There is a rapid onset of organizing alveolitis/fibrosis at 120-140 d after whole lung irradiation of C57BL/6J mice. To test the hypothesis that circulating cells of bone marrow origin contribute to irradiation fibrosis, irradiated chimeric green fluorescent protein (GFP)+ C57BL/6J mice were followed for GFP+ cells in areas of lung fibrosis. In a second experimental model, C57BL/6J female mice received 20 Gy total lung irradiation, and after 60 or 80 d were intravenously injected with cells from a clonal GFP+ male bone marrow stromal cell line or male GFP+ whole bone marrow, respectively. The mice were then followed for the development of pulmonary fibrosis, and the contribution of Y-probe-positive, GFP+ cells to fibrotic areas was quantitated. Bromodeoxyuridine labeling of developing fibrotic areas showed that the cell division occurred predominantly in GFP+, Y-probe-positive, and vimentin-positive cells. Immunohistochemistry demonstrated that these cells were macrophages and fibroblasts, not endothelial cells. Mice that received manganese superoxide dismutase-plasmid/liposome intratracheal injection 24 h before total lung irradiation demonstrated a decrease in GFP+ fibroblastic cells in the lung. Thus, pulmonary irradiation fibrosis contains proliferating cells of bone marrow origin, and gene therapy prevention of this condition acts in part by decreasing the migration and proliferation of marrow origin cells.

We investigated the importance of mitochondrial localization of the SOD2 (MnSOD) transgene product for protection of 32D cl 3 hematopoietic cells from radiation-induced killing. Four plasmids containing (1) the native human copper/zinc superoxide dismutase (Cu/ZnSOD, SOD1) transgene, (2) the native SOD2 transgene, (3), the SOD2 transgene minus the mitochondrial localization leader sequence (MnSOD-ML), and (4) the SOD2 mitochondrial leader sequence attached to the active portion of the SOD1 transgene (ML-Cu/ZnSOD) were transfected into 32D cl 3 cells and subclonal lines selected by kanamycin resistance. Clonogenic in vitro radiation survival curves derived for each cell clone showed that Cu/ZnSOD- and MnSOD-ML-expressing clones had no increase in cellular radiation resistance (D0=0.89 +/- 0.01 and 1.08 +/- 0.02 Gy, respectively) compared to parent line 32D cl 3 (D0=1.15 +/- 0.11 Gy). In contrast, cell clones expressing either SOD2 or ML-Cu/ZnSOD were significantly radioresistant (D0=2.1 +/- 0.1 and 1.97 +/- 0.17 Gy, respectively). Mice injected intraesophageally with SOD2-plasmid/liposome (MnSOD-PL) complex demonstrated significantly less esophagitis after 35 Gy compared to control irradiated mice or mice injected intraesophageally with Cu/ZnSOD-PL or MnSOD-ML-PL. Mice injected with intraesophageal ML-Cu/ZnSOD-PL showed significant radioprotection in one experiment. The data demonstrate the importance of mitochondrial localization of SOD in the in vitro and in vivo protection of cells from radiation-induced cellular damage.

Background-Diagnosing acute coronary syndrome in patients presenting with chest discomfort is a challenge. Because acute myocardial ischemia/reperfusion is associated with endothelial upregulation of leukocyte adhesion molecules, which persist even after ischemia has resolved, we hypothesized that microbubbles designed to adhere to endothelial selectins would permit echocardiographic identification of recently ischemic myocardium. Methods and Results-Lipid microbubbles (diameter, 3.3Ϯ1.7 m) were synthesized. The selectin ligand sialyl Lewis

Radiation of the esophagus of C3H/HeNsd mice with 35 or 37 Gy of 6 MV X rays induces significantly increased RNA transcription for interleukin 1 (Il1), tumor necrosis factor alpha (Tnf), interferon gamma inducing factor (Ifngr), and interferon gamma (Ifng). These elevations are associated with DNA damage that is detectable by a comet assay of explanted esophageal cells, apoptosis of the esophageal basal lining layer cells in situ, and micro-ulceration leading to dehydration and death. The histopathology and time sequence of events are comparable to the esophagitis in humans that is associated with chemoradiotherapy of non-small cell lung carcinoma (NSCLC). Intraesophageal injection of clinical-grade manganese superoxide dismutase-plasmid/liposome (SOD2-PL) 24 h prior to irradiation produced an increase in SOD2 biochemical activity in explanted esophagus. An equivalent therapeutic plasmid weight of 10 microgram ALP plasmid in the same 500 microliter of liposomes, correlated to around 52-60% of alkaline phosphatase-positive cells in the squamous layer of the esophagus at 24 h. Administration of SOD2-PL prior to irradiation mediated a significant decrease in induction of cytokine mRNA by radiation and decreased apoptosis of squamous lining cells, micro-ulceration, and esophagitis. Groups of mice receiving 35 or 37 Gy esophageal irradiation by a technique protecting the lungs and treating only the central mediastinal area were followed to assess the long-term effects of radiation. SOD2-PL-treated irradiated mice demonstrated a significant decrease in esophageal wall thickness at day 100 compared to irradiated controls. Mice with orthotopic thoracic tumors composed of 32D-v-abl cells that received intraesophageal SOD2-PL treatment showed transgenic mRNA in the esophagus at 24 h, but no detectable human SOD2 transgene mRNA in explanted tumors by nested RT-PCR. These data provide support for translation of this strategy of SOD2-PL gene therapy to studies leading to a clinical trial in fractionated irradiation to decrease the acute and chronic side effects of radiation-induced damage to the esophagus.