REPORT

Effect of Adenovirus-Mediated Expression of Sonic Hedgehog Gene on Hair Regrowth in Mice With Chemotherapy-Induced Alopecia

Noboru Sato, Philip L. Leopold, Ronald G. Crystal

Affiliations of authors: N. Sato, Division of Pulmonary and Critical Care Medicine, Weill Medical College of Cornell University, New York, NY; P. L. Leopold, R. G. Crystal, Division of Pulmonary and Critical Care Medicine and Institute of Genetic Medicine, Weill Medical College of Cornell University.

Correspondence to: Ronald G. Crystal, M.D., Weill Medical College of Cornell University, Institute of Genetic Medicine, 520 East 70th St., Starr 505, New York, NY 10021 (e-mail: geneticmedicine{at}med.cornell.edu).


    ABSTRACT
 Top
 Notes
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Background: The Sonic hedgehog (Shh) gene is involved in the initiation of hair growth. We have shown that localized, transient, enhanced expression of the Shh gene in mouse skin mediated by an adenovirus (AdShh) vector accelerates initiation of the anagen (i.e., growth) phase of hair follicle development. Because hair regrowth in chemotherapy-induced alopecia is associated with follicle cell proliferation and active melanogenesis similar to that observed in the anagen phase of normal hair growth, we examined whether AdShh-mediated Shh expression would accelerate hair regrowth in the skin of mice with chemotherapy-induced alopecia. Methods: After establishment of cyclophosphamide-induced alopecia, in either 3- or 7-week-old mice, AdShh or a control vector (AdNull) was delivered to dorsal skin by intradermal injection. Hair regrowth and melanogenesis were assessed by histology and gross morphology. Fisher's exact test was used to compare differences in outcomes between AdShh-treated and control (AdNull-treated or not injected with any vector [naive]) mice. All statistical tests were two-sided. Results: Northern blot analysis confirmed enhanced Shh expression after AdShh administration in 7-week-old mice. Two weeks after AdShh administration, the injection site (all of five mice) showed large, anagen-phase hair follicles with a normal distribution of melanin. In contrast, both skin treated with AdNull (all of five mice) and skin from naive mice (all of five mice) showed dystrophic hair follicles with irregular distribution of melanin (P<.001 in both comparisons). Gross morphologic observations confirmed that AdShh-treated mice, but not naive mice or AdNull-treated mice, showed skin darkening at the injection site indicative of entry into anagen phase (P<.001 in both comparisons). AdShh treatment of 3-week-old mice with cyclophosphamide-induced alopecia was followed by accelerated hair follicle recovery (19 of 22 mice); such recovery was not observed at this rate in AdNull-treated or naive skin (P<.001 for both comparisons). Conclusion: Localized, transient, enhanced expression of Shh gene in skin, mediated by an adenovirus vector, might be a future strategy to accelerate hair follicle regrowth after chemotherapy-induced alopecia.



    INTRODUCTION
 Top
 Notes
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Hair loss, or alopecia, is a distressing side effect for individuals undergoing chemotherapy. In the United States, it is estimated that 85% of chemotherapy patients experience some degree of alopecia (16). Hair regrowth after chemotherapy can take from 3 to 6 months, and a small percentage of patients fail to experience complete recovery (4). Chemotherapy-induced alopecia is particularly devastating because it is an outward sign of an otherwise hidden disease, leading some patients to refuse systemic chemotherapy (2,6).

We hypothesized that it may be possible to accelerate recovery of damaged hair follicles given the appropriate molecular signal(s). Hair regrowth following chemotherapy-induced alopecia is associated with hair follicle cell proliferation and active melanogenesis similar to that observed in the normal anagen phase of hair growth (7,8). Among a variety of hormones, growth factors, and development-related molecules identified as being involved in normal hair follicle growth, Sonic hedgehog (Shh) expression appears to play an important role as an initiator of hair follicle growth during both fetal hair follicle development and the postnatal hair cycle (914). Shh, first identified as Hedgehog, a segment polarity gene in Drosophila development, is involved in pattern formation of vertebrate organs, including brain, heart, lung, gut, skin, and skeleton (1523). Shh functions in association with a complex signaling pathway that includes Patched 1 (Ptc 1) and Patched 2 (Ptc 2), the Shh receptor; Smoothened (Smo; a putative transmembrane G-protein coupled receptor that interacts with Ptc); Gli 1, Gli 2, and Gli 3 (a family of transcription factors); and a variety of other signaling molecules that include the Wnt and transforming growth factor-{beta} family (2244). Previously, we demonstrated that localized, transient, enhanced expression of Shh in murine postnatal skin by the intradermal administration of an adenovirus gene transfer vector expressing the mouse Shh complementary DNA (cDNA) (AdShh) accelerates the initiation of the anagen growth phase of hair follicles (12).

