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Am J Physiol Lung Cell Mol Physiol 286: L1188-L1193, 2004; doi:10.1152/ajplung.00288.2003
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Adenovirus-mediated transfer of the 1-cys peroxiredoxin gene to mouse lung protects against hyperoxic injury

Yan Wang, Yefim Manevich, Sheldon I. Feinstein, and Aron B. Fisher

Institute for Environmental Medicine, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19104-6068

Submitted 22 August 2003 ; accepted in final form 27 January 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
1-Cys peroxiredoxin (1-cysPrx) is a novel antioxidant enzyme that has been shown to reduce a broad spectrum of peroxides including phospholipid hydroperoxides. We tested the hypothesis that adenovirus-mediated transfer of the 1-cysPrx gene can protect lungs of mice from oxidant injury. Mice infected with AdLacZ/AdNull were used as a control (AdCon). X-galactosidase staining revealed widespread expression of the LacZ gene in airways and lung alveoli. Compared with AdCon, 1-cysPrx expression was increased about twofold at 3 days after adenovirus infection. Mice with increased Prx expression showed less loss of body weight and longer survival during exposure to 100% O2 or to 85% O2 for 4 days followed by 100% O2. At 72 h of 100% O2 exposure, AdPrx infection protected mouse lungs from injury as indicated by less pleural effusion, lower lung wet/dry weight, less protein and fewer nucleated cells in bronchoalveolar lavage fluid, and lower content of thiobarbituric acid-reactive substances and protein carbonyls in lung homogenate. These findings show that increased expression of 1-cysPrx through adenovirus-mediated gene transfer protects mouse lungs from hyperoxic injury and delays death.

peroxiredoxin 6; LacZ gene; protein carbonyls; thiobarbituric acid-reactive substances; gene therapy; bronchoalveolar lavage fluid

HYPEROXIC LUNG INJURY IS CHARACTERIZED by injury to epithelial and capillary endothelial cells resulting in increased pulmonary capillary permeability, inflammation, cell death, and respiratory failure (10, 18). Current evidence indicates that the damaging effects of breathing gas with high oxygen concentration are caused in large part by increased formation of reactive oxygen species (ROS) resulting in an imbalance between the rate of ROS generation and their scavenging by antioxidant defenses (10). The latter include both enzymatic and nonenzymatic pathways. Primary attention for the role of antioxidant enzymes in hyperoxic injury has focused on catalase, glutathione peroxidases, and superoxide dismutases (SOD) (6, 10, 11).

Recently, it has been recognized that peroxiredoxins (Prx) may play a critical role in antioxidant defense. Prx are a recently described superfamily of nonseleno-peroxidases that catalyze the reduction of a broad spectrum of peroxides. Of the six mammalian members of this family, five contain two reactive cysteines and utilize thioredoxin as the reductant (21). Peroxiredoxin VI or 1-cys peroxiredoxin (1-cysPrx) has a single redox-active cysteine and utilizes GSH to catalyze the reduction of H2O2 and organic hydroperoxides including phospholipid hydroperoxides (8). 1-cysPrx is highly enriched in the lung compared with other organs and is especially expressed in Clara and alveolar epithelial type II cells (14). Previous studies have demonstrated that overexpression of 1-cysPrx in NIH 3T3 cells enhances their ability to reduce H2O2 and protects their glutamine synthetase against H2O2-mediated inactivation (12). Our laboratory has shown that 1-cysPrx can reduce peroxidized membrane phospholipids in H441 cells, a lung epithelial cell line (17), whereas an antisense-mediated decrease in expression of 1-cysPrx in L2 cells, another lung epithelial cell line, resulted in apoptotic cell death (20). Recently, gene-targeted mice with absent 1-cysPrx have been shown to be more sensitive to paraquat-induced oxidative injury (23). These data indicate that 1-cysPrx can play an important role in cellular defense against oxidative stress in lung-derived cells. In this study, the effect of overexpression of 1-cysPrx using adenovirus-mediated transfer on the pulmonary response to hyperoxia was evaluated.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Adenovirus construction. The adenoviral constructs with the 1-cysPrx (AdPrx), LacZ gene (AdLacZ), or empty vector (AdNull) were kindly provided by the Institute of Human Gene Therapy, University of Pennsylvania (2). The assembly and production of recombinant adenovirus were performed with the Adeno-X Expression System (Clontech, Palo Alto, CA) as described previously (20). Briefly, a shuttle vector containing a cytomegalovirus immediate-early promoter-driven expression cassette with full-length cDNAs for rat 1-cysPrx or LacZ genes was cotransfected with a plasmid that contains first-generation replication-incompetent human type 5 adenovirus genome into competent TOP10F' Escherichia coli cells (Invitrogen, Carlsbad, CA). An XbaI restriction site was introduced into the upstream primer, and a KpnI site was introduced into the downstream primer. The plasmids containing the insert of 1-cysPrx or LacZ genes were verified by DNA sequencing and stored at –80°C. The titers were 1.5 x 1011 plaque-forming units (PFU)/ml for AdPrx, 1.9 x 1010 PFU/ml for AdLacZ, and 9 x 1010 PFU/ml for AdNull.

