Published online before print November 11, 2004, doi: 10.1161/01.RES.0000150373.15149.ff
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© 2004 American Heart Association, Inc.
Integrative Physiology |
Molecular Imaging of Atherosclerotic Plaques Using a Human Antibody Against the Extra-Domain B of Fibronectin
From the Cardiovascular Research, Institute of Physiology (C.M.M., P.K.S., P.M., R.R., D.Z., T.F.L.), University of Zurich and Cardiovascular Center, Zurich University Hospital, Switzerland; Department of Chemistry and Applied Biosciences (P.A., C.H., D.N.), Swiss Federal Institute of Technology, Zurich, Switzerland; Laboratory of Cell Biology (P.C.), Istituto Nazionale per la Ricerca sul Cancro, Genoa, Italy; Unit of Innovative Therapies (L.Z.), Department of Experimental and Clinical Immunology, Istituto Giannina Gaslini, Genoa, Italy; Institute of Anaesthesiology (C.K.H.), Triemli City Hospital, Zurich, Switzerland; and the Institute of Pathology (M.M.), Zurich University Hospital, Switzerland.
Correspondence to Dario Neri, Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology, Wolfgang-Pauli Strasse 10, CH-8093 Zurich, Switzerland. E-mail neri{at}pharma.ethz.ch
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Abstract |
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Current imaging modalities of human atherosclerosis, such as angiography, ultrasound, and computed tomography, visualize plaque morphology. However, methods that provide insight into plaque biology using molecular tools are still insufficient. The extra-domain B (ED-B) is inserted into the fibronectin molecule by alternative splicing during angiogenesis and tissue remodeling but is virtually undetectable in normal adult tissues. Angiogenesis and tissue repair are also hallmarks of advanced plaques. For imaging atherosclerotic plaques, the human antibody L19 (specific against ED-B) and a negative control antibody were labeled with radioiodine or infrared fluorophores and injected intravenously into atherosclerotic apolipoprotein E–null (ApoE–/–) or normal wild-type mice. Aortas isolated 4 hours, 24 hours, and 3 days after injection exhibited a selective and stable uptake of L19 when using radiographic or fluorescent imaging. L19 binding was confined to the plaques as assessed by fat staining. Comparisons between fat staining and autoradiographies 24 hours after 125I-labeled L19 revealed a significant correlation (r=0.89; P<0.0001). Minimal antibody uptake was observed in normal vessels from wild-type mice receiving the L19 antibody and in atherosclerotic vessels from ApoE–/– mice receiving the negative control antibody. Immunohistochemical studies revealed increased expression of ED-B not only in murine but also in human plaques, in which it was found predominantly around vasa vasorum and plaque matrix. In summary, we demonstrate selective targeting of atheromas in mice using the human antibody to the ED-B domain of fibronectin. Thus, our findings may set the stage for antibody-based molecular imaging of atherosclerotic plaques in the intact organism.
Key Words: vascular targeting • angiogenesis • apolipoprotein E–null mice • near infrared
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Introduction |
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X-ray–based contrast angiography visualizes the vessel wall indirectly, allowing only a quantification of luminal stenosis. More information about the architecture of the vessel wall has been gained with the addition of intravascular ultrasound (IVUS).1 Novel, experimental imaging techniques, such as thermography2 and optical coherence tomography,3,4 provide additional insight into vessel function and morphology, respectively. However, all these techniques are invasive and provide exclusively morphological or functional imaging without giving details about the molecular components of the vessel wall.
Major advances in the field of noninvasive plaque imaging have been obtained in recent years with ultrafast computed tomography (CT)5 and MRI.6 Although these techniques have further enriched our tools for morphological plaque imaging, our means to visualize plaques at the molecular level are still sparse.
