single cytokine could be increased by cotransfer of a second cytokine gene. To test this approach, CMS-5, a murine fibrosarcoma, was transduced with retroviral vectors carrying interleukin-2 (IL-2), interferon-gamma (IFN-gamma), or granulocyte-macrophage-colony-stimulating factor (GM-CSF) cDNA alone or IL-2 cDNA in combination with IFN-gamma or GM-CSF cDNA. Single cytokine-secreting clones were selected to match levels of cytokine production by double cytokine-secreting clones so that similar amounts of cytokine were secreted. IFN-gamma- and IL-2/IFN-gamma-secreting CMS-5 cells showed increased levels of major histocompatability complex class I expression compared with IL-2- and GM-CSF-secreting or parental CMS-5 cells, IL-2/IFN-gamma-secreting CMS-5 cells were always rejected by syngeneic mice, whereas the same number of CMS-5 cells secreting only one of these cytokines or mixtures of single cytokine-secreting CMS-5 cells were not rejected. In vivo depletion of CD4+, CD8+, or natural-killer effector cell subpopulations showed that CD8+ cytotoxic T cells were primarily responsible for rejection of IL-2/IFN-gamma-transduced tumor cells. Our data show the successful use of a single retroviral vector to stably transduce two cytokine genes into the same tumor cell, leading to an increased effect on the in vivo induction of antitumor immunity.

Animals↗
PubMed1994

Human lung tumor cell secretion of interleukin-2 for protection against tumor engraftment.

BACKGROUND: Lung cancer continues to claim large numbers of human lives each year despite advances made in conventional therapies. The use of biologic response modifiers to modulate the immune system against human tumors is an alternate form of immunotherapy. Interleukin-2 (IL-2), or T-cell growth factor, is an important modulator of activated T cells. We show here that tumor cells transduced with human IL-2 cDNA provide protective immunity against engraftment of IL-2-secreting, as well as parental non-IL-2-secreting, tumor cells in vivo. METHODS: In an attempt to increase the antigen-induced proliferation and cytotoxicity T cells within the vicinity of tumor antigen, we have transduced human lung tumor cell lines (generated from whole tumor specimens obtained fresh from the operating room) with a vector containing the IL-2 gene. Cell lines secreting 0.5-20 Cetus units/ml of IL-2 were generated. Control cell lines were similarly established using the same retroviral vector containing the gene for adenosine deaminase (ADA). The growth of tumor xenografts of the vector-modified cell lines was observed in severe combined immunodeficient (scid) mice. RESULTS: Using C.B-17 scid mice, we have observed that the local secretion of IL-2 by these human lung tumor cell lines will prevent engraftment of that tumor into scid mice. The parental tumor as well as the tumor containing the ADA gene grow aggressively in the scid mouse. Growth arrest also correlated strongly with the amount of IL-2 secreted by the tumor cells. The local secretion of IL-2 by the transduced cell line will abrogate the tumorigenicity of the parental cell line as well as an allogeneic tumor. The inhibition of growth occurs only when the tumors are placed in close proximity to each other. After gamma irradiation, transduced tumor cells will continue to secrete IL-2. CONCLUSION: These results indicate that (a) human lung tumor cell lines can be transduced with IL-2-containing retroviral vectors; (b) local and sustained release of IL-2 will induce an antitumor response by the host against the IL-2-secreting as well as the control tumor cells; (c) secretion of IL-2 continues after the cells are irradiated. This study suggests that cytokine-secreting human lung tumors may be used in vaccination protocols for cancer patients.

Animals↗
PubMed1994

Human tumor vaccines and genetic engineering of tumors with cytokine and histocompatibility genes to enhance immunogenicity.

Genetically engineered tumor cells can be used as vaccines in order to stimulate an immune response. To date, tumor cells have been modified in vitro so that they secrete cytokines or express histocompatibility molecules that they naturally fail to express. These tumor cells differ in the types of immune responses they induce and in whether the responses have local or systemic efficacy. Many questions have been raised during the past year, including whether allogeneic or autologous tumor cells should be employed and whether there may be a risk of inducing autoimmune disease along with the antitumor response. Nevertheless, because of the paucity of available therapies for patients with advanced cancer, investigators must attempt to refine the approaches used in order to minimize patient risk while maximizing tumor cell destruction.

Cytokines↗