Adenovirus infection protocol. The use of animals for these studies was approved by the University of Pennsylvania Animal Use and Care Committee. Male C57BL/6 mice, free of specific pathogens, weighing 24–28 g and age 8–11 wk, were anesthetized intraperitoneally with ketamine (50 mg/kg) and xylazine (10 mg/kg). Adenovirus stock was diluted with Tris-buffered saline (TBS) to 50 µl and was administered intranasally in four aliquots via a pipette tip followed by circumferential compression of the thorax to facilitate adenoviral dispersion to the distal air spaces (1). This method has been found previously to produce widespread expression of LacZ gene in mouse and rat lungs (1, 22). Infected animals were maintained in separate isolator cages for 3 days before hyperoxic exposure. Three experimental groups of mice were studied, an AdPrx experimental group (n = 48) and AdLacZ (n = 50) and AdNull control groups (n = 16).

In situ {beta}-galactosidase expression. X-galactosidase (X-gal) staining was used as described previously to provide a qualitative measurement of gene transfer (1, 16, 20, 22). The X-gal solution (pH 7.9) (Roche Diagnostics, Indianapolis, IN) was used to detect the activity of E. coli {beta}-galactosidase ({beta}-gal). Fresh lungs from mice infected with AdLacZ were washed repeatedly with ice cold phosphate-buffered saline (PBS), fixed with 0.2% glutaraldehyde and 2% formaldehyde in PBS for 30 min at 4°C, washed again with PBS, and incubated at 37°C with solution containing 0.5 mg/ml of X-gal for ~8 h until the appearance of a blue color. The tissues were then photographed with a digital camera.

For the study of sectioned slides, the stained tissues were immersed in 10, 20, and 30% sucrose sequentially for postfixation and paraffin embedded; longitudal sections were cut at a thickness of 6 µm. Sections were collected on slides, counterstained with Nuclear Fast Red (Vector Laboratories, Burlingame, CA) for 10 min, and mounted on coverslips with cedarwood oil.

Exposure of mice to hyperoxic stress. Mice were exposed to either 85 or 100% O2 (medical grade) at 1 atmosphere absolute in a Plexiglas chamber (Braintree Scientific, Braintree, MA). Oxygen was passed through a bubble humidifier and introduced into the sealed chamber at 6–8 l/min to provide ~5 gas exchanges per hour. The chamber oxygen concentration was measured continuously with a VTI O2 gas analyzer (Vacumed, Ventura, CA) and exceeded 97% for an input gas of 100% O2. For convenience, exposure under these conditions is termed 100% O2. Chamber CO2 was absorbed with a soda lime filter and was maintained at <0.2%. Humidity in the chamber was maintained at 45–50% by the gas flow. Mice were allowed food and water ad libitum and maintained on a 12-h dark-light cycle. Food and paper bedding were the same as for mice maintained in the University of Pennsylvania Animal Facility. Cages were opened daily for ~5 min for change of water, food, and bedding and removal of dead mice.

Survival study. Mice treated with AdPrx (n = 32), AdLacZ (n = 34), or AdNull (n = 16) were exposed to hyperoxia for evaluation of survival. The conditions used for study of survival were continuous 100% O2 exposure or 85% O2 exposure for 4 days followed by 100% O2. The number of surviving mice was determined at 12-h intervals.