Atherosclerosis is recognized as a chronic inflammatory disease7 with a prominent angiogenic component, leading to the proliferation of vasa vasorum that perfuse the plaque.8 Imaging of plaque-associated angiogenesis is of considerable interest as rupture of vasa vasorum appears to be directly related to plaque instability and sudden cardiac death.9 In line with this concept, treatment of atherosclerotic mice with antiangiogenic compounds such as endostatin or fumagillin resulted in decreased plaque progression.10
The extra-domain B (ED-B) of fibronectin is one of the best characterized markers of tissue remodeling and angiogenesis.11,12 This 91-aa domain, the sequence of which is conserved from mouse to man,13 is normally absent in fibronectin but can be inserted into the fibronectin molecule by a mechanism of alternative splicing of the primary transcript. Because of the sequence conservation of ED-B throughout evolution, it has been impossible so far to raise monoclonal antibodies to this antigen by hybridoma technology.14 However, in the recent past, we isolated a number of anti–ED-B human monoclonal antibodies using synthetic antibody libraries, which were biopanned using phage display methodologies11,15,16 or iterative colony filter screening.17 In particular, the human antibody L19 (specific against ED-B), which displays a high binding affinity to ED-B,16 has been shown to selectively target neovasculature in animal models of angiogenesis-related diseases18 and in patients with cancer.19 The tumor-targeting properties of the L19 antibody have been characterized by quantitative biodistribution analysis for a variety of antibody formats, for example, as monomeric scFv (single-chain Fv) antibody fragment, as noncovalent homodimeric scFv fragment, as miniantibody (or small immune protein [SIP]) in which the scFv moiety is fused to a CH4 domain of a human IgE serving as dimerization domain,20 and as full IgG.18 Furthermore, several derivatives of the scFv(L19) antibody fragment have been shown to target neovasculature in animal models with promising therapeutic results.21,22
In our study, we investigated the ability of the L19 miniantibody, hereafter termed SIP(L19), recombinant miniantibody (SIP) format of L19, to image plaques in atherosclerotic mice using antibody labeling with radioiodine or near-infrared fluorophores and evaluated the expression of fibronectin ED-B in murine and human plaques.
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Materials and Methods |
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Antibodies
Cloning and purification of the L19 antibody in miniantibody format has been described previously.23 The cloning of the HyHEL-10 antibody in miniantibody format was achieved by appending the gene coding for the human CH4 domain of an IgE to the C terminus of scFv (HyHEL-10).24 The CH4 domain was amplified by polymerase chain reaction (PCR) with primers CtermHyHel10SacIICH4 (5'-GAAATAAAACCGCGGGGCTCTGGGGGCCCGCGTGCT–3'), which anneals to the 3'-end of the HyHEL-10 sequence and appends a restriction site for the endonuclease SacII, and the primer CtermCH4EcoRI (5'-CTGGAATTCGAGCTCGGTACCTAGCAGCCACCC–3'), which anneals to the 3' of the CH4 and appends a restriction site for the endonuclease EcoRI. The sequence of the CH4 was then assembled to scFv (HyHEL-10) by PCR with the pull-through oligonucleotides HindIII-SIP (5'-CTGAAGCTTGTCGACCATGGGCTGGAGCC–3'), which anneals to the 5' of the sequence of the signal secretion leader peptide of the scFv and introduces a restriction site for the endonuclease HindIII and CtermCH4EcoRI. As a result of this strategy, the scFv and the CH4 moieties were joined by a linker with sequence GGSG. The gene coding for the miniantibody was cloned into the HindIII and EcoRI sites of plasmid pcDNA3.1 (Invitrogen) and used to obtain stable transfectants in human embryonic kidney 293 cells (American Type Culture Collection). The miniantibody HyHEL-10 was purified from the culture supernatant by affinity chromatography using lysozyme-Sepharose resin as described.24 For autoradiography studies, L19 and HyHEL-10 were radiolabeled with iodine-125 as described,23 and 10 µg of the resulting radiolabeled proteins (10 µCi) were injected in mice from the tail vein. For fluorescence microscopy studies, antibodies were labeled with Cy5-NHS (Amersham Pharmacia) as described,25 yielding an antibody–dye molar ratio of 1:5. A total of 100 µg of Cy5-labeled antibody were injected in the tail vein. For fluorescence in in situ and ex vivo studies, the antibody L19 was labeled with the infrared fluorophore Cy7-NHS (Amersham Pharmacia) as described,26 with protein:Cy7 ratios of 2:1. A total of 100 µg of Cy7-labeled antibody was injected into the tail vein. After 24 hours, mice were imaged using a home-built infrared fluorescence imager with exposure times between 0.5 and 2 seconds.26
Animals
Plaques were analyzed in male atherosclerotic apolipoprotein E knockout (ApoE–/–) mice27 (C57BL/6J background) fed a high-cholesterol diet (1.25% total cholesterol; RD12108 from Research Diets) for 2 months starting at 8 weeks of age. Corresponding wild-type male C57BL/6J mice on normal chow were used as negative controls. All animal experiments were performed in accordance with institutional guidelines and approved by the local animal committee.