Since hair follicle regeneration in chemotherapy-induced alopecia is accompanied by hair follicle growth similar to that observed in the normal anagen phase of postnatal hair growth (7), we hypothesized that overexpression of Shh in skin by the intradermal administration of AdShh may accelerate hair regrowth from chemotherapy-induced alopecia. To test this hypothesis, hair regrowth following cyclophosphamide-induced alopecia was evaluated in two murine models after administration of AdShh.


    METHODS
 Top
 Notes
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Adenovirus Vectors

To induce expression of Shh, a replication-deficient adenovirus-5 (Ad5)-based recombinant adenovirus with E1 and E3 deletions in which the murine Shh cDNA was constructed with transgene expression under control of the cytomegalovirus (CMV) immediate-early promoter and enhancer (AdShh) (12). The control vector (AdNull) is structurally similar but contains no transgene (45). The preparation and titer determination of the adenovirus vectors were as described previously (46,47). All vectors used were free of replication-competent adenovirus (48).

Experimental Models

All of the animal experiments were approved by the Weill Medical College of Cornell University Institutional Animal Care and Use Committee pursuant to National Institutes of Health Guidelines. Two complementary experimental models for chemotherapy-induced alopecia were used: 1) chemotherapy-induced alopecia in 7-week-old mice that had previously undergone hair depilation (7); and 2) chemotherapy-induced alopecia in 3-week-old mice with previously clipped hair (49) (Table 1Go). In both models, the skin is in the anagen phase at the time of administration of the chemotherapeutic agent. The models differ in the mechanism of anagen phase induction. In the 7-week-old mouse model, the anagen phase is induced by a physical stimulus (depilation) (7), while in the 3-week-old mouse model, the anagen phase occurs naturally on postnatal day 28 (50). For both models, the chemotherapeutic agent was cyclophosphamide, and the AdShh or AdNull vectors were administered by intradermal injection to the mouse dorsal skin. The extent of hair follicle maturation was evaluated for histologic evidence for morphologic changes in the hair follicles in the anagen phase and for gross melanogenesis.


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Table 1. Comparison of experimental models for chemotherapy-induced alopecia in studies evaluating the effect of expression of Sonic hedgehog gene (administered through adenovirus vectors) on hair regrowth
 
The 7-week-old mouse alopecia model is based on the observation that cyclophosphamide-induced alopecia is most effective when it is administered during rapid hair growth (e.g., the anagen phase) (7). Depilation was used to induce hair growth from resting follicles in the telogen phase. The dorsal skin of the 7-week-old C57BL/6 female mice (Jackson Laboratories, Bar Harbor, ME) in the telogen phase was depilated with a wax/rosin mixture (experimental day 0) (7). When the depilated skin entered the late anagen phase at experimental day 9 after depilation (skin color change from pink to black), cyclophosphamide (freshly prepared as a stock solution at 10 mg/mL in phosphate-buffered saline [PBS] [pH 7.4]) was injected by the intraperitoneal route (125 mg/kg of body weight; Sigma Chemical Co., St. Louis, MO). By experimental day 14, the mice showed complete alopecia on the depilated dorsal skin. The Ad vectors were delivered by intradermal injection into the caudal region of the dorsal skin on experimental day 15. Skin specimens were taken on day 22 for histologic evaluation. In a second set of mice, the mouse dorsal skin was clipped and photographed on experimental day 29. The 3-week-old mouse alopecia model takes advantage of the coordinated entry of murine juvenile skin from the telogen phase into the second anagen phase during the fourth postnatal week (50). The dorsal skin of postnatal day-21 (experimental day 0) mice was clipped. On postnatal days 33–35 (experimental days 12–14), after the dorsal skin entered the late anagen phase, cyclophosphamide (100 mg/kg of body weight) was delivered by intraperitoneal injection leading to severe hair loss evident on the dorsal skin in approximately 7 days. To evaluate the effect of Shh on hair regrowth, Ad vectors (AdShh or AdNull [control vector], 5 x 108 plaque-forming units [pfu], 50 µL) were delivered by intradermal injection into the medial region of the dorsal skin 1–2 days after cyclophosphamide treatment. The decision to administer Ad vectors closer to the time of cyclophosphamide treatment in this 3-week-old mouse model compared with the 7-week-old model was derived from preliminary studies (data not shown) showing that the skin of male mice entered the anagen phase approximately 18–20 days after cyclophosphamide treatment (experimental days 29–35). To provide 14 days to observe the gross morphologic effects of AdShh administration (12), the vector was delivered within 4 days of cyclophosphamide treatment. For gross morphologic examination, mice were graded as positive for anagen phase induction if an area of skin 3 mm or greater in diameter at the site of the injection was distinctly darkened relative to the adjacent skin at 20 days after Ad vector administration. Specimens were taken 14 days after vector administration for histologic evaluation. Because of prior reports of sex-specific differences in the response of the 3-week-old mouse model to chemotherapeutic agents (49), male and female mice were evaluated separately.

