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Although long-term expression of therapeutic molecules is necessary for the treatment of permanent deficiencies, short-term expression of therapeutic molecules inducing local or systemic effects is preferable in clinical situations where temporary substitution is the goal. One such clinical setting is the administration of hematopoietic growth factors in cancer chemotherapy-induced myelosuppression. Several plasmid vectors containing the human granulocyte colony-stimulating factor (G-CSF) gene under transcriptional control of different regulatory elements were constructed. In vitro production of G-CSF by nonvirally transfected murine fibroblast clones initially increased after lethal irradiation and was detectable for at least 12 days. We also demonstrate that a single injection of irradiated G-CSF-secreting fibroblasts leads to accelerated hematopoietic recovery and mobilization of committed peripheral blood progenitor cells equivalent to that achieved by twice daily s.c. administration of high doses of recombinant human G-CSF. Using dicistronic vectors, high levels of G-CSF secretion were also obtained in human fibroblasts.

Recent experiments have shown that gamma interferon (IFN-␥), either administered or induced in vivo, e.g., by certain bacteria, is a key mediator in inducing hypersensitivity to bacterial lipopolysaccharides. The source of endogenous IFN-␥ in this context (natural killer versus T H 1 cells) has not been investigated yet. In order to investigate the role of antigen-specific, IFN-␥-producing T H 1 cells in murine Pseudomonas aeruginosa infection, a murine T H 1 cell line was propagated in vitro by using recombinant P. aeruginosa outer membrane protein I. Adoptive transfer experiments were performed by intravenous injection of various amounts of T H 1 cells into P. aeruginosa-challenged SCID mice. Adoptive transfer of 5 ؋ 10 6 T cells into SCID mice followed by an intraperitoneal challenge with 1.4 ؋ 10 6 CFU of live P. aeruginosa resulted in the rapid death of the animals within 12 h postchallenge, whereas transfer of lower T-cell doses and saline as a control did not cause any detrimental effects. After challenge with 2.8 ؋ 10 6 CFU of P. aeruginosa, similar results were obtained 18 h postchallenge; however, at the end of the 72-h observation period, no significant differences in survival rates were obtained between the groups treated with different amounts of T cells. The rapid death of mice treated with 5 ؋ 10 6 T cells was reflected by 860-fold-elevated levels of tumor necrosis factor alpha (TNF-␣) present in serum 2 h postchallenge, whereas no significant differences in TNF-␣ serum levels were detectable in mice treated with lower doses of T cells or with saline. Pretreatment of T-cell-reconstituted SCID mice with neutralizing anti-IFN-␥ monoclonal antibodies completely protected mice from bacterial challenge and reduced TNF-␣ levels in serum. We conclude that under the experimental conditions described here, IFN-␥-and interleukin-2-producing T H 1 cells represent an important trigger mechanism inducing TNF-␣-mediated hypersensitivity to bacterial endotoxin.

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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.