Harvest and Tissue Processing
Animals were euthanized 4 hours, 24 hours, and 3 days after injection of the antibody. Each animal experiment was performed in triplicates and repeated at least 2x (n
6 animals for each time point). Briefly, after puncture of the left ventricle, vessels were rinsed with PBS followed by pressure fixation at 100 mm Hg using 4% paraformaldehyde in PBS for 4 minutes. The whole aorta was then excised. For en face analysis, the aorta was opened longitudinally and postfixed in paraformaldehyde for 2 hours. For analysis of cross-sections, vessels were postfixed in paraformaldehyde for 2 hours and then embedded in OCT compound (Tissue-Tek).
For immunohistochemical analyses of ED-B expression in normal and atherosclerotic murine and human arteries (n=3 for each), vessels were embedded in OCT compound without previous fixation. Human tissue samples were obtained from the dilated atherosclerotic abdominal aorta of a patient who underwent aneurysm repair. Additional human tissue from plaque-bearing coronary and iliac arteries was obtained from autopsies within 24 hours after patient death. Samples of normal internal mammary arteries were retrieved from patients <70 years of age who underwent coronary artery bypass grafting.
Tissue Analysis
Plaque imaging was performed in 3 dimensions using at least 5 serial cross-sections (5-µm thickness) of the proximal aorta combined with en face analyses of the descending and abdominal aorta. Plaque area was visualized by fat staining (Oil red O) and photographed with a digital camera (Olympus C-4040 Zoom; spatial resolution 4.1 megapixels) mounted on an Olympus SZX9 microscope. Nuclei were identified by 4'-6-diamidino-2-phenylindole (DAPI) staining. Endothelial cells were identified by anti-von Willebrand Factor (A0082; DAKO). Anti-rabbit Alexa Fluor 488 (Molecular Probes) was used as a secondary antibody. Immunohistochemical detection of ED-B fibronectin was performed using SIP(L19) at a concentration of 0.06 µg/mL as described previously.23 At least 5 serial sections (5-µm thickness) of 3 different tissue samples from each source were analyzed. After exposing longitudinally opened aortas overnight, autoradiographic imaging (Phosporimager 400B; Molecular Dynamics; spatial resolution set at 88 µm) was used for detection of antibodies labeled with 125I. Signal/noise ratio in autoradiographies of longitudinally opened aortas was determined using linear integration of signal intensity (ImageQuaNT) over corresponding areas of aortas taking the mean of 3 measurements. Comparisons between fat staining and corresponding autoradiography 24 hours after injection of 125I-labeled SIP(L19) were determined after converting color images into black and white, tracing all positive areas (Analysis 3.2 software; SoftImaging System) and correlating percentage of positive out of total vessel areas using linear regression. Macroscopic near-infrared imaging was performed using a home-built infrared fluorescence imager and a CCD camera (C5985; Hamamatsu; 800x600 pixels, spatial resolution 
125 µm), with exposure times between 0.5 and 2 seconds.26 Signal/noise ratio in fluorescence images of longitudinally opened aortas was determined using linear integration of signal intensity (NIH Image 1.63f software) over corresponding areas of aortas, taking the mean of 3 measurements. Correlations between fat staining and corresponding fluorescence 24 hours after administration of Cy7-labeled SIP(L19) were determined as described above for autoradiography. Confocal microscopy (Leica DM IRBE with Confocal Software 97 to 03) was performed to detect the Cy5-linked antibody in aortic cross-sections.
Statistics
Correlations between fat staining and L19 uptake as assessed by autoradiography or fluorescence were performed by using linear regression (GraphPad Prism V4). Values are given as mean or mean±SD, respectively.
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Labeling of Antibodies With Radioiodine or Fluorescent Dyes
For plaque targeting studies, SIP(L19) and SIP(HyHEL-10), recombinant miniantibody HyHEL-10 (directed against hen egg lysozyme), were purified to homogeneity and labeled either with radioiodine (125I) or near-infrared fluorescent dyes. We checked the purity of the antibody preparations by gel electrophoresis, confirming that the recombinant miniantibodies were able to form disulfide-linked covalent homodimers when analyzed in nonreducing conditions (Figure 1). The fluorophore Cy5 was chosen for confocal laser microscopic fluorescence analysis. The related fluorescent dye Cy7 was used for near-infrared imaging applications because its light absorption and emission properties provide a more favorable tissue penetration with reduced background.
