Affiliations of authors: M. E. Wilcox, W. Yang, D. Senger, Z. Q. Shi, P. A. Forsyth, Departments of Oncology and Clinical Neurosciences, University of Calgary, and Tom Baker Cancer Centre, Alberta, Canada; N. B. Rewcastle, Pathology Department, Foothills Hospital, Calgary; D. G. Morris (Departments of Medicine and Oncology), P. M. A. Brasher (Department of Epidemiology, Prevention and Screening), Tom Baker Cancer Centre; R. N. Johnston (Departments of Medical Biochemistry and Oncology), S. Nishikawa, P. W. K. Lee (Departments of Microbiology and Infectious Diseases), University of Calgary.
Correspondence to: Peter A. Forsyth, M.D., F.R.C.P.C., Department of Oncology, Tom Baker Cancer Centre, 1331 29 St., N.W., Calgary, AB, T2N 4N2, Canada (e-mail: peter.forsyth{at}cancerboard.ab.ca).
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ABSTRACT |
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INTRODUCTION |
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We evaluated as a potential anti-glioma therapy a replication-competent virus that replicates in tumor cells and lytically kills them. This strategy contrasts with several other viral therapies that use replication-defective viruses as vehicles for gene delivery (e.g., suicide genes, tumor suppressors, or genes influencing immune functions) (811). Other replication-competent viruses used to kill tumor cells directly by oncolysis include adenovirus (12), herpes simplex virus (HSV) 1 (13), Newcastle disease virus (14), vesicular stomatitis virus (15), and the poliovirus (16). We discovered that reovirus (respiratory enteric orphan), a double-stranded RNA virus commonly isolated from the respiratory and gastrointestinal tracts of humans (17), infects and lyses tumor cells but not normal cells (6). Reovirus does not cause disease in humans, but it produces a lethal infection in neonatal (18) and SCID (severe combined immunodeficient) NOD (19) mice.
Reovirus usurps the activated Ras-signaling pathway of the host tumor cell for its own replication and targets malignant tumor cells with activated Ras (2022). The restriction of reovirus replication in untransformed cells is due to the activation of the double-stranded RNA-activated protein kinase (PKR) by early viral transcripts, which inhibits the translation of viral proteins (22). Activated Ras (or an activated element of the Ras pathway) inhibits (or reverses) PKR activation and allows viral protein synthesis and a lytic infection to occur. Tumors in SCID NOD mice treated with a single intratumoral injection of reovirus regressed; in most cases, no viable tumor remained (6). The purpose of this study was to determine the effectiveness of reovirus as an experimental treatment in malignant gliomas.
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MATERIALS AND METHODS |
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Established cell lines were obtained from the American Type Culture Collection, Manassas, VA. The cells were grown in Dulbecco's modified Eagle medium (DMEM)/F12 containing 10% fetal bovine serum (FBS) at 37 °C in a humidified 5% CO2 incubator; the cells were passaged when they reached approximately 80% confluence, harvested by trypsin treatment, and replated in DMEM/F12 containing 10% FBS. Each cell line was tested routinely for Mycoplasma contamination.
Virus.
Reovirus serotype 3 (strain Dearing) was grown and purified as described previously (23), except that -mercaptoethanol was omitted from the extraction buffer. Reovirus was propagated in suspension cultures of L929 cells and purified. Reovirus labeled with [35S]methionine was grown and purified; the particle/plaque-forming unit (PFU) ratio for purified reovirus was typically 100/1. Dead virus was prepared by exposing live virus to UV light for 45 minutes.
Radiolabeling of reovirus-infected cells and preparation of lysates.
Confluent monolayers of cell lines were infected with reovirus (multiplicity of infection [MOI] of approximately 40 PFUs per cell). At 4648 hours after infection, the medium was replaced with methionine-free DMEM containing 10% dialyzed FBS and 0.1 mCi/mL of [35S]methionine. After further incubation for 24 hours at 37 °C, the cells were washed in phosphate-buffered saline (PBS) and lysed in the same buffer containing 1% Triton X-100, 0.5% sodium deoxycholate, and 1 mM EDTA. The nuclei were then removed by low-speed centrifugation at 6000g for 10 minutes at 4 °C, and the supernatants were stored at -80 °C.
