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Phase II Trial of Autologous Tumor Vaccination, Anti-CD3-Activated Vaccine-Primed Lymphocytes, and Interleukin-2 in Stage IV Renal Cell Cancer

Alfred E. Chang, Qiao Li, Guihua Jiang, Donna M. Sayre, Thomas M. Braun, Bruce G. Redman

From the Departments of Surgery, Biostatistics, and Internal Medicine, University of Michigan, Ann Arbor, MI.

Address reprint requests to Alfred Chang, MD, 3302 Cancer Center, 1500 E Medical Center Dr, Ann Arbor, MI 48109; email: aechang{at}umich.edu.

	ABSTRACT

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Purpose: Previous preclinical and clinical studies have demonstrated that autologous tumor vaccines can induce relatively specific tumor-reactive T cells in draining lymph nodes. The adoptive transfer of these cells can result in tumor regression.

Patients and Methods: Patients with stage IV renal cell cancer (RCC) were vaccinated with irradiated autologous tumor cells admixed with Calmette-Guérin bacillus. Approximately 7 days later, vaccine-primed lymph nodes (VPLNs) were harvested and the lymphoid cells secondarily activated with anti-CD3 monoclonal antibody and expanded in interleukin 2 (IL-2). The activated cells were subsequently infused intravenously along with the concomitant administration of bolus IL-2 (360,000 U/kg intravenously x 15 doses).

Results: Thirty-nine patients were entered onto the study, of whom 34 completed an initial course of cell therapy consisting of a mean (SEM) number of 4.3 (2.2) x 10¹⁰ VPLN cells. Among subjects who received cell therapy, there were nine responses (four complete responses [CRs] and five partial responses [PRs]), for an overall response rate of 27%. The durations of the CRs were > 48, 45, > 35, and 12 months, and the durations of the PRs were > 63, 48, 15, 12, and 4 months. Cultured tumor cells were available to assess in vitro cytokine release of VPLN cells in 24 subjects. The median cytokine release ratio of interferon gamma (IFN) to IL-10 for responders and nonresponders was 992 and 5, respectively, which was significantly different (P = .047).

Conclusion: The treatment protocol resulted in durable tumor responses in patients with advanced RCC. The ratio of IFN and IL-10 cytokines released in response to tumor by the VPLN cells was a significant correlate with tumor response.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
THE ADOPTIVE TRANSFER of tumor-reactive lymphoid cells has been one of the most effective immunotherapies for eradicating advanced tumor burdens in animal models.^1,2 However, there are theoretical obstacles in attempting to translate the principles defined in animal studies to clinical therapy of human cancers. One of these obstacles is the concern of some investigators that many of the chemically induced animal tumors that have been studied are inappropriate models of the human condition.^3,4 Human cancers are of spontaneous origin and are believed by some individuals to be poorly immunogenic in contrast to most animal tumors. Nevertheless, the relative immunogenicity of tumors can be defined experimentally, and there exists a subgroup of murine tumors that can be classified as poorly immunogenic. Using stringent murine tumor models, we have demonstrated that tumor-reactive T cells can be generated against poorly immunogenic tumors.^5–7 Furthermore, the efficacy of adoptive immunotherapy has correlated with the immunologic function of the cellular reagent and the number of cells transferred. Methodologies that will allow ex vivo expansion of tumor-reactive T cells in large numbers while maintaining their immune function is an important requirement for clinical cellular therapy.

We have focused our attention on the lymph node as a reliable tissue compartment to retrieve tumor-reactive T cells.⁸ This is the site where dendritic cells migrate to sensitize naïve T cells to newly processed antigen. We have been able to elicit effector T cells reactive to poorly immunogenic murine tumors in lymph nodes after vaccination.^5–7 The use of a bacterial adjuvant as a component of the vaccine has been key to inducing T-cell responses against these immunogenic tumors. The nodal effector cells can be activated in vitro with anti-CD3 monoclonal antibody (mAb), which upregulates the expression of interleukin-2 (IL-2) receptors and facilitates expansion in IL-2-containing media.⁵ This short-term activation procedure has permitted the expansion of sensitized T cells that retain their immunologic specificity.⁹

On the basis of our animal models, we have conducted preliminary studies to assess the feasibility of this approach in patients with metastatic disease.^10–12 An autologous tumor cell vaccine was used to induce tumor-reactive cells in vaccine-primed lymph nodes (VPLNs). Anti-CD3/IL-2 activation of the VPLN cells resulted in significant expansion of CD8⁺ T cells with relative specificity against autologous tumor cells. Antigen reactivity was restricted to major histocompatibility class I. Furthermore, the infusion of these cells with IL-2 administration resulted in significant tumor responses, mainly in patients with renal cell carcinoma (RCC). As a result of these observations, we have conducted a phase II trial of this cellular approach in patients with stage IV disease. The end points of this study were to determine the response rate of the treatment and to identify potential laboratory correlates to tumor response.

	PATIENTS AND METHODS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Study Population
Patients with metastatic RCC were eligible to participate in the study. All subjects were required to have disease evaluable by physical or radiographic examination and life expectancies of at least 2 months. Subjects were excluded if there was evidence of brain metastases by computed tomographic scans, which were routinely performed before protocol entry. Individuals on corticosteroids were also excluded. No subject received other therapies during the protocol follow-up period. Thirty-nine individuals fulfilled the eligibility criteria and form the basis of this report. This protocol was approved by the institutional review board of the University of Michigan Medical Center and the Food and Drug Administration. Informed consent was obtained from each subject.

Tumor Vaccination
All patients were required to have cryopreserved autologous tumor cells for tumor vaccination. Freshly resected tumors were dissociated under sterile conditions into single-cell suspensions by mechanically mincing tumor into 5-mm³ pieces, followed by enzymatic digestion. In general, 1 g of tumor was digested in a minimum volume of 40 mL of an enzyme mixture that consisted of Hanks’ balanced salt solution (HBSS; BioWhittaker, Walkersville, MD), which contained 2.5 U/mL hyaluronidase type V, 0.5 mg/mL collagenase type IV, and 0.05 mg/mL deoxyribonuclease type I (all from Sigma Chemical Co, St. Louis, MO). The digestion was performed at room temperature with constant stirring in a trypsinizing flask for 2 to 6 hours. The resulting cell suspension was filtered through a layer of no. 100 nylon mesh (Nytek; TETKO Inc, Briarcliff Manor, NY) and cryopreserved in 90% human AB serum (GIBCO, Grand Island, NY) plus 10% dimethylsulfoxide (Sigma) at -178°C in liquid nitrogen for subsequent immunization and immunologic assay.

For vaccination, cryopreserved tumor cells were thawed and washed twice in Dulbecco’s phosphate-buffered saline (DPBS 1X without calcium and magnesium; BioWhittaker). Viable tumor cells were irradiated to a dose of 25 Gy, counted by trypan blue exclusion, and resuspended in DPBS so that a volume of 0.2 to 0.4 mL contained 1 to 2 x 10⁷ viable tumor cells and 1 to 8 x 10⁷ colony-forming units of Calmette-Guérin bacillus (BCG; Organon Teknika Corporation, Durham, NC). Subjects were vaccinated intradermally at two sites approximately 10 cm below the inguinal crease in both thighs.

T Cell Activation and Expansion
Approximately 7 to 10 days later, the enlarged, vaccine-primed, tumor-draining lymph nodes (ie, VPLNs) were removed for culture. A single-cell suspension of VPLN cells was obtained by mechanical dissociation. Briefly, VPLNs were minced with a scalpel in HBSS. The fragments were then pressed through a stainless steel mesh with a glass syringe plunger. The resultant cell suspension was filtered through nylon mesh and washed in HBSS. The cells were counted using trypan blue exclusion and suspended in serum-free medium (X-Vivo-15; BioWhittaker) at 2 x 10⁶ cells/mL. The cells were placed on precoated, 24-well tissue culture plates at 2 mL/well for 48 hours in a 37°C, humidified incubator with 5% carbon dioxide. The plates were coated with 600 µL of OKT-3 (Ortho Pharmaceutical Corporation, Raritan, NJ) at a concentration of 10 µg/mL in DPBS overnight at 4°C. The plates were washed three times with DPBS before use. After 48 hours of exposure to the immobilized anti-CD3 antibody, the cells were harvested and counted. The cells were diluted with serum-free X-Vivo-15 medium at 3 x10⁵/mL containing 60 U IL-2/mL (Chiron Therapeutics, