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Ann Thorac Surg 2001;71:1817-1823
© 2001 The Society of Thoracic Surgeons
a Division of Cardiothoracic Surgery, Washington University School of Medicine, St. Louis, Missouri, USA
Address reprint requests to Dr Patterson, Division of Cardiothoracic Surgery, Washington University School of Medicine, 3108 Queeny Tower, One Barnes-Jewish Hospital Plaza, St. Louis, MO 63110
Presented at the Poster Session of the Thirty-sixth Annual Meeting of The Society of Thoracic Surgeons, Fort Lauderdale, FL, Jan 31–Feb 2, 2000.
Background. Multiple gene cotransfection may be an effective strategy to modulate concurrent pathologic events after lung transplantation. We investigated in vivo naked plasmid lung cotransfection during cold preservation and the role of lung parenchyma/naked plasmid exposure time.
Methods. F344 rats underwent left main bronchus instillation of pCF1-CAT (chloramphenicol acetyl transferase) (130 µg) ± pCF1-ß-Gal (ß-galactosidase) (130 µg) in saline. Part Ia: 4°C preservation versus cotransfection. Lung isografts (4 groups, n = 8) were stored after transfection for 1 (2 groups: one received only pCF1-CAT), 6, and 18 hours. Recipient sacrifice was after 48 hours. Part Ib: 4°C preservation versus transgene expression. Rats were sacrificed 48 hours after transfection in a nontransplant setting (2 groups, n = 8; one received only pCF1-CAT). In a third group (n = 8) lungs were harvested 24 hours after transfection, stored for 18 hours, and recipients were sacrificed after 24 hours. The CAT and ß-Gal enzymatic-linked immunosorbent assays were performed. Part II: Lung/plasmid exposure time. In three groups (n = 6) after pCF1-CAT transfection the left main bronchus was not clamped, clamped for 10 minutes, or clamped for 1 hour. Sacrifice was after 48 hours.
Results. Part Ia: Lung CAT protein was (in picograms per 100 µg of total protein): median, 42 (range, 25 to 95) after 1 hour (only CAT); 67 (19 to 296) after 1 hour, 32 (6 to 157) after 6 hours; and 9 (5 to 243) after 18 hours. Lung ß-Gal protein was (in picograms per 100 µg of total protein): median, 20 (range, 5 to 353) after 1 hour; 17 (6 to 157) after 6 hours; 4 (1 to 74) after 18 hours (1 hour versus 18 hours, p = 0.04 for both proteins). CAT and ß-Gal production were significantly correlated (p = 0.0001, r = 0.924). Part Ib: Lung CAT protein was (in picograms per 100 µg of total protein): median, 2 (range, 0.6 to 10) no transplant, only CAT; 7 (0.3 to 13) no transplant; 3 (0.9 to 14) transplant. Part II: Left lung CAT protein was (in picograms per 100 µg of total protein): median, 31 (range, 6 to 83) no clamp; 74 (25 to 430) 10 minutes of clamp; 111 (30 to 263) 1 hour of clamp. Right lung CAT protein was (in picograms per 100 µg of total protein): median, 0.06 (range, 0 to 0.9) no clamp; 1 (0 to 6) 10 minutes of clamp; 1 (0 to 18) 1 hour of clamp.
Conclusions. Efficient lung isograft endobronchial cotransfection results from using naked plasmid. Cold preservation affects transfection efficiency but not transgene expression. Lung parenchyma/naked plasmid exposure time determines transfection efficiency.
Related Article
Ann. Thorac. Surg. 2001 71: 1823-1824.
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