Histologic Analysis

Skin was harvested, fixed in 4% paraformaldehyde in PBS (pH 7.4) and embedded in paraffin. Transverse 5-µm paraffin sections were stained with hematoxylin–eosin. The specimens were graded as positive for anagen phase according to the following criteria: 1) thickened dermis, 2) hair follicle invagination deep into the adipose tissue layer, and 3) evidence of melanin synthesis in the hair follicle matrix.

Gene Expression

Northern blotting in the 7-week-old model was carried out with samples from mice on experimental day 18 (day 3 after Ad vector injection) by purifying total RNA from dorsal skin taken from the area of injection (1 cm x 1 cm) by use of the TrizolTM RNA preparation kit (Life Technologies, Inc. [GIBCO BRL], Rockville, MD). Isolated RNA (10 µg/lane) was separated on a 1% agarose gel, transferred to a nylon membrane (Stratagene, La Jolla, CA) and hybridized with 32P-deoxycytidine triphosphate (dCTP)-labeled probes synthesized from cDNA by random hexamer priming (Stratagene). After detection of target RNA, filters were stripped with 0.1 x saline-sodium citrate at 95 °C and re-probed with 32P-dCTP labeled probe against glyceraldehyde phosphate dehydrogenase to ensure equal RNA loading.

Statistical Methods

Statistical evaluations employed Fisher's exact test. All P values were two-sided.


    RESULTS
 Top
 Notes
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Chemotherapy-Induced Alopecia in 7-Week-Old Mice

Administration of cyclophosphamide to 7-week-old mice after depilation reliably resulted in alopecia on the depilated dorsal skin within 5 days after cyclophosphamide injection. No alopecia occurred in mice receiving only depilation, only cyclophosphamide injection, or no treatment (data not shown). The presence of alopecia was supported by histologic examination. Mouse skin was harvested 6 days after cyclophosphamide injection when all mice with depilation showed complete alopecia on the dorsal skin (data not shown). Mouse skin without treatment showed hair follicles in the telogen phase. Similarly, skin of mice without depilation, but injected with cyclophosphamide, showed hair follicles in the telogen phase without obvious tissue damage. Depilated mouse skin without cyclophosphamide injection showed growing hair follicles, indicating entry into the anagen phase. In contrast, the mouse skin treated with both depilation and cyclophosphamide showed remarkably dystrophic hair follicles and an irregular pattern of melanin deposition resulting from damage to melanocytes (data not shown).

