Annals of Surgical Oncology 9:94-103 (2002)
© 2002 Society of Surgical Oncology
Sentinel Node Mapping Identifies Vaccine-Draining Lymph Nodes With Tumor-Specific Immunological Activity
Cynthia S. Chin, MD and
Harry D. Bear, MD, PhD
From the Department of Surgery and the Massey Cancer Center, Medical College of Virginia at Virginia Commonwealth University, Richmond, Virginia.
Correspondence: Address correspondence and reprint request to: Dr. H. D. Bear, Division of Surgical Oncology, Box 980011, Medical College of Virginia at Virginia Commonwealth University, Richmond, VA 23298; Fax: 804-828-4808; E-mail: hbear{at}hsc.vcu.edu
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ABSTRACT
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Background: Adoptive immunotherapy (AIT) with 4T07-IL2 vaccine-draining lymph node (DLN) cells induced regression of established 4T07 mammary carcinomas, but contralateral non-DLN were inactive. These experiments were performed to determine whether mapping with isosulfan blue (IB), as described for identification of sentinel nodes, would identify vaccine-DLN with antitumor activity.
Methods: Ten days after vaccination with 4T07-IL-2, .1 ml of 1% IB was injected into the vaccination site (footpads or flanks). After 3 minutes, mice were euthanized, and the blue-stained nodes were collected. With flank vaccination, IB identified both an inguinal and an axillary node. We also collected DLNs blindly in mice not receiving IB dye. DLN cell suspensions were then activated with bryostatin 1, ionomycin, and IL-2, expanded in culture, and adoptively transferred to mice bearing established 4T07 flank tumors.
Results: Complete tumor regression occurred in nearly all mice treated with popliteal or inguinal DLNs collected with or without IB. IB-stained axillary DLNs cured 100% of tumor-bearing mice, whereas none of the mice treated with blindly collected axillary DLNs were cured.
Conclusion: We have shown that IB identifies immunologically active DLNs, does not interfere with expansion of lymphocytes in vitro, and, more importantly, has no detrimental effect on the ability of lymphocytes to induce tumor regression in vivo. For axillary DLNs, use of IB mapping identified immunologically active lymph nodes that could not otherwise be found.
Key Words: Bryostatin 1 • Ionomycin • Adoptive immunotherapy • Cyclophosphamide • Sentinel node • Isosulfan blue
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INTRODUCTION
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We have shown that adoptive transfer of vaccine-draining lymph node (DLN) cells after ex vivo pharmacological activation and expansion can induce tumor regressions in tumor-bearing hosts.1,2 Pharmacological activation is achieved with bryostatin 1 and ionomycin (B/I). Bryostatin 1, a macrocyclical lactone derived from a marine invertebrate (Bugula neritina) activates protein kinase C, whereas ionomycin increases intracellular calcium. This combination of B/I mimics an important physiologic T-cell activation pathway.3,4 The adoptive transfer of activated and expanded tumor-sensitized lymphocytes has also been shown to mediate potent antitumor effects in several other animal models.5–8 We have also been able to demonstrate antitumor reactivity in DLN cells collected from breast cancer patients.9 In our murine studies, routine collection of DLNs involves removal of all lymph nodes in the draining lymphatic basin. However, in human clinical trials, collection of DLNs has generally involved random collection of a few nodes in a basin, guided only by identifying enlarged or reactive nodes. The alternative, collecting all of the regional lymph nodes (i.e., a complete lymph node dissection) would probably cause unacceptable morbidity for clinical protocols investigating adoptive immunotherapy (AIT) or monitoring postvaccination immunological activity in tumor antigen-vaccinated patients. Being able to distinguish active nodes in a particular basin from nonactive or less active nodes would allow a more focused, less intrusive lymphadenectomy procedure that would reduce the potential for postoperative morbidity associated with a more extensive lymphadenectomy procedure and increase the likelihood of antitumor effects.