Immunoprecipitation and sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDSPAGE).
Immunoprecipitation of 35S-labeled reovirus-infected cell lysates with a polyclonal anti-reovirus serotype 3 serum was done as described previously (24).
Immunofluorescence analysis of reovirus infection.
The cells were grown on coverslips and infected with reovirus (MOI of approximately 10 PFUs per cell) or mock-infected. At 48 hours after infection, the cells were fixed in an ethanol/acetic acid (20/1) mixture for 5 minutes and then rehydrated by sequential washes in 75%, 50%, and 25% ethanol, followed by four washes with PBS. For paraffin-embedded tumor sections, slides were treated with xylene and then rehydrated by sequential washes in 75%, 50%, and 25% ethanol followed by four washes with PBS. The sections and/or cells were then exposed to the primary antibody (rabbit polyclonal anti-reovirus type 3 serum diluted 1/100 in PBS) for 2 hours at room temperature. After three washes with PBS, the cells were exposed to the secondary antibody (goat anti-rabbit immunoglobulin [Ig] G [whole molecule] fluorescein isothiocyanate [FITC] conjugate diluted 1/100 in PBS containing 10% goat serum and 0.005% Evan's blue counterstain) for 1 hour at room temperature. Finally, the cells were washed three more times with PBS, followed by one wash with double-distilled water, dried, mounted on slides in 90% glycerol containing 0.1% phenylenediamine, and viewed with a Zeiss Axiophot microscope mounted with a Carl Zeiss camera (Carl Zeiss Inc., Thornwood, NY).
Assay to detect mitogen-activated protein kinase (MAPK).
The "PhosphoPlus" p44/42 MAPK (Thr202/Tyr204) antibody kit was used for the detection of MAPK in cell lysates according to the manufacturer's instructions (New England Biolabs Inc., Beverly, MA). Briefly, monolayer cultures were lysed with the recommended SDS-containing sample buffer and subjected to SDSPAGE, followed by electroblotting onto nitrocellulose paper. The membrane was then probed with the primary antibody (anti-total MAPK or anti-phospho-MAPK), followed by the horseradish peroxidase-conjugated secondary antibody as described in the manufacturer's instruction manual.
Animals.
Five- to 8-week-old female and male SCID NOD mice were purchased from the Cross Canada Institute, Edmonton, Canada; female and male CD-1 nude mice were purchased from Charles River Canada, Constant, Canada. The animals were housed in groups of three to five in a vivarium maintained on a 12-hour light/dark schedule with a temperature of 22 °C ± 1 °C and a relative humidity of 50% ± 5%. Food and water were available ad libitum. All procedures were reviewed and approved by the University of Calgary Animal Care Committee.
In vivo studies in a subcutaneous glioma model.
Actively growing U87 or U251N cells were harvested, washed, and resuspended in sterile PBS at a density of 2 x 107 cells/mL. SCID NOD mice received a subcutaneous injection of 2.0 x 106 cells (20 mice each for U251N and U87) suspended in 100 µL of PBS in the hind flank. Tumors grew for 23 weeks until they were clearly growing, and palpable tumors measuring approximately 0.25 cm2 were obtained. Then a single intratumoral injection of 1.0 x 107 PFUs of either live or dead (UV radiation-inactivated) reovirus in 20 µL of sterile PBS was administered. Tumor size (length x width) was measured twice weekly. All animals were killed when they lost 25% of their body weight (which was measured twice a week) or had difficulty ambulating, feeding, or grooming.
In vivo studies in an SCID NOD intracranial glioma model.