Hair Regrowth in the 7-Week-Old Mouse Alopecia Model

In the 7-week-old mouse alopecia model, hair follicles in mouse dorsal skin typically recover normal morphology approximately 20 days after cyclophosphamide treatment (7). The potential of Shh to initiate hair follicle regeneration was evaluated by administration of AdShh to the skin of mice on experimental day 15, 6 days after cyclophosphamide treatment. To evaluate Shh gene expression in the skin after the intradermal administration of an adenovirus vector encoding Shh (AdShh), mouse skin was harvested on experimental day 18, 3 days after adenovirus vector injection. RNA was extracted and evaluated by Northern blot analysis with the use of a mouse Shh probe. Compared with controls, including naive mouse skin or skin injected with an adenovirus vector lacking an expression cassette (AdNull), the skin injected with AdShh showed a strong vector-derived Shh expression of 3.0 kilobases, as described previously (Fig. 1Go) (12). Consistent with the data, expression of the endogenous Patched gene (the Shh receptor gene) was increased by AdShh.



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Fig. 1. Expression of Sonic hedgehog (Shh) messenger RNA and Patched1 (Ptc) gene (Ptc gene expresses a protein that acts as a receptor for Shh protein) messenger RNA in mouse skin with alopecia after local administration with an adenovirus vector expressing Shh (AdShh). Seven-week-old C57BL/6 mice were depilated (experimental day 0). Cyclophosphamide (125 mg/kg) was injected intraperitoneally into the mice on experimental day 9. On experimental day 15, AdShh or an adenovirus vector lacking the transgene (AdNull) (5 x 108 plaque-forming units in 50 µL each) was injected intradermally. Total RNA was isolated from the skin on day 18 after depilation (day 3 following vector treatment), and evaluated by Northern blot analysis. Glyceraldehyde-phosphate dehydrogenase (GAPDH) messenger RNA was evaluated as a control for RNA loading. kb = kilobases.

 
Natural hair regrowth after the complete alopecia on the dorsal skin began as early as day 9 after cyclophosphamide injection (experimental day 18) in this model. Previous experiments have shown that macroscopic effects mediated by the administration of AdShh (e.g., skin color change resulting from active melanogenesis) in normal mouse skin were observed 10–14 days after injection (12). Administration of AdShh by intradermal injection was performed in the caudal region of the mouse back so that the AdShh-mediated effect was not masked by the naturally occurring hair regrowth that proceeds from anterior to posterior (7,51). Control skin showed deformed catagen or telogen hair follicles with an abnormal distribution of melanin granules resulting from destruction of melanocytes (Fig. 2, A and BGo) (7,52). In contrast, the AdShh-injected skin showed large hair follicles in anagen phase that extended deep into the dermis accompanied by a regular distribution of melanin granules within the hair follicles at the injection site (Fig. 2, CGo). Areas outside the injection site showed damaged hair follicles typical of the morphology observed in control skin. Consistent with the histologic observations, neither naive nor AdNull-injected skin developed an obvious skin color change at the site of injection 29 days after depilation (Fig. 2, D and EGo). In contrast, AdShh-injected skin showed a remarkable darkening in skin color at the injection site (Fig. 2, F and GGo).



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Fig. 2. Accelerated hair follicle growth after intradermal administration of adenovirus vector expressing the Sonic hedgehog gene (AdShh) in chemotherapy-induced alopecia induced in 7-week-old mice. Mice were depilated and treated with cyclophosphamide as described in Fig. 1Go. AdShh or control vector lacking transgene (AdNull) (5 x 108 plaque-forming units) was injected intradermally into the skin on day 15 after depilation. Histologic analysis was performed on day 22 after depilation, and gross morphologic analysis was performed on day 29 after depilation. A–C) Histology. A) Mouse skin without vector treatment. B) AdNull-treated mouse skin. C) AdShh-treated mouse skin. Bar in C = 200 µm. D–G) Gross morphology. D) Naive mice. E) AdNull-treated mice. F and G) AdShh-treated mice.

 
Histologic analysis on experimental day 22 showed that the onset of the anagen phase was accelerated at the site of injection in all of the mice (five of five) injected with AdShh, while none of the naive mice (none of five) or AdNullinjected mice (none of five) showed acceleration of the hair-growth phase (P<.001) comparing AdShh-treated mice with either AdNull-treated mice or naive mice. In addition, intradermal administration of AdShh induced macroscopic melanogenesis in all the mice tested at both the histologic and macroscopic levels. Macroscopic observations on experimental day 29 in naive or AdNull-injected mice showed no melanogenesis at the site of injection (naive, 0 of 9; AdNull, 0 of 9; AdShh, 9 of 9 [P<.001]) comparing AdShh-treated mice with either AdNull-treated mice or naive mice by Fisher's exact test.

