Chest 1997 Jun;111(6 Suppl):137S
PMID: 9184564, UI: 97328006
Chest 1997 Jun;111(6 Suppl):128S-129S
PMID: 9184560, UI: 97328002
Hum Gene Ther 1997 Mar 20;8(5):545-553
Daily systemic administration of hematopoietic growth factors can be associated with dose-limiting systemic side effects. To overcome this, we have investigated hematopoietic cytokine gene transfer to the marrow cavity of dogs by direct intramarrow injection of adenoviral vectors. In marrow culture, replication-deficient (E1-deleted) adenoviral vectors were able to transduce marrow stromal cells, demonstrating 30-fold greater expression than from other marrow cell types. High-level (ng/ml) cytokine production from transduced stromal cells persisted for 14 days in culture. Because adenovectors could efficiently transduce marrow stromal cells in culture, we investigated if stromal cells could also be transduced in vivo following direct intramarrow vector injection. Adenovectors with genes for interleukin 6 (IL-6) and Lac Z (beta-galactosidase) were injected directly into the marrow cavity of dogs resulting in protein expression localized to within the treated marrow. To evaluate this approach further in dogs, we constructed a vector expressing biologically active canine granulocyte-macrophage colony stimulating factor (GM-CSF). 293 cells infected with ADGM-CSF demonstrated prevalent GM-CSF mRNA by Northern blot and 135 +/- 30 ng/ml of protein as measured by enzyme-linked immunosorbent assay (ELISA). In vitro bioactivity of protein expressed was confirmed by canine GM colony-forming assay (CFU-GM). In vivo high-level protein production was noted in supernatants of marrow aspirates 72 hr following direct intramarrow administration of ADGM-CSF (baseline mean +/- SEM, 27 +/- 22 ng/ml, 72-hr sample 921 +/- 461 ng/ml). A localized myeloid expansion of marrow and significant peripheral leukocytosis (neutrophilia) were noted in all ADGM-CSF-treated dogs. Peripheral blood changes lasted for up to 3 weeks in dogs following single intramarrow injection. Thus, adenoviral cytokine expression from the marrow of a single large bone (ilium) led to compartmentalized expression of growth factor and an increase of hematopoiesis sufficient to cause peripheral blood changes in a large animal model.
PMID: 9095406, UI: 97249520
Ann N Y Acad Sci 1996 Oct 31;796:235-244
In summary, we have been able to demonstrate that adenovirus vectors are valuable tools in examining the roles played by individual cytokines in lung responses and inflammation. These viral vectors have marked trophism for the epithelial cells of the lung and are highly efficient in transferring the genes into these lining cells. This results in significant expression of cytokines both within the lumen and the parenchyma of the lung. As a result of an individual cytokine being overexpressed, there are cytokine-specific changes seen-IL-6 resulting in lymphocytosis, MIP2 resulting in neutrophil accumulation, RANTES resulting in monocyte accumulation, TGF beta resulting in monocytosis but no fibrosis, and GM-CSF most surprisingly resulting in tissue eosinophilia, granuloma formation, and subsequently the onset of fibrosis. These vectors have helped pinpoint the role of a number of the cytokines in inducing chronic inflammatory changes to the lung and imply that a single cytokine may not be the only trigger resulting in chronic changes within the lung parenchyma.
Am J Pathol 1996 Oct;149(4):1395-1403
Replication-defective adenoviral vectors are capable of localized transfer and expression of incorporated gene product in lung tissue. We have constructed an adenoviral vector that expresses rat macrophage inflammatory protein (MIP)-2, a C-X-C chemokine specifically chemotactic for neutrophils, Supernatants from 293 cells, infected with the adenoviral MIP-2 (ADMIP-2) construct, showed potent chemotactic activity and the ability of the ADMIP-2 vector to transcribe and make functional protein was confirmed. In vivo analysis of bronchoalveolar lavage fluid from rats after intratracheal instillation of ADMIP-2 (10(9) plaque-forming units) showed a 10-fold increase in the absolute number of neutrophils in bronchoalveolar lavage fluid as opposed to rats treated with an equal titer of an E1-disabled control virus expressing firefly luciferase (ADCA-18). Neutrophils constituted 65% of total BAL cells with alveolar macrophages being the other major cell type recovered. Rat MIP-2 protein was increased (nanograms per milliliter) in bronchoalveolar lavage fluid over a period of 7 days in ADMIP-2-treated animals. MIP-2 mRNA was demonstrated by Northern blot analysis in lung tissue, and histological analysis confirmed the presence of massive localized tissue neutrophilia. Evidence of chronic tissue injury and repair (ie, fibrosis) was not detected up to 2 weeks after the neutrophil infiltrate had resolved, subsequent to decreased chemokine presence. Adenoviral gene transfer proved an effective tool for the assessment of lung tissue expression of this chemokine in vivo and is useful in developing rodent models of tissue neutrophilia.
PMID: 8863686, UI: 97017073
Ann Surg Oncol 1996 May;3(3):317-324
BACKGROUND: Transfection of the costimulatory molecule B7-1 into some murine tumors can increase antitumor immunity and eradicate tumor growth. The purpose of this work was to construct an adenovirus-B7 (Ad-B7) expression vector and study B7-1 gene transfer into human cancer cells. METHODS: The human B7-1 cDNA was ligated into an expression cassette containing the human cytomegalovirus immediate early gene promoter and then inserted into the E1 region of the Ad5 genome by homologous recombination. The resulting Ad-B7 vector was used to infect established cancer cell lines and freshly resected cancers. Resected tumors were disaggregated into single cell suspensions by mechanical mincing and enzymatic digestion. Surface expression of B7-1 after infection was verified by flow cytometry. RESULTS: Expression kinetics in three cell lines showed that infected cells began to express B7-1 within 24 h. The proportion of B7-1+ cells continued to increase during the next 48 h, after which expression remained relatively constant during the next 5 days (up to 98% B7-1+ cells). Fresh tumor cells from various cancers displayed similar kinetics, but with greater variability in the proportion of cells expressing B7-1 (13% to 95% B7-1+ cells). Cancers which were successfully infected included 3 colorectal adenocarcinomas, 2 leiomyosarcomas, 2 lung squamous cell carcinomas, and 1 renal cell carcinoma. CONCLUSIONS: The Ad-B7 vector is a rapid and efficient means of gene transfer which does not require host cell proliferation. The ultimate objective is to engineer autologous tumors to express B7-1 and vaccinate cancer patients in an adjuvant or palliative setting.
PMID: 8726189, UI: 96292653
J Clin Invest 1996 Feb 15;97(4):1102-1110
Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic cytokine whose expression is increased in numerous respiratory diseases, particularly in asthma. However, the role of GM-CSF in the pathogenesis of these conditions in vivo remains unclear. Here, we report the functional activities of GM-CSF highly expressed in rat lung after intrapulmonary transfer of the gene coding for murine GM-CSF by using an adenoviral vector. This high, transient expression of GM-CSF led to the sustained but self-limiting accumulation of eosinophils and macrophages associated with tissue injury in the lung followed by varying degrees of irreversible fibrotic reactions observed in later stages. These results suggest that GM-CSF plays a previously unrealized role in the development of respiratory conditions characterized by eosinophilia, granuloma and/or fibrosis and provide the rationale for targeting this molecule in these diseases.
PMID: 8613534, UI: 96193895