Harvesting and preparation of lung tissue. Mice were evaluated for lung injury following exposure to a sublethal duration of hyperoxia. Mice were weighed on an electrical scale (TL-602; Denver Instruments, Denver, CO) before and after exposure to O2. After induction of anesthesia (50 mg/kg pentobarbital intraperitoneally) at the end of exposure, a midline laparotomy-thoracotomy was performed, and immediately the pleural effusion was collected with a 1-ml syringe. The trachea was cannulated for continuous ventilation, and mice were exsanguinated by laceration of the left renal artery and vein. The pulmonary vasculature was flushed by cannulation of the pulmonary artery via the right ventricle with PBS followed by en bloc removal of the heart and lungs. The heart and large airways were dissected away from the lungs and discarded. The hilum of the right upper and middle lobes was ligated and removed for measurement of the wet-to-dry weight ratio. Specimens were lightly blotted, weighed immediately, and then placed in a heated vacuum chamber (Scientific Glass Apparatus, Bloodfield, NJ) until repeated weighing demonstrated no change in weight (~3 days). The remaining lung lobes were lavaged by repeated instillation and aspiration of 1.5 ml (0.5 ml x 3) PBS containing 0.5 mM EDTA, pH 8.0. The bronchoalveolar lavage fluid (BALF) was placed on ice for cell counting and analysis of protein concentration. In some experiments, the left lung was used to evaluate tissue X-gal staining and histology. In other experiments, the lung tissue was frozen in liquid nitrogen and stored at –80°C for immunoblot analysis and measurement of thiobarbituric acid-reactive substances (TBARS) and protein carbonyls.

Immunoblot analysis of 1-cysPrx expression. Frozen lung tissue was thawed in 0.05% Tween 20-TBS buffer containing 10 mM Tris·HCl, pH 7.5, 150 mM NaCl and homogenized with a Potter-Elvehjem homogenizer at 4°C in the presence of Complete Protease Inhibitor cocktail (Boehringer Mannheim, Indianapolis, IN). Tissue extracts were then sonicated and centrifuged at 10,000 g for 20 min, and protein concentration was determined. Protein samples (10 µg) were subjected to a 12% SDS-PAGE gel on a XCell electrophoresis apparatus (Invitrogen, Carlsbad, CA) and then were transferred to a polyvinylidene fluoride membrane (Millipore, Bedford, MA). Membranes were incubated in blocking solution (0.1% Tween 20-TBS buffer containing 10% nonfat dry milk; Bio-Rad, Richmond, CA) for 1 h and then were probed with a polyclonal antibody (1:2,000 dilution) to a 1-cysPrx peptide (1:2,000 dilution) followed by peroxidase-conjugated secondary antibody as described previously (17, 20). The reaction was detected by enhanced chemiluminescence (ECL; NEN Life Science, Boston, MA) and quantitated by densitometric scanning of X-ray film using the FluorS multi-imager (Bio-Rad).

BALF analyses. BALF analyses were performed immediately after collection. Total nucleated cells were counted on a Coulter Counter (Coulter Electronics). The remaining BALF was centrifuged for 20 min at 1,000 rpm and 4°C. Protein concentration of the supernatant was measured by Coomassie blue (Bio-Rad) dye binding with bovine gamma globulin as the standard.

Biochemical measurements. For tissue analysis, lung (1:10) in PBS containing 0.01% butylated hydroxytoluene was homogenized under N2. TBARS, protein carbonyls, and total protein in tissue homogenates were determined by spectrophotometric assay as previously described (3, 25).