The ability of reovirus to cause regression of human glioma cells xenotransplanted into the right cerebral hemisphere was tested in female SCID NOD mice. Actively growing U87lacZ (or U251N) cells were prepared as described above, and 2.0 x 106 cells in 2 µL of PBS were injected intracerebrally into the right putamen. A 0.5-mm burr hole was made 1.52 mm right of the midline and 0.51 mm posterior to the coronal suture through a scalp incision. Stereotactic injection used a 5-µL syringe (Hamilton Co., Reno, NV) with a 30-gauge needle, inserted through the burr hole to 3 mm, mounted on a Kopf stereotactic apparatus (Kopf Instruments, Tujanga, CA). Sixty seconds later, the needle was withdrawn and the incision was sutured. Fourteen days later, when these tumors typically occupied 10% of the cerebral hemisphere and the animals were asymptomatic, a single intratumoral injection of 1 x 107 PFUs of either live or dead (UV radiation-inactivated) virus in 2 µL of PBS was then administered stereotactically. All mice were anesthetized by intraperitoneal administration (ketamine [85 mg/kg] plus xylazine [15 mg/kg); MTC Pharmaceuticals, Cambridge ON, Canada). Animals were killed when they lost 20% of their body weight or had difficulty ambulating, feeding, or grooming. All SCID NOD mice treated with live virus became sick and needed to be killed. (In comparison, animals treated with dead virus remained healthy.) Animals were then killed in pairs; i.e., when an animal treated with live virus needed to be killed, an animal treated with dead virus was killed at the same time and vice versa. Animals were anesthetized, perfused intracardially with PBS and then by 4% paraformaldehyde.
In vivo studies in a CD-1 nude mouse intracranial glioma model.
Actively growing U87lacZ (or U251N) cells were prepared and injected intracerebrally into female nude mice as described above, with the following exceptions: 1) Reovirus was administered intracerebrally 19 days after tumor implantation, and 2) as with any survival experiment, each animal was killed when it became sick (i.e., lost >20% of its body weight or had difficulty ambulating, feeding, or grooming) or when the experiment was terminated at 90 days. All of the brains of the live-virus-treated animals were examined, but only 70% of the brains of the dead-virus-treated animals were available for histologic examination. (Seven animals treated with dead virus died when technical staff were unavailable to process the brains.)
Preparation of brains and other organs for analysis.
Major organs (brains, hearts, lungs, livers, kidneys, and spleens) from three mice in each group were collected at the time that the animal was killed after intracardiac perfusion with PBS. Organs were fixed in formalin and embedded in paraffin.
Cell lines for tumor specimens.
A number of low-passage, human glioma cell lines were established from tumor specimens that were transported directly from the operating room to the laboratory in DMEM. This study was approved by the Conjoint Medical Ethics Committee. A neuropathologist (N. B. Rewcastle) confirmed the histopathologic diagnosis. The specimen was split in half and fixed in formalin (to confirm its identity) or placed in DMEM with 10% FBS. The tissue was then washed in DMEM, cut into pieces of approximately 12 mm in diameter, passed through a cell screen, and then further disaggregated by sequential 20-minute exposures to trypsin (0.5%) and EDTA (0.53 mM) in Dulbecco's PBS. The cells were pelleted through FBS (200g for 8 minutes), resuspended in DMEM/F12 plus 20% FBS, and cultured in 150-cm2 flasks. Cultures became confluent 34 weeks later and were subsequently harvested with trypsinEDTA. The cells were replated for infectibility assays at 104 cells per well in DMEM/F12 (plus 5% FBS plus L-glutamine: 300 µL/well) and on glass coverslips.
Statistical analyses.
Statistical analyses were carried out with the use of the statistical procedures of the Statistical Analysis Software (SAS Institute, Inc., Cary, NC). Continuous and binary measurements were compared between groups with the Wilcoxon rank sum test and Fisher's exact test, respectively. Survival curves were generated by the KaplanMeier method. The log-rank statistic was used to compare the distributions of survival times. All reported P values were two-sided and were considered to be statistically significant at <.05.