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The Anti–ED-B Miniantibody SIP(L19) Selectively Targets Murine Plaques
To assess plaque targeting ability, we labeled the specific miniantibody SIP(L19) and the control antibody SIP(HyHEL-10) with 125I and injected it in equal amounts (10 µg, 10 µCi) into the tail vein of wild-type mice with normal vessels or ApoE–/– mice with plaques. The mouse aortas were harvested at different time points, opened longitudinally, imaged by autoradiography, and stained by Oil red O. Wild-type mice without plaques showed no preferential antibody uptake in the aorta, whereas ApoE–/– mice revealed a strong and selective uptake of L19 in plaques already at 4 hours (Figure 2a). Aortic autoradiographies obtained with the specific L19 or the control HyHEL-10 miniantibodies at 4 hours, 24 hours, and 3 days after injection demonstrated that only L19 showed a strong and selective uptake in atherosclerotic plaques that remained elevated for at least 3 days (Figure 2b). The percentage of injected antibody (dose per gram of blood) obtained by gamma counter analysis of blood specimens was 0.58±0.19 for L19 and 0.26±0.22 for HyHEL-10 at 24 hours, indicating a similar clearance profile. In addition, the L19 miniantibody revealed an excellent signal/noise ratio for detecting atherosclerotic plaques using the region-of-interest integration of autoradiographic data. Indeed, the plaque/normal vessel ratios were 17:1 at 4 hours, 27:1 at 24 hours, and 105:1 at 3 days for SIP(L19); in contrast, the corresponding values for SIP(HyHEL-10) were <6:1, <5:1, and <3:1, respectively.
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For quantifying the ability of SIP(L19) to bind to atherosclerotic plaques, we analyzed longitudinally opened aortas from ApoE–/– mice that received an intravenous injection of 125I-labeled SIP(L19) 24 hours before harvesting. En face autoradiographic signals and fat stainings were traced after converting them to black-and-white images. Comparisons of these corresponding signals in four different animals using linear regression (Figure 2c) demonstrated a significant correlation (r=0.89; P<0.0001; y=0.96x–0.05).
To characterize the plaque structures targeted by the L19 miniantibody in more detail, we injected ApoE–/– mice with Cy5-labeled SIP(L19). Subsequent analyses of aortic cross-sections showed that SIP(L19) targeted portions of atherosclerotic plaques, which colocalized with von Willebrand staining (Figure 3a through 3c). In other plaques, we observed the above-mentioned staining pattern together with targeting of plaque matrix adjacent to endothelial cells (Figure 3d through 3f). In wild-type mice with normal arteries, no accumulation of SIP(L19) was detectable using the same experimental conditions (Figure 3g and 3h). Together, these findings demonstrated specific targeting of the SIP(L19) miniantibody to atherosclerotic plaques in ApoE–/– mice.
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Cy7-Labeled SIP(L19) Allows Macroscopic Fluorescence Detection of Murine Plaques
Because of its high sensitivity and resolution, near-infrared fluorescence imaging is likely to be the technique of choice for detecting plaques in small vessels such as human coronary arteries.28 Accordingly, we linked L19 to the near-infrared fluorophore Cy7 and injected it into ApoE–/– mice. Twenty-four hours after injection of Cy7-labeled SIP(L19), plaques could be visualized in animals by epi-illumination after removing major organs (Figure 4a). Subsequent excision and longitudinal opening of aortas confirmed an intense and selective signal (Figure 4c) corresponding to atherosclerotic plaques (Figure 4d). The signal/noise ratio 24 hours after Cy7-labeled SIP(L19) was excellent with a plaque/normal vessel ratio of 18:1. In contrast, aortic plaques from ApoE–/– mice injected with saline (Figure 4b, 4e, and 4f) displayed no relevant autofluorescence (Figure 4b and 4f). Comparisons between corresponding en face fluorescence signals and fat stainings in three different animals using linear regression (Figure 4g) revealed a highly significant correlation (r=0.83; P<0.0005; y=0.80x–3.72). These results showed that labeling of L19 with a near-infrared fluorophore provided macroscopic fluorescence detection of murine atherosclerotic plaques.
