This report details the transfer of a human epidermal growth factor (hEGF) expression plasmid to porcine partial-thickness wound keratinocytes by particle-mediated DNA transfer (Accell). After gene transfer an external sealed fluid-filled wound chamber was used to protect the wound, provide containment of the exogenous DNA and expres peptide, and permit saing of the wound fluid. Analysis of wound fluid for hEGF and total protein, an indicator of reformation of the epithelal barrier, showed that wounds bombarded with the hEGF plasmid exhibited a 190-fold increase in EGF concentration and healed 20% (2.1 days) earier than the controls. EGF concentrations in wound fluid persisted over the entire 10-day monitored period, decreasing from 200 pg/ml to 25 pg/ml over the first 5 days. Polymerase chain reaction results showed that plasmid DNA was present in the wound for at least 30 days. These findings demonstrate the possible utility of in vivo gene transfer to enhance epidermal repair.

At 60-801% confluence keratinocyte cultures were assigned as controls or for gene transfer by infection with one or the other of two recombinant retrovirus vectors: (i) an amphotropic helper-free murine leukemia virus vector, MFG-lacZ, containing the (-galactosidase gene, lacZ (R.C.M., unpublished), or (ii) a vector, a-SGC-hGH, containing the gene sequence from hGH (L. Cohen and R.C.M., unpublished). Subconfluent keratinocyte cultures were incubated on 3 consecutive days with the retrovirus in Polybrene (8 pg/ml) and 10% fetal bovine serum containing Dulbecco's modified Eagle's medium (DMEM; GIBCO).During transfection the modified Waymouth medium was removed and replaced daily by an equal amount of DMEM containing the retrovirus at a concentration of 1-2 x 107 plaque-forming units/ml. After 6 hr the virus-containing medium was removed and replaced by Waymouth medium. This procedure was repeated on 3 consecutive days. At confluence, keratinocytes were released from the flasks with 0.01% trypsin and resuspended in normal unbuffered saline (0.9%O) containing 100 international units ofpenicillin and 100 pg of streptomycin per ml, at a concentration of 3 x 106 cells per ml. Keratinocytes resuspended in saline were found to be >90% viable, as determined by trypan blue staining.The procedure for making 170 reproducible standardized f-thickness skin wounds on the backs of 13 anesthetized pigs for these experiments was as follows. Under aseptic conditions using a 2.25-cm2 square template the skin was excised with a scalpel to the level ofthe panniculus carnosus muscle with careful hemostasis. Wound margins were tattooed with India ink, allowing for photoplanimetric evaluation of the surface area on sequential standardized photographs. The chambers, serving as an in vivo cell culture device, were applied to each wound (1, 4). The chamber (P. A. Medical, Columbia, TN) consists of a flexible transparent vinyl top bonded to an adhesive base. The base has a central opening fitting the wound margins. Chambers were filled with 1.2 ml of isotonic saline containing 100 pg of streptomycin and 100 international units penicillin per ml (1, 4). Twenty-two wounds were treated with lacZ transfected Abbreviations: hGH, human growth hormone; H&E, hematoxylin/ eosin; X-Gal, 5-bromo-4-chloro-3-indolyl -D-galactoside. t~o whom reprint requests should be addressed. 9307The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Prolonged exposure to supraphysiological oxygen concentrations results in the generation of reactive oxygen species, which can cause significant lung injury in critically ill patients. Supplementation with human recombinant antioxidant enzymes (AOE) may mitigate hyperoxic lung injury, but it is unclear which combination and concentration will optimally protect pulmonary epithelial cells. First, stable cell lines were generated in alveolar epithelial cells (MLE12) overexpressing one or more of the following AOE: Mn superoxide dismutase (MnSOD), CuZnSOD, or glutathione peroxidase 1. Next, A549 cells were transduced with 50-300 particles/cell of recombinant adenovirus containing either LacZ or each of the three AOE (alone or in combination). Cells were then exposed to 95% O(2) for up to 3 days, with cell number and viability determined daily. Overexpression of either MnSOD (primarily mitochondrial) or CuZnSOD (primarily cytosolic) reversed the growth inhibitory effects of hyperoxia within the first 48 h of exposure, resulting in a significant increase in viable cells (P < 0.05), with 1.5- to 3-fold increases in activity providing optimal protection. Protection from mitochondrial oxidation was confirmed by assessing aconitase activity, which was significantly improved in cells overexpressing MnSOD (P < 0.05). Data indicate that optimal protection from hyperoxic injury occurs in cells coexpressing MnSOD and glutathione peroxidase 1, with prevention of mitochondrial oxidation being a critical factor. This has important implications for clinical trials in preterm infants receiving SOD supplementation to prevent acute and chronic lung injury.

v-Ha-ras has been demonstrated previously to induce neuroendocrine di erentiation of medullary thyroid carcinoma (MTC, malignant C cell tumor) cell lines. The potential role of ras mediated signaling in neuroendocrine cells in vivo has been investigated by expressing v-Ha-ras under control of the neural/ neuroendocrine speci®c calcitonin/calcitonin gene-related peptide (CGRP) promoter. Five independent mouse lineages were derived following germ line insertion of the transgene. Four of the ®ve lineages consistently express the transgene; neuroendocrine expression is found in three of the ®ve lineages as both spliced and full length messages. Phenotypically, the mice expressing rascal have shortened lifespans primarily due to the high incidence of MTCs between 6 months to a year of age. C-cell hyperplasia is demonstrated in several mice in the absence of gross evidence of tumor formation. Histopathological and ultrastructural analyses demonstrate typical features of MTCs including prominent immunohistochemical staining for calcitonin and dense-core neurosecretory-type granules. In addition, four of 22 tumors co-express thyroglobulin (a non-neuroendocrine follicular epithelial cell marker) and calcitonin (a neuroendocrine marker) in a subset of the tumor cells. The rascal transgenic mouse provides a unique model for investigating the sequential pathogenesis of MTC and possibly also for elucidating the relationship between MTC and mixed medullary-follicular carcinomas.