Therefore, we first determined whether lymphocytes from non-DLNs (NDLN) collected from the same tumor-sensitized animal would exhibit the same antitumor effects after ex vivo B/I activation, 10-day culture, and adoptive transfer to a tumor-bearing host as DLN cells. If these experiments indicated that there was a difference in immunological activity between DLN cells and NDLN cells collected from the same animal, we would then test the hypothesis that sentinel node mapping, which has been described in recent years as a way to identify the first node or nodes in the pathway from a primary tumor site to the regional lymphatic basin,10–13 might identify the vaccine-DLNs harboring most of the tumor-specific immunological activity. Current sentinel node techniques use isosulfan blue (IB) dye, radioactive tracers, or a combination of the two. For this study we have chosen to use IB dye to distinguish vaccine-DLNs in a regional lymphatic basin from NDLNs within the same basin. Finally, we wanted to determine whether the use of IB had any adverse effects on the ex vivo activation and expansion of these lymphocytes and, most importantly, whether it would have any adverse effects on the efficacy of AIT in treatment of tumor-bearing animals.
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MATERIALS AND METHODS
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Mice
Virus-free BALB/c mice (Charles River Laboratories, Cambridge, MA) were used between 8 and 12 weeks of age, caged in groups of six or fewer, and provided food and water ad libitum. All guidelines at Virginia Commonwealth University, which conform to the American Association for Accreditation of Laboratory Animal Care and the US Department of Agriculture recommendations for the care and humane experimental use of animals, were followed.
Tumor Cell Lines
The 4T07 mammary tumor and its interleukin (IL)-2 transduced counterpart (4T07-IL-2) were kindly provided by Dr. Jane Tsai at the Michigan Cancer Foundation, Detroit, MI. Cells were maintained in Dulbecco’s modified Eagle’s medium with 10% heat-inactivated fetal calf serum (Hyclone, Logan, UT), 1 mM of sodium pyruvate, 100 U/ml of penicillin, and 100 µg/ml of streptomycin (Sigma, St. Louis MO). G418 (Sigma) 600 µg/ml was added to 4T07-IL-2 cultures. Meth A sarcoma (American Type Culture Collection, Rockville, MD) was maintained in RPMI 1640 with 10% fetal calf serum, 1 mM of sodium pyruvate, .1 mM of nonessential amino acids, 2 mM of L-glutamine, 100 U/ml of penicillin, 100 µg/ml of streptomycin, 10 mM of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer, and 5 x 10-5 M of 2-mercaptoethanol (Sigma; complete RPMI). All cells were incubated in 250-ml T flasks (PGC, Gaithersburg, MD) at 37°C in humidified air with 5% CO2. Tumor cells were collected for passage or for inoculation of mice with .05% trypsin/ethylenediaminetetraacetic acid (Fisher, Pittsburgh, PA).
Sensitization of DLNs and Preparation of Lymphoid Cell Suspension
Mice were inoculated in either one hind footpad or subcutaneously (SC) in one shaven flank with 1 x 106 viable 4T07-IL-2 cells or irradiated (10,000 rad) Meth A sarcoma cells. 4T07-IL-2 tumors regress spontaneously by 15 days after inoculation. Ten days after sensitization, half of the mice were injected into their vaccination site with .1 ml of 1% IB (US Surgical Corporation, Norwalk, CT). After 3 minutes, mice were killed by CO2 inhalation, and the IB-stained DLNs (SDLNs) were collected under sterile conditions. The remaining mice, not injected with IB, had all of the lymph nodes in the draining basins collected blindly (BDLN; i.e., ipsilateral popliteal nodes were collected after footpad vaccination and ipsilateral axillary and inguinal nodes were collected after flank vaccination). In experiments with NDLN lymphocytes, the contralateral popliteal nodes were collected 10 days after footpad vaccination with 4T07-IL-2.
Pharmacological Activation and Expansion of Lymphocytes in Culture
NDLNs, BDLNs, and SDLNs were dispersed into single-cell suspensions, washed, and resuspended in complete RPMI at 1 x 106 cells per milliliter. Lymph node cells were then incubated for 18 hours with 5 nM of bryostatin 1 (kindly provided by the National Cancer Institute, Bethesda, MD), 1 µM of ionomycin (Calbiochem, San Diego, CA), and 80 IU/ml of recombinant interleukin (rIL)-2 (Chiron, Emeryville, CA) at 37°C in humidified air with 5% CO2 (B/I activated). After incubation, cells were washed three times with warm (37°C) RPMI 1640 media and cultured for up to 10 days in complete RPMI with 40 IU/ml of rIL-2 in 50-ml T flasks. Cells were split to 1 x 106 cells per milliliter and re-fed with medium and 40 IU/ml of rIL-2 every other day. After 10 days of expansion (generally resulting in a 10- to 20-fold expansion), cells were washed, resuspended in plain RPMI, and adoptively transferred intravenously (IV) to tumor-bearing hosts.