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ED-B Domain of Fibronectin Is Highly Expressed in Murine and Human Plaques
To investigate the expression of ED-B in normal and diseased murine and human vessels, we performed immunohistochemical stainings of L19. The expression of ED-B–containing fibronectin was minimal in normal aortas from wild-type mice (Figure 5a). However, we found a markedly enhanced expression in plaques from ApoE–/– mice (Figure 5b). Similar findings were observed in healthy and atherosclerotic human arteries. Indeed, normal internal mammary arteries showed virtually no ED-B expression (Figure 5c), whereas sections through human atheroma from an abdominal aortic aneurysm revealed high levels of ED-B (Figure 5d). Specifically, we observed prominent ED-B expression around vasa vasorum as well as in plaque matrix. We detected a similar staining pattern in sections of human atherosclerotic coronary and iliac arteries obtained at autopsy. ED-B was only minimally expressed in a disease-free area of a plaque-bearing coronary artery (Figure 5e), whereas on the same section, the plaque and the underlying media revealed marked overexpression of ED-B (Figure 5f).
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These immunohistochemical stainings revealed a markedly enhanced expression of the ED-B domain of fibronectin in murine and human atherosclerotic plaques compared with minimal ED-B expression in normal vessels.
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Discussion |
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In clinical practice, our tools for imaging atherosclerosis, such as angiography, ultrasound, and CT, still rely on morphological criteria. To visualize plaques at the molecular level, targeting agents such as specific antibodies or low-molecular weight contrast agents can be applied.
In this study, we demonstrate that SIP(L19), a human recombinant antibody specific against the ED-B domain of fibronectin, is able to selectively target atherosclerotic plaques in ApoE–/– mice using radioiodine or fluorescent dyes.
Particularly, our findings demonstrate a good signal/noise ratio and a high specificity of the antibody. The signal/noise ratio in isolated, atherosclerotic aortas ranged from 17:1 at 4 hours to 105:1 at 3 days after radioiodine-labeled antibody injection. This improvement in signal/noise ratio over time reflects the time the antibody needs for reaching its target and engaging a specific binding. A similar signal/noise ratio was obtained 24 hours after administration of the antibody labeled with a near-infrared fluorophore. The highly significant correlations between autoradiographic or fluorescence signals and fat staining 24 hours after antibody injection further support the specificity of the L19 antibody for plaque recognition.
Our study indicates that the L19 antibody can be used to deliver either radionuclides or infrared fluorophores to atherosclerotic plaques in vivo. Therefore, these findings open novel opportunities for radioactive plaque imaging (eg, by positron emission tomography [PET]) or for near-infrared fluorescence detection of atherosclerosis.
Plaque imaging using PET has been reported previously by Dinkelborg et al, who have demonstrated a selective uptake of technetium (99mTc)-labeled endothelin derivatives in plaques of Watanabe rabbits.29 However, this study did not address the specificity of their approach by testing a labeled unspecific derivative. Relying on the increased metabolic activity of inflammatory cells within the advanced plaque, application of 18F-fluorodeoxyglucose (FDG) has been shown to image human carotid plaques in patients.30 Very recently, this approach has been further validated in Watanabe rabbits where increased 18F-FDG uptake in atherosclerotic lesions correlated with the number of plaque macrophages.31 Furthermore, 99mTc-labeled annexin V has been reported recently to target apoptotic macrophages in experimental atheroma.32 Thus, PET imaging is attractive being a noninvasive approach, but its spatial resolution down to few millimeters may exhibit limitations for plaque imaging. In addition, for using L19-based PET imaging in the clinical setting, the antibody blood clearance profile would impose image acquisitions at late time points with critical consequences for the radioprotection of patients.
Therefore, near-infrared imaging appears to be a more promising modality for clinical applications because of its high sensitivity and resolution.28 Recent advances in the miniaturization of endoscopes33 suggest that it should be possible to apply intravascular near-infrared fluorescence imaging for the detection of plaques, even in small vessels such as human coronary arteries. Such angioscopic imaging modalities would provide molecular information about plaque biology and may be competitive with IVUS imaging techniques in terms of resolution, sensitivity, and real-time imaging.