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RESULTS |
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We analyzed the susceptibility of 24 established glioma cell lines to reovirus. Dramatic and widespread cell killing after exposure to live (but not dead) reovirus occurred in 20 (83%) cell lines. (Fig. 1, A, shows representative examples.) After 48 hours of infection, widespread cell death was found in U87, U251N, and A172 cell lines; almost complete cell death was seen after 72 hours. In contrast, cells receiving either dead or no virus (data not shown) remained healthy. U118 was the most poorly infectible cell line and was not killed with reovirus. To ensure that cell lysis was due to viral replication, cells were reacted with rabbit anti-reovirus antibody, followed by FITC-conjugated goat anti-rabbit IgG. Susceptible lines (U87, U251N, and A172) expressed viral antigens, whereas nonsusceptible lines (e.g., U118) did not (Fig. 1
, B). Replication of reovirus in susceptible lines was further confirmed by metabolic labeling with [35S]methionine. Lysates were immunoprecipitated with a polyclonal anti-reovirus type 3 serum and analyzed by SDSPAGE (Fig. 1
, C). Eighty-three percent of cell lines were susceptible to reovirus infection (Table 1
). Reovirus replication, however, was restricted in U118, U178, U343, and UC18 (data not shown). Since MAPK occurs downstream of Ras, we evaluated whether MAPK phosphorylation predicted susceptibility to reovirus infection. MAPK was activated in 90% of the cell lines susceptible to reovirus infection (Fig. 1
, D and E). No MAPK phosphorylation was found in the resistant lines U118, U178, U343, or UC18. It is interesting that SF126 and U373 cell lines were susceptible to reovirus infection but showed no MAPK activation.
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SCID NOD mice bearing subcutaneous human malignant gliomas were treated with a single injection of live or dead (UV radiation-inactivated) reovirus (Fig. 2, A and B). U251N xenografts demonstrated a striking regression of the live-virus-treated tumors. A statistically significant difference in tumor size was apparent by day 7 and persisted until day 21 (0.11 cm2 versus 1.49 cm2; Wilcoxon rank sum test, P = .0002) when both groups needed to be killed. Similar results were found with U87 (Fig. 2
, B; 0.38 cm2 versus 3.39 cm2, Wilcoxon rank sum test, P = .0002).
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As expected, we observed substantial toxicity in SCID NOD mice; reovirus is known to cause a fatal infection in these severely immunocompromised mice. Eighty percent of the live-virus-treated group had bilateral hind limb necrosis after approximately 21 days (at which time the tumors had regressed and been "cured" [i.e., a sustained and long-lived absence of tumor]). In contrast, animals treated with dead virus needed to be killed because of tumor growth. Initially, we wondered if this toxicity was specific for glial tumors, but we found the same toxic effects in mice with a breast cancer xenograft. Histologic analysis of the affected limbs showed necrosis of unknown cause involving all tissues in the limb. In addition, all animals treated with live virus developed small foci of myocarditis without obvious symptoms. These foci consisted of two or three necrotic myocytes and a few polymorphonuclear and mononuclear cells.
In Vivo Oncolysis in an Intracerebral Model of Human Malignant Gliomas in SCID NOD Mice
Although we knew reovirus infection would be fatal in SCID NOD mice, we investigated whether reovirus was effective against intracerebral gliomas in these mice. Reovirus caused dramatic tumor regression in orthotopic gliomas followed 14 days later with a single intratumoral injection of 1 x 107 PFUs of live virus. Representative brain cross-sections are shown in Fig. 3, A. The percentage of the brain hemisphere occupied by tumor was dramatically reduced in the live-virus-treated group compared with the dead-virus-treated control group (U251N: 2.5% versus 38.3%, respectively; Wilcoxon rank sum test, P = .0004. U87lacZ: 2.4% versus 40.8%, respectively; Wilcoxon rank sum test, P = .0009) (Fig. 3
, A and B). As expected, all animals treated with live virus became sick and needed to be killed 514 days after intracerebral reovirus inoculation, whereas all animals treated with dead virus remained healthy. Animals treated with live virus died approximately 15 days before their tumors would have become symptomatic. Animals were killed in pairs (i.e., a dead-virus-treated animal was killed at the same time as a live-virus-treated animal), so that differences in tumor size would not be confounded by differences in survival. In some animals, the live-virus-treated brains had necrotic tumor and a few scattered viable tumor cells. Mice treated with live reovirus intracerebrally also developed hind limb necrosis. Diffusely scattered microhemorrhages of unknown cause (possibly from a diffuse encephalitis) likely contributed to the animals' death. There was no brain inflammation in animals treated with dead virus.