Treatment of Established Tumors
Mice were inoculated SC into their shaven left flank with 3 x 105 wild-type 4T07 cells in .05 ml of Dulbecco’s modified Eagle’s medium. Three days later, some mice were given cyclophosphamide (CYP, 100 mg/kg) intraperitoneally. Lymphocytes (10 x 106) for AIT, in .5 ml of RPMI, were infused IV 24 hours later (i.e., on day 4).
Protection Experiments
Naïve mice received adoptive transfer of 10 x 106 4T07-IL-2-sensitized, B/I-activated, and expanded popliteal SDLN or BDLN cells. One month later, mice that had received tumor-sensitized activated T cells and age-matched control mice were inoculated SC with 3 x 105 wild-type 4T07 cells into the shaven left flank.
Tumor Measurements
In all experiments, tumor growth was monitored with twice a week measurements of perpendicular diameters. When the tumor area was >144 mm2 or if the mouse seemed ill, the animal was euthanized by CO2 inhalation. Complete tumor regression was defined as an absence of a measurable tumor on consecutive measurements.
Statistical Analysis
Differences in tumor growth were assessed by analysis of variance and Tukey-Kramer honestly significant difference test by use of JMPTMIN software (SAS Institute Inc., Cary, NC). Results of tumor growth are presented as the means and SE of tumor area in each treatment group. Each experiment included at least six mice per group and was repeated at least twice. An 
< .05 was used throughout to determine significant differences.
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RESULTS
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Treatment of 4T07 Flank Tumors With DLN, But Not NDLN, Lymphocytes Induces Tumor Regression
Mice with 3-day 4T07 tumors either were not treated (control) or were treated with CYP alone, CYP plus AIT with NDLN lymphocytes, or CYP plus AIT with DLN lymphocytes. As shown in Fig. 1, only CYP plus AIT with DLN cells induced complete tumor regression in all mice. Tumor growth in mice treated with CYP plus AIT with DLN cells was significantly different from that in untreated mice, mice treated with CYP alone, or mice treated with CYP plus AIT with NDLN cells [F(3,20) = 8.2026, P = .0009]. Tumor growth in groups treated with CYP plus AIT with NDLN cells was not significantly different from that of either control or CYP-alone groups. Thus, lymphocytes from vaccine-DLNs were immunologically active in AIT, whereas AIT with NDLN lymphocytes was not effective for inducing tumor regressions.
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FIG. 1. Treatment of 4T07 flank tumors with draining lymph nodes (DLNs), but not non-DLN (NDLN), bryostatin 1 and ionomycin-activated, 10-day expanded lymphocytes induces tumor regression. Mice with 3-day 4T07 tumors either were not treated (control) or were treated with cyclophosphamide alone (CYP), CYP plus adoptive immunotherapy (AIT) with NDLN lymphocytes, or CYP plus AIT with DLN lymphocytes. Mean tumor area (±SE) is shown over time. Tumor growth in mice treated with CYP plus DLNs was significantly different from that in mice that were not treated, were treated with CYP alone, or were treated with CYP plus NDLN AIT [F(3,20) = 8.2026, P = .0009]. The control, CYP alone, and CYP plus NDLN AIT groups were not significantly different from each other. Numbers to the right indicate the number of mice with complete tumor regression per total number of mice in the group.
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Ex Vivo Expansion of 4T07-IL-2-Sensitized SDLN or BDLN Lymphocytes After B/I Activation
IB injection into the footpad 10 days after mice were vaccinated with 4T07-IL-2 resulted in a blue-stained popliteal node in all mice. In mice vaccinated in the flank and then injected with IB at that site, both an axillary and an inguinal node stained blue. Cells from 4T07-IL-2 tumor-sensitized DLN basins collected blindly (BDLN) expanded approximately 11-fold in the 10-day ex vivo expansion period after B/I-activation. Lymphocytes derived from lymph nodes collected after IB dye staining (SDLN) expanded approximately 18-fold during the 10-day ex vivo culture period (Fig. 2).