Hair Regrowth in the 3-Week-Old Mouse Alopecia Model

To explore further the effects of AdShh on chemotherapy-induced alopecia, we examined a second alopecia model by using younger mice (3 weeks old). In this model, both male and female mice were evaluated, since a previous report that had used the same model had noted genderspecific responses (49). Mice were clipped on postnatal day 21 (experimental day 0) to aid in observing the skin color change that is associated with the telogen-to-anagen phase transition. Cyclophosphamide was administered during the second anagen phase leading to alopecia after approximately 1 week. AdShh was intradermally injected into the dorso-medial skin. A dorso-medial injection site, rather than a posterior injection site, was used because the natural hair regrowth observed in this model did not interfere with the ability to observe the effects of AdShh as was the case in the 7-week-old mouse model.

Similar to the 7-week-old mouse model, the naive and AdNull-injected skin showed dystrophic hair follicles with an extracellular distribution of melanin granules on day 27 (Fig. 3, A and BGo). In contrast, AdShh-injected skin contained larger growing hair follicles with a regular distribution of melanin granules in matrix cells of anagen phase hair follicles at the injection site (Fig. 3, CGo). Gross morphologic analysis correlated with histologic findings (Fig. 3, D–GGo). The occurrence of melanogenesis and anagen phase hair follicles at the injection site was specific to AdShh-treated mice. With follow-up on experimental day 33, both male and female mice responded to AdShh treatment (melanogenesis observed: all mice— naive [none of 22], AdNull [none of 22], and AdShh [19 of 22; P<.001]; mice by sex—naive [none of 12 male mice and none of 10 female mice]; AdNull [none of 12 male mice and none of 10 female mice]; AdShh [11 of 12 male mice and eight of 10 female mice; P<.001 comparing male AdShh-treated mice with either male AdNull-treated mice or male naive mice and P<.001 comparing female AdShh-treated mice with either female AdNull-treated mice or female naive mice by Fisher's exact test]). Although there was no significant difference in the response of male versus female mice to AdShh administration (P = .27), the intradermal administration of AdShh appeared to induce onset of anagen phase more rapidly in male mice compared with female mice. At experimental day 27, the majority of AdShh-treated male mice showed melanogenesis at the injection site, while only 10% of the female mice had responded.



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Fig. 3. Accelerated hair follicle growth after intradermal administration of adenovirus vector expressing the Sonic hedgehog gene (AdShh) in 3-week-old mice. In the 3-week-old mouse model, the skin enters the anagen phase in a highly predictable manner in the fourth postnatal week, so depilation is not necessary to induce anagen phase. To clearly observe the telogen-to-anagen phase transition, the skin was clipped to remove existing hair at postnatal day 21 (experimental day 0). After the anagen phase occurred, cyclophosphamide (100 mg/kg intraperitoneally) was administered (postnatal days 33–35; experimental days 12–14). To evaluate the effect of AdShh administration, AdShh or AdNull (5 x 108 plaque-forming units) was injected intradermally 1–2 days after cyclophosphamide treatment. Histologic evaluation was performed 14 days after vector administration. Gross morphologic analysis was performed on day 14 after vector administration. A–C) Histology. A) Mouse skin without vector treatment showing dystrophic hair follicles. B) AdNull-treated mouse skin showing dystrophic hair follicles. C) AdShh-treated mouse skin showing large growing hair follicles. Bar in C = 200 µm. D–G) Gross morphology. D) Naive mice. E) AdNull-treated mice. F and G) AdShh-treated mice.