Statistical analysis. Data are expressed as means ± SE. Statistical significance was assessed by using SigmaStat software (Jandel Scientific, San Jose, CA). Group differences were evaluated by one-way ANOVA or Student's t-test. Survival curves in O2 were compared by log-rank analysis. Differences between mean values were considered statistically significant at P < 0.05.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Lung distribution of gene transfer. By Western blot, the expression of 1-cysPrx in AdPrx-treated mice approximately doubled compared with control mice (AdLacZ/AdNull-treated) during 72 h of room air breathing after adenovirus administration (Fig. 1). This time point represents the start of O2 exposure. With continued room air breathing, the differential expression level decreased back to baseline (Fig. 1). Hyperoxia (100% O2) beginning 3 days after adenovirus treatment resulted in a further increase in the relative 1-cysPrx expression (compared with AdLacZ/AdNull control) in the AdPrx-infected mice (Fig. 1). At day 1 of hyperoxia (4 days after adenovirus administration), the expression of 1-cys Prx in the O2-exposed mice was 2.5 times the control value; at day 4 of hyperoxia (7 days after adenovirus administration), the differential expression level had decreased but was still ~60% above control (Fig. 1). For mice exposed to 85% O2, 1-cysPrx expression was increased by 75 ± 11% at 4 days of exposure (7 days after adenovirus administration) (n = 3).



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  		Fig. 1. The effect of infection with adenoviral construct with 1-cys peroxiredoxin vector (AdPrx) on 1-cys peroxiredoxin (1-cysPrx) expression in mouse lung. Adenovirus was administered at 3 days (D–3) before the start of hyperoxia (D0). Lungs were evaluated at days 1–4 (D1–D4) of hyperoxia. A: representative immunoblots for 1-cysPrx protein (10 µg per lane). Mice infected with AdPrx or adenoviral construct with LacZ gene (AdLacZ) vector were exposed to hyperoxia (100% O2) or room air. Lungs were harvested at indicated time points and probed with a polyclonal antibody to 1-cysPrx peptide. B: quantitation of 1-cysPrx expression at each time point. Hyperoxic exposure was begun at D0, which represented the peak 1-cysPrx expression under room air. Western blots for 1-cysPrx protein were quantified for 1-cysPrx expression with the FluorS MultiImager software. Data points represent means ± SE for 3 experiments.

 
To assess the distribution of transgene expression, we harvested lungs 3 days after AdLacZ vector delivery and examined them by X-gal staining. {beta}-gal expression was distributed throughout each of the lung lobes (Fig. 2A) and was detected in both distal airways and the alveolar region (Fig. 2B). Epithelial cells lining the small airways and alveolar type 2 epithelial cells showed expression of the transgene although possible expression in alveolar type I epithelial and endothelial cells could not be determined at this level of resolution (Fig. 2B).



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  		Fig. 2. Expression of LacZ gene in mouse lung following intranasal delivery of 4 x 109 plaque-forming units AdLacZ. LacZ gene expression was examined by X-galactosidase (X-gal) staining, which results in a blue color. AdPrx-infected lungs are shown as a control for LacZ expression. Lungs were harvested at 3 days after vector administration. A: stained whole lungs. B: photomicrographs of 6-µm lung sections. AdNull, adenoviral construct with empty vector.

 
General response and mortality in hyperoxia. No differences in response or mortality to 100% O2 were noted for AdLacZ- vs. AdNull-infected mice, and these groups were combined as a control (AdCon). AdCon mice appeared lethargic and responded poorly to stimulation subsequent to 24 h of hyperoxia; by 48 h, cyanosis was noted around the anterior nares. The general response of mice in the AdPrx group was similar to control but was delayed by ~24 h. At 72 h, AdCon mice lost ~25% of their body weight compared with 16% for mice infected with AdPrx (Table 1).


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  		Table 1. Body weight and pleural fluid in mice exposed to 100% O2 for 72 h

 
AdCon mice exposed to 100% O2 began to die at ~64 h, whereas the initial death in the AdPrx group did not occur until 84 h of hyperoxia (Fig. 3A). The time to 50% lethality (LT50) for the AdPrx group (92.4 ± 2.9 h, n = 14) was significantly longer (P < 0.05) than that observed for the AdCon group (78.2 ± 5.6 h, n = 15). In a second experimental protocol, mice were exposed initially to 85% O2, and 4 days later the chamber O2 was increased to 100%. With this protocol, all of the AdCon mice died on day 5 of exposure, whereas >50% of the AdPrx animals survived for 7 days. The LT50 for AdPrx was significantly longer (P < 0.05) compared with AdCon mice (Fig. 3B). Subsequent studies of pulmonary injury were done only for the 100% O2 protocol.