An elegant approach of noninvasive, near-infrared–based plaque imaging has been reported recently by Weissleder’s group: Taking advantage of the combination of MRI and diffuse optical tomography,34 fluorescence-mediated tomography visualized noninvasively proteolytic activities within murine plaques.35 The favorable image resolution of near-infrared imaging makes this approach an attractive experimental research tool.28 However, the limited penetration depth of infrared light of 10 cm may limit its noninvasive application in patients to relatively superficial structures. Another MRI-based molecular imaging approach has been described recently using nanoparticles coated with an anti-
vß3 integrin peptidomimetic in rabbits.36 This modality is attractive, and the 2-fold signal enhancement in rabbit lesions is remarkable, but it remains to be seen whether it can be translated into a useful application in patients.
Beyond its diagnostic applications, L19 offers promising therapeutic opportunities on the basis of the selective delivery of bioactive molecules to the target tissue. In contrast to the above-mentioned low-molecular weight contrast agents used by other groups, recombinant antibodies such as L19 allow the construction of a variety of derivatives, either by chemical modification or as fusion proteins. Indeed, several L19 derivatives have been produced and tested in tumor animal models. They include fusion proteins with cytokines,22 vascular endothelial growth factor,37 procoagulant factors,38 as well as conjugates with photosensitizers.21 These antibody derivatives are available for testing therapeutic applications in animal models of atherosclerosis. If successful in animals, anti–ED-B targeted therapeutic strategies may be of considerable clinical relevance given the conserved sequence of ED-B from mouse to man.13 In line with this premise, we demonstrated that ED-B is virtually undetectable in normal human adult vessels, whereas a markedly enhanced expression of this antigen was found around vasa vasorum of human aortic plaques and plaque matrix. Interestingly, sections of diseased coronary arteries showed not only enhanced ED-B expression in plaques but also in the adjacent diseased media. In contrast, ED-B expression was barely detectable in disease-free portions of the same vessel. These findings are in line with our previous studies showing increased expression of ED-B in angiogenesis18,19 and tissue repair39 of animals and patients.
On the basis of our findings, we will pursue the development of L19 for antibody-based atherosclerotic plaque targeting in the following diagnostic and therapeutic areas. Using near-infrared–based angioscopy, we will evaluate Cy7-labeled SIP(L19) for the in vivo visualization of plaques in larger experimental animals. If positive, imaging studies will be extended to patients because the L19 antibody is already used for imaging solid tumors in patients.19 At the therapeutic level, we will use L19 derivatives currently available in our laboratory and test their ability to modulate plaque progression in the experimental animal.
In the clinical context, there is still an unmet need for detailed visualization of atherosclerotic plaque components, ideally those that indicate instability such as thrombosis, inflammation, or formation of new blood vessels.40 Indeed, the medical relevance of plaque angiogenesis is underlined by the fact that rupture of vasa vasorum leads to intraplaque hemorrhage, which may promote atheroma progression.9 We anticipate that molecular imaging of critical components of the vulnerable plaque will have a considerable impact on improving the diagnosis of this relevant condition.
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Acknowledgments |
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This work was funded in part by European Union grant G5RD-CT-2001-00532 and by Bundesamt für Bildung und Wissenschaft 02.0057 (C.M.M., T.F.L.), the Swiss Heart Foundation (C.M.M.), Jomed/Abbott, Germany (C.M.M.), Deutsche Forschungsgemeinschaft grant 1587/1-1 (P.K.S.), the Swiss National Science Foundation grants 3100-068118 (T.F.L., C.M.M.) and 3100-064912 (D.N.), and by the Associazione Italiana per la Ricerca sul Cancro (L.Z.).
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Footnotes |
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D.N. and L.Z. are consultants and shareholders of Philogen, a biotechnology company that has acquired the rights to the L19 antibody from the academic institutions of these authors (the Swiss Federal Institute of Technology Zurich and the National Cancer Research Institute, Genoa, respectively). D.N. and L.Z. declare competing financial interests.
*These authors contributed equally to this work. 
Presented in part at the Scientific Sessions of the American College of Cardiology, March 7–11, 2004, New Orleans, La.
Original received June 25, 2004; resubmission received October 7, 2004; revised resubmission received November 1, 2004; accepted November 1, 2004.
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