 

    DISCUSSION
 Top
 Notes
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
This study demonstrates that localized, enhanced, transient expression of Shh in mouse skin accelerates hair follicle regrowth after chemotherapy-induced alopecia in mice. In general, strategies to develop treatments for patients experiencing chemotherapy-induced alopecia have taken two distinct routes: prevention of hair loss and promotion of hair recovery. Efforts to prevent hair loss after the administration of chemotherapeutic agents include physical methods, such as hypothermic treatment of the scalp, that seek to decrease adverse effects by slowing cellular metabolism and decreasing local blood flow (1,35,53). Biologic strategies, including administration of minoxidil, ImuVert (a biologic response modifier extracted from the bacterium Serratia marcescens), interleukin 1, and keratinocyte growth factor, prevent chemotherapy-induced alopecia in mice after treatment with cytarabine, but these strategies fail to prevent hair loss induced by cyclophosphamide (35,5457). 1,25-Dihydroxyvitamin D3 has been reported to show protection in experimental cyclophosphamide-induced alopecia, leading to a phase I trial (52,58). Cyclosporine A also shows protection in a similar experimental model but may be dangerous to use in humans in the oncology clinical setting, since cyclosporine A might promote cancer metastases (59,60). A promising report focused on a new class of drugs that inhibit cyclin-dependent kinases (61). The inhibitors prevented hair loss after administration of different chemotherapy regimens into two mouse models and may well contribute to prevention of hair loss in patients, provided that this new class of drugs has an appropriate safety profile. As efforts to prevent chemotherapy-induced alopecia improve, strategies to accelerate recovery of hair follicles are also progressing. Topical minoxidil reduced the period of alopecia in individuals undergoing chemotherapy (62). Similarly, 1,25-dihydroxyvitamin D3 improved hair regrowth after chemotherapy-induced tissue damage (52). The present study utilizes Shh, a secreted protein with signaling capability that functions as part of the normal mechanism for controlling the hair growth cycle. Ultimately, a combination of treatments that prevent hair loss and accelerate hair follicle recovery may prove to be the most effective for chemotherapy patients.

In addition to Shh, localized, transient expression of other signaling molecules may also aid in regeneration of hair follicles after chemotherapy-induced alopecia. Secreted growth factors and morphogens, such as keratinocyte growth factor (54,63), fibroblast growth factor 5 (50,64), transforming growth factors-{alpha} (65), epidermal growth factor (66), and hox gene products (67), have been implicated in control of the hair follicle growth cycle. An in vitro experiment using isolated mouse dermal papilla showed that Wnt 3a, a secreted signaling molecule, induced anagen phase with subsequent hair growth after implantation in mouse skin (68). Given the ability of these gene products to control downstream gene expression, incorporation of the genes into treatments must be approached with caution. For example, constitutive expression of Shh can induce atypical morphologies in mouse and human skin that resemble basal cell nevus syndrome (69,70). In contrast, transient expression of Shh failed to produce the same pathology (12). Therefore, the timing and extent of gene expression may be critical, and the ability to achieve local, transient expression of the genes by using vectors like adenovirus may be essential to achieve constructive biologic activity of the gene products.

The present study compared Shh-induced hair follicle recovery in male and female mice. A previous report suggested that 1,25-dihydroxyvitamin D3-mediated protection against chemotherapy-induced hair loss was less effective in the female mice than in the male mice, possibly related to P450-mediated metabolism of 1,25-dihydroxyvitamin D3 (71,72). In addition, activation of estrogen receptors in the dermal papilla may slow progression into anagen phase, introducing another sex-specific variable in hair cycle biology (73), although no direct link between Shh and 1,25-dihydroxyvitamin D3 or estrogen signaling is postulated here. The differences may relate to the rate of hair follicle recovery rather than to the ability to recover. These data suggest that therapeutic intervention after chemotherapy may exhibit sex-specific pharmacokinetics.

In conclusion, the Shh signaling pathway is involved in the initiation of the hair follicle growth phase during hair follicle regeneration after chemotherapy-induced tissue injury. The present data support the idea that the Shh signaling pathway acts as a biologic switch that accelerates the initiation of the hair growth phase and that the use of transient, localized gene transfer may prove to be a useful technology for elucidating general mechanisms controlling hair regeneration in a variety of disease states.


    NOTES
 
Supported in part by Public Health Service grant R01AR46282–01A1 from the National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Department of Health and Human Services; by the Will Rogers Memorial Fund, Los Angeles, CA; and by GenVec, Inc., Gaithersburg MD.

We thank Neil R. Hackett for helpful discussions and N. Mohamed for help in preparing this manuscript.


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 Abstract
 Introduction
 Methods
 Results
 Discussion
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Manuscript received December 20, 2000; revised August 29, 2001; accepted October 9, 2001.


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