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  		Fig. 3. Effect of AdPrx infection on the survival of mice exposed to 100% O2. Groups of mice infected with AdPrx or AdCon (combined results for AdLacZ and AdNull) were exposed to O2 under 2 different protocols. Mice were evaluated at 12 h intervals for time of death. The difference between AdPrx vs. AdCon is statistically significant (P < 0.01) for both experimental protocols. A: continuous 100% O2 exposure. B: mice were initially exposed to 85% O2 for 4 days and then to 100% O2.

 
Pleural effusion and lung wet-dry weight ratio. At 72 h of hyperoxia, bilateral pleural effusion was noted in all mice, but its volume in mice infected with AdPrx was significantly less compared with AdCon mice (Table 1). Hyperoxic exposure resulted in a time-related increase in lung wet-dry ratio (P < 0.05) in AdCon lungs at 24 h that increased further at 48 and 72 h of hyperoxia. The wet-dry weight ratio from mouse lungs infected with AdPrx also was increased at 48 and 72 h of hyperoxia, but the change was significantly less compared with AdCon (Fig. 4A).



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  		Fig. 4. The effect of AdPrx infection on lung injury with hyperoxia. Results are means ± SE (n = 4) for AdPrx and AdCon infection at varying times of exposure to 100% O2. *P < 0.05 vs. AdCon group. A: ratio of wet to dry weight of mouse lung. B: protein concentration in bronchoalveolar lavage fluid (BALF). Inset: values for 24 and 48 h of exposure with an expanded y-axis. C: nucleated cells in BALF.

 
BALF analysis. Hyperoxia resulted in an increased protein content in BALF from AdCon mice that was a function of oxygen exposure time (Fig. 4B). The number of nucleated cells showed a similar time-dependent increase (Fig. 4C). BALF protein and nucleated cells were significantly less at each time point of hyperoxia for the AdPrx compared with the AdCon group (Fig. 4, B and C).

Lung TBARS and protein carbonyls. The level of both TBARS and protein carbonyls in lung homogenates was increased significantly in the AdCon group at 24 h of hyperoxia and showed further increases at 48 h and 72 h (Fig. 5, A and B). In the AdPrx-treated group, TBARS were increased only slightly and protein carbonyls were unchanged at 24 h of 100% O2. TBARS and protein carbonyls increased at 48 h and 72 h of hyperoxia in the AdPrx group, but their levels were significantly below those for AdCon mice (Fig. 5, A and B).



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  		Fig. 5. Effect of AdPrx infection on thiobarbituric acid-reactive substance (TBARS, A) and protein carbonyls (B) in mouse lung homogenate during hyperoxia. Values are means ± SE (n = 4). *P < 0.05 vs. AdCon group.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
1-cysPrx is a recently described antioxidant enzyme member of the Prx family that is expressed at relatively high levels in the lung (14). In lung epithelial cell lines, overexpression of 1-cysPrx protein was shown to increase the ability of these cells to degrade H2O2 and to protect them against oxidant-induced plasma membrane damage (17), whereas treatment with an antisense oligonucleotide to 1-cysPrx resulted in lipid peroxidation and apoptosis (20). 1-cysPrx knockout mice showed increased sensitivity to paraquat-induced oxidative lung injury compared with wild-type mice (23). These results indicate that 1-cysPrx can function as an antioxidant enzyme, can reduce the accumulation of phospholipid hydroperoxides, and can prevent apoptotic cell death. However, the effect of 1-cysPrx overexpression on oxidative stress in intact animals has previously not been evaluated.