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Because of serious toxic effects in SCID NOD mice, we tested reovirus in the less immunocompromised nude mice. We compared the survival of nude mice with intracerebral gliomas treated with a single intratumoral inoculation of live (107 PFUs) virus administered 19 days after tumor implantation with that of nude mice treated with dead virus. U251N tumor-bearing animals had statistically significantly longer survival than control animals treated with dead virus (P<.0001). The median survival of animals treated with dead virus was 42 days, and the median survival was not reached in the group treated with live virus. At 90 days, eight (67%) of 12 live-virus-treated animals were still alive (Fig. 4, A). Similarly, with U87LacZ tumors, the median survival for the dead-virus-treated group was 48 days and not reached in the live-virus-treated group. At 90 days, nine (82%) of 11 animals treated with live virus were still alive (Fig. 4
, B), whereas all of the group treated with dead virus had died. No toxic effects, such as hind limb necrosis or myocarditis, were found in nude mice. Importantly, animals treated with live virus appeared to be healthy and gained weight, whereas the group treated with dead virus all lost weight and appeared to be ill. U251N dead-virus-treated mice, on average, lost 18.7% of their baseline body weight, whereas live-virus-treated animals gained 8.9% of their body weight (Wilcoxon rank sum test, P = .0001). Similar results were found with the U87LacZ-bearing mice: The group treated with dead virus lost 19.6% of their body weight, whereas the animals treated with live virus showed a gain of 12.5% of their body weight (Wilcoxon rank sum test, P = .0001).
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Malignant Glioma Surgical Specimens
To determine whether reovirus oncolysis also occurred in primary cultures from brain tumor surgical specimens, we tested 16 ex vivo brain tumor surgical specimens (Fig. 6) derived from four glioblastoma multiformes, three anaplastic astrocytomas, one astrocytoma, one oligodendroglioma, and seven meningiomas. Reovirus infected and killed all nine (100%) primary glioma cultures but had no effect on cultured meningiomas. Viral proteins were detected in live-virus-treated glioma cells with the use of indirect immunofluorescence microscopy. Glial fibrillary acidic protein staining of cultured gliomas detects glial lineage. Dead-virus-treated tumor specimens remained healthy and continued to proliferate. The number of specimens was small, but this evidence suggests that reovirus oncolysis might be effective in a substantial portion of gliomas.
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DISCUSSION |
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The most important features of this potential treatment for gliomas are its efficacy in an intracerebral location and in surgical glioma specimens, its targeting of signaling pathways that are commonly activated in gliomas, and its ability to kill remote and invasive tumor cells (6). Live-virus-treated nude mice with orthotopic tumors not only had dramatically prolonged survival but also were objectively healthier (by body weight) than their dead-virus-treated counterparts. There was no evidence of diffuse viral encephalitis either morphologically or in terms of gross behavior or weight loss. Several animals had moderate hydrocephalus, but this condition occurred as commonly in the dead-virus-treated group as in the live-virus-treated group and the potential clinical significance of this phenomenon is unclear. The ability of reovirus to lyse all glioma cell lines derived from surgical specimens suggests that a substantial proportion of gliomas may respond to this treatment clinically, although the number of specimens available to us for testing was relatively small and these primary cultures do not completely reflect the tumor environment in patients.
The basis of the ability of the reovirus to target and kill tumor cells but not to infect nonproliferating normal cells lies in its ability to usurp the highly activated signaling pathways found in tumor cells. This ability is most clearly established for Ras or elements in its downstream pathway, and Ras activation is very common in malignant gliomas. We found a strong but not perfect association between Ras activation (as measured indirectly by MAPK activation) and host infectibility; 83% of established glioma cell lines susceptible to reovirus oncolysis had MAPK phosphorylation. The mechanism of selective replication in tumor cells with low MAPK activity is unknown and is being investigated; however, it may involve activation of parallel signaling pathways, such as Jun-N-terminal kinase (JNK). We have been unsuccessful in critically testing the hypothesis that MAPK activation is critical for in vivo oncolysis, since the glioma cell lines that we tested with low or absent MAPK activation would not form tumors in mice.