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FIG. 2. Ex vivo expansion of 4T07-interleukin-2-sensitized isosulfan blue-stained draining lymph nodes (SDLNs) or blindly collected draining lymph nodes (BDLNs) after bryostatin 1 and ionomycin activation. Expansion of cell numbers over time is shown. Lymphocytes from SDLNs expanded 18-fold by day 10 in culture. Lymphocytes derived from BDLNs expanded 10-fold in the same 10-day period.
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Treatment of 4T07 Flank Tumors With 4T07-IL-2-Sensitized SDLNs or BDLNs After Ex Vivo B/I Activation and 10-Day Expansion
Mice with 3-day 4T07 tumors either were not treated (control) or were treated with CYP alone, CYP plus AIT with popliteal BDLN lymphocytes, or CYP plus AIT with popliteal SDLN lymphocytes (Fig. 3). None of the mice treated with CYP alone demonstrated tumor regression, and only one of the six mice in the control group showed complete tumor regression. All of the mice treated with CYP plus AIT with either SDLN or BDLN lymphocytes exhibited complete tumor regressions, and tumor growth in these two treatment groups was not significantly different. However, these two groups were both significantly different from the control group and the group treated with CYP alone [F(3,20) = 9.1395, P = .0005]. Similar results were observed with inguinal SDLN and BDLN lymphocytes from mice vaccinated in the flank [Fig. 4; F(3,20) = 8.2789, P = .0009].
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FIG. 3. Regression of 4T07 flank tumors after adoptive immunotherapy (AIT) with 4T07-interleukin-2-sensitized popliteal isosulfan blue-stained draining lymph node (SDLN) or blindly collected draining lymph node (BDLN) cells activated ex vivo with bryostatin 1 and ionomycin. Mice with 3-day 4T07 tumors either were not treated (control) or were treated with cyclophosphamide alone (CYP), CYP plus AIT with popliteal BDLN lymphocytes, or CYP plus AIT with popliteal SDLN lymphocytes. Mean tumor area (±SE) is shown over time. Tumor growth in mice treated with CYP plus either SDLN- or BDLN-based AIT were not significantly different from each other, but were significantly different both from control mice and from mice treated with CYP alone [F(3,20) = 9.1395, P = .0005]. The control group and the CYP group were not significantly different from each other. Numbers to the right indicate the number of mice with complete tumor regression per total number of mice in the group.
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FIG. 4. Regression of 4T07 flank tumors after adoptive immunotherapy (AIT) with 4T07-interleukin-2-sensitized inguinal isosulfan blue-stained draining lymph node (SDLN) or blindly collected draining lymph node (BDLN) cells activated ex vivo with bryostatin 1 and ionomycin. Mice with 3-day 4T07 tumors either were not treated (control) or were treated with cyclophosphamide alone (CYP), CYP plus AIT with inguinal BDLN lymphocytes, or CYP plus AIT with inguinal SDLN lymphocytes. Mean tumor area (±SE) is shown over time. Tumor growth in mice treated with CYP plus either SDLN- or BDLN-based AIT was not significantly different, but was significantly different in control mice or mice treated with CYP alone [F(3,20) = 8.2789, P = .0009]. The control group and the CYP group were not significantly different from each other. Numbers to the right indicate the number of mice with complete tumor regression per total number of mice in the group.
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Treatment of 4T07 Flank Tumors With 4T07-IL-2-Sensitized Axillary SDLN or BDLN Lymphocytes After Ex Vivo B/I Activation and 10-Day Expansion
Mice with 4T07 tumors were either not treated (control) or were treated with CYP alone, CYP plus AIT with axillary BDLN lymphocytes, or CYP plus AIT with axillary SDLN lymphocytes (Fig. 5). The control group and the CYP group were not significantly different from each other. None of the mice in the group treated with CYP plus AIT with axillary BDLN cells exhibited complete tumor regression, and this group was not significantly different from either the control or CYP-alone groups. Conversely, all mice treated with CYP plus AIT with lymphocytes from axillary SDLNs exhibited complete tumor regression; this was significantly different from the control, CYP-alone, and CYP plus axillary BDLN groups [F(3,20) = 5.8549, P = .0049].
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FIG. 5. Treatment of 4T07 flank tumor with 4T07-interleukin-2-sensitized axillary isosulfan blue-stained draining lymph node (SDLN) or blindly collected draining lymph node (BDLN) cells activated ex vivo with bryostatin 1 and ionomycin. Mice with 3-day 4T07 tumors either were not treated (control) or were treated with cyclophosphamide alone (CYP), CYP plus adoptive immunotherapy (AIT) with axillary BDLN lymphocytes, or CYP plus AIT with axillary SDLN lymphocytes. Mean tumor area (±SE) is shown over time. The control group and the CYP group were not significantly different from each other. The group treated with CYP plus AIT with axillary BDLN cells was not significantly different from the control or CYP groups. The group of mice treated with CYP plus AIT with lymphocytes from axillary SDLNs had significantly decreased tumor growth compared with the control and the CYP-alone groups [F(3,20) = 5.8549, P = .0049]. Numbers to the right indicate the number of mice with complete tumor regression per total number of mice in each group.
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Treatment of 4T07 Flank Tumors With Popliteal SDLN or BDLN Lymphocytes From Either Meth A- or 4T07-IL-2-Sensitized Mice
To address any concerns that the IB dye may somehow activate lymphocytes nonspecifically, an experiment was performed in which 4T07 tumor-bearing mice were treated with B/I-activated, expanded DLN cells collected from either 4T07-IL-2- or Meth A-sensitized mice, with or without IB dye. Mice with 3-day 4T07 tumors either were not treated (control) or were treated with CYP alone or with CYP plus AIT with 4T07-IL-2-sensitized BDLN or SDLN cells or Meth A-sensitized BDLN or SDLN cells (Fig. 6). All adoptively transferred cells were activated with B/I and expanded in culture for 10 days. Tumor regressed in one of the six control mice and in none of the mice treated with CYP alone, CYP plus Meth A BDLN, or CYP plus Meth A SDLN. Treatment with popliteal 4T07-IL-2 BDLN or SDLN lymphocytes induced complete tumor regression in all treated mice. The 4T07-IL-2 BDLN and SDLN groups were not significantly different from each other but were significantly different from the control, CYP, CYP plus Meth A BDLN, and CYP plus Meth A SDLN groups [F(5,30) = 12.1506, P < .0001]. The IB dye does not seem to induce nonspecific antitumor activity in DLN cells collected with this technique.
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FIG. 6. Specificity of adoptive immunotherapy (AIT) effect: treatment of 4T07 flank tumors with popliteal isosulfan blue-stained draining lymph node (SDLN) or blindly collected draining lymph node (BDLN) lymphocytes from either Meth A- or 4T07-sensitized mice. Mice with 3-day 4T07 tumors either were not treated (control) or were treated with cyclophosphamide only (CYP) or CYP plus AIT with either 4T07-interleukin (IL)-2-sensitized BDLN or SDLN cells or Meth A-sensitized BDLN or SDLN cells activated with bryostatin 1 and ionomycin. Mean tumor area (±SE) is shown over time. All of the mice treated with the 4T07-IL-2 BDLN or SDLN cells had complete tumor regression. The 4T07-IL-2 BDLN and SDLN groups were not significantly different from each other. Tumor growth in those two groups was significantly different from the control, CYP alone, Meth A BDLN, and Meth A SDLN AIT groups [F(5,30) = 12.1506, P < .0001]. The control, CYP alone, Meth A draining lymph nodes, and Meth A SDLN groups were not significantly different from each other. Numbers to the right indicate the number of mice with complete tumor regression per total number of mice in each group.
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Adoptive Transfer of 4T07-IL-2 Sensitized Popliteal SDLNs or BDLNs to Naïve Mice Confers Resistance to 4T07 Tumor Challenge 1 Month Later
In our experience, naïve mice receiving adoptively transferred tumor-sensitized lymphocytes after B/I activation and expansion were resistant to tumor growth when challenged 1 month later with wild-type 4T07 tumor cells (data not shown). As described in Materials and Methods, age-matched controls and naïve mice that had received an IV infusion of 4T07-IL-2-sensitized, B/I-activated, 10-day expanded lymphocytes were inoculated SC 1 month later with 4T07 tumor cells. All mice that received tumor-sensitized, B/I-activated DLN cells, regardless of whether IB was used or not, exhibited complete tumor regression (Fig. 7). These groups both were significantly different from the age-matched control group, in which progressive tumor growth was observed [F(4,25) = 9.6505, P < .0001]. Thus, the IB did not adversely affect the ability of B/I-activated DLN lymphocytes to confer long-lasting immunity.
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FIG. 7. Adoptive transfer of 4T07-interleukin (IL)-2 sensitized popliteal isosulfan blue-stained draining lymph node (SDLN) or blindly collected draining lymph node (BDLN) lymphocytes to naïve mice protects against 4T07 tumor challenge 1 month later. Naïve mice received 4T07-IL-2-sensitized, bryostatin 1 and ionomycin (B/I)-activated, 10-day expanded lymphocytes, which had been collected with or without isosulfan blue staining. One month later, these mice, as well as age-matched controls, were inoculated subcutaneously with 4T07 tumor cells. All mice treated with tumor-sensitized, B/I-activated draining lymph node cells, regardless of whether isosulfan blue was used, exhibited complete tumor regression. These groups were both significantly different from the age-matched control group [F(4,25) = 9.6505, P < .0001]. Numbers to the right indicate the number of mice with complete tumor regression per total number of mice in each group.
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DISCUSSION
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In this murine mammary carcinoma model, we first observed that cells from the NDLNs of vaccine-sensitized mice had significantly less antitumor activity than DLN cells adoptively transferred to a tumor-bearing host after ex vivo B/I activation and expansion. Therefore, it is important to distinguish draining and immunologically active lymph nodes from NDLNs to identify those nodes with the most potent antitumor immunological activity. With the IB technique, we were able to distinguish the draining and immunologically active nodes from the other nodes in a vaccine-draining basin. IB was able to identify, 100% of the time, a popliteal SDLN or an axillary and inguinal SDLN in mice vaccinated in the footpad or flank, respectively. After ex vivo B/I activation, the lymphocytes from IB-stained lymph nodes expanded as well as BDLN cells. The popliteal and inguinal SDLN cells, after B/I activation and expansion, were just as efficient as popliteal and inguinal BDLN cells in mediating tumor regression after adoptive transfer to tumor-bearing mice. It is interesting to note that cells from axillary SDLN lymphocytes were significantly more effective in the treatment of tumor-bearing mice than was AIT with axillary BDLN cells. Both ipsilateral popliteal and inguinal nodes are markedly enlarged after tumor vaccination in the footpad or flank, respectively. However, the ipsilateral axillary nodes, after flank vaccination, are not noticeably enlarged at the time of collection. We believe that the clinical enlargement of popliteal and inguinal nodes at the time of collection enables us to retrieve immunologically active lymph nodes without the need for IB dye. It should be noted, moreover, that there is virtually always only one node identifiable in either of these areas of the mouse. Yet the lack of obvious nodal enlargement in the ipsilateral axilla after flank vaccination reinforces the need for a method that can identify the lymph nodes draining a vaccination site that contains immunologically active lymphocytes with potent antitumor activity. We were able to identify the immunologically active axillary DLNs with IB, and these lymphocytes were significantly more effective in inducing 4T07 tumor regression than axillary node lymphocytes collected blindly. The use of IB also did not interfere with long-lasting immunological memory.
Clinical application of the IB sentinel node technique for identification of potent antitumor lymph nodes draining a vaccination site may allow a more focused and precise lymphadenectomy procedure in clinical protocols evaluating AIT with vaccine-DLN cells. Moreover, this technique may also have clinical relevance in other protocols in which postvaccination antitumor immunological activity is monitored. The use of IB mapping provides a simple method for performing a limited lymphadenectomy procedure, allowing the investigator to obtain lymphocytes for ex vivo analysis with less morbidity than would be associated with a more extensive procedure. It has recently been reported that fine-needle aspirates of tumors in patients with metastatic melanoma have been cultured ex vivo for the study of host-tumor interaction pre- and postvaccination.14 The IB mapping technique could be used to guide postvaccination collection of lymphatic tissue with the greatest potential of exhibiting antitumor activity and could be used to facilitate monitoring of antitumor activity induced by vaccination, as well as for AIT applications.
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Acknowledgments
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This work was supported by Grants R01 CA48075 and T32 CA09573 from the National Cancer Institutes, National Institutes of Health, and Department of Health and Human Services.
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Footnotes
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Presented at the 54th Annual Meeting of the Society of Surgical Oncology, Washington, DC, from March 15–18 2001.
Received for publication March 9, 2001.
Accepted for publication July 16, 2001.
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ABSTRACT
INTRODUCTION