In the present study, we utilized exposure to hyperoxia as a mouse model of acute oxidant stress (6, 7, 11, 19). Hyperoxia is an extensively studied oxidant insult characterized by increased alveolar permeability, pulmonary edema, lung inflammation, and death (7, 18). Adenovirus-mediated delivery was used for overexpression of 1-cysPrx before O2 exposure. We have previously reported that adenoviruses can efficiently transduce the 1-cysPrx gene in a lung-derived cell line (L2) in vitro (20). Adenovirus-mediated transfer of several other genes including Na,K-ATPase subunit genes (7) or antioxidants, catalase, and SOD (6) has been shown previously to protect against hyperoxic injury. We compared the effects of pretreatment using an adenovirus vector with either a 1-cysPrx or control (LacZ or null) transgene inserted. X-gal staining after AdLacZ delivery revealed widespread {beta}-gal transgene expression distributed in both distal airway and alveoli. Alveolar type 2 cells expressed the transgene, as indicated by LacZ expression, but expression in the type 1 epithelial and endothelial cells could not be determined reliably at this level of resolution. We did not directly evaluate the expression of 1-cysPrx transgene since the available antibodies do not differentiate the native and overexpressed proteins. Adenovirus-mediated gene transfer increased lung 1-cysPrx expression relative to control infection during air breathing with a further increase during hyperoxia. We previously have shown induction of endogenous 1-cysPrx during O2 exposure (13). The change in lung 1-cysPrx expression of AdPrx-infected mice with 100% O2 was ~2.5-fold (Fig. 2) compared with our previous demonstration of ~1.5-fold induction in the lungs of normal (noninfected) mice (13). Thus overexpression of the transgene and induction of the native gene during hyperoxia appear to be additive.

We measured survival rates of mice exposed to hyperoxia to ascertain whether increased expression of 1-cysPrx in the mouse lung had an impact on the outcome from acute lung injury. We chose two models of hyperoxia. In model one, mice were exposed to 100% oxygen. In the second model, mice were exposed first to 85% O2, a dose in the middle of the lethal range of oxygen exposure, and then after 4 days to 100% oxygen. This protocol may be expected to accentuate possible differences in oxidant sensitivity of the mice. In both models, the AdPrx group has a significant survival advantage over controls. In addition, we observed that compared with AdCon, AdPrx mice showed delayed constitutional effects of O2 exposure, less pulmonary edema and pleural effusion, and lower lung lavage protein and cell count. These data indicate that overexpression of 1-cysPrx partially protected mice from the lung damage and lethality associated with oxygen exposure.

Further evidence that overexpression of 1-cysPrx protected mouse lungs in hyperoxia was obtained by measurement of biochemical indexes of oxidative lung injury. TBARS are a well-accepted marker of oxidative modification of lipids, and the presence of carbonyl groups is taken as presumptive evidence of oxidative modification of proteins (3, 5, 15, 25). In the present study, the increased lung content of TBARS and protein carbonyls in mice exposed to hyperoxia provides evidence of oxidative stress. The level of TBARS and protein carbonyls was decreased significantly in mouse lung infected with AdPrx compared with AdCon.

The issue of the proper control for these studies was considered. Because we were interested in the effect on 1-cysPrx on resistance to hyperoxia, we used adenovirus-treated mice as a control. Adenovirus, as expected, caused lung inflammation, which would be reflected in the indexes of lung injury that were measured in this study (24). Furthermore, previous studies have indicated that inflammation, including that due to viral infections of the lung, may alter sensitivity to oxidant stress (4, 9). Thus the present study indicates that 1-cysPrx overexpression can protect during hyperoxic exposure but does not indicate that delivering it by adenovirus would be effective gene therapy or that 1-cysPrx would protect uninfected lungs.

In summary, the results of this study show that adenovirus-mediated 1-cysPrx overexpression in the lung compared with a control adenovirus promotes resistance to oxidative stress. Thus 1-cysPrx plays an important role in lung antioxidant defense.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
Support was provided by National Heart, Lung, and Blood Institute Grant HL-65543 and Cystic Fibrosis Foundation Grant CFFS886 (VectorCore Component III).


   ACKNOWLEDGMENTS
 
We thank Drs. Melpo Christofidou-Solomidou and Steven Albelda for advice regarding adenovirus delivery and oxygen exposure, Kathy Notarfrancesco for assistance with histology, Lu Lu for assistance with adenovirus vectors, and Jennifer Rossi for typing the manuscript.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. B. Fisher, Inst. for Environmental Medicine, Univ. of Pennsylvania Medical Center, 1 John Morgan Bldg., Philadelphia, PA 19104-6068 (E-mail: abf{at}mail.med.upenn.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 RESULTS
 DISCUSSION
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 REFERENCES
 

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