The search for more effective cancer therapies has focused on viruses as potential therapeutic agents. Those evaluated either have direct oncolytic properties (1216,25) or serve as delivery vehicles for foreign genetic material (2628). Advantages and shortcomings exist for each of these strategies. Virally mediated direct oncolysis may overcome the difficulties of low rates of infection and gene transfer that have plagued virus-mediated gene transfer approaches in glioma patients (29). In contrast, reovirus rapidly destroys tumor cells by direct oncolysis and does not require the expression of foreign genes. In addition, reovirus should target most malignant glioma cells, since its receptor (the sialic acid receptor) is ubiquitously expressed and it does not require down-regulation (i.e., reduced activity) of specific tumor suppressor genes that other viruses do (12,30). Tumor suppressors like p53 or p16/pRB are commonly down-regulated in gliomas, but a substantial number of glioma cells lack these alterations. Instead, reovirus targets tumors with activated Ras-signaling pathways or its downstream elements (22). The importance of this mechanistic distinction is exemplified by the demonstration that the U87 glioblastoma cell line, which contains functional p53, is resistant to the EB1 gene-attenuated adenovirus ONYX-0155 but is very sensitive to reovirus treatment.
Important questions remain regarding both the ultimate clinical efficacy and the safety of this virus. In terms of efficacy, it is reassuring that intracerebral reovirus did not affect the animals' health but usually "cured" their tumors and caused lysis of gliomas derived from patients. In addition, to our knowledge, the survival advantage with reovirus treatment in our orthotopic model is equivalent or superior to any published report of viral therapy for brain tumors in animal models. The clinical efficacy of reovirus treatment will remain unknown until it is tested in a proper clinical trial. Since a small number of animals treated with live virus had persistent or recurrent tumor, multiple viral administrations or combinations with conventional chemotherapy (25) or radiotherapy (31) may be needed in the clinical application of reovirus. The occurrence of hydrocephalus, which seemed to be related to the tumor and not to the viral therapy, suggests the possibility that some patients may require a cerebrospinal fluid shunt after intracerebral inoculation.
The short-term as well as the long-term safety of delivering a replication-competent virus into the brain is the most important question. We have not definitively answered this question here, and it is the subject of ongoing investigations in our laboratories. We cannot prove that a diffuse encephalitis was not present after intracerebral administration of reovirus. However, since the animals treated with live virus looked healthier, gained more body weight, and survived longer than the animals treated with dead virus, encephalitis would have to have been mild if it had occurred. The findings of persistent chronic inflammation after intracerebral administration of adenovirus (32) or HSV-1 (32,33) highlight the importance of comprehensive neurovirulence testing of intracerebrally administered reovirus before it is considered for a clinical trial in glioma patients. A phase I trial of intratumoral reovirus in patients with cutaneous metastases from systemic cancer is currently under way and is a first step in determining its toxicity in humans.
Here, we investigated the use of reovirus as an oncolytic agent in animal models of malignant gliomas. Our study suggests that reovirus has very potent oncolytic activity against experimental gliomas. If a comprehensive evaluation of its short-term and long-term side effects in the brain in nonhuman primates is favorable, it should be considered for clinical trials in glioma patients.
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NOTES |
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M. E. Wilcox and W. Yang share first authorship. P. W. K. Lee and P. A. Forsyth share senior authorship.
Supported by a grant from the National Cancer Institute of Canada (with funds raised by the Canadian Cancer Society). Other funding included the Alberta Cancer Board, Partners in Health, the Canadian Brain Tumor Foundation, and generous donations by the Dr. Michael Longinotto Molecular Neuro-Oncology Fellowship Fund and Mr. Clark Smith.
We thank Ms. Eve Lee for her expert preparation of the manuscript and Drs. Gavin Stuart and Thomas Feasby for their leadership and support.
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Manuscript received October 6, 2000; revised April 12, 2001; accepted April 27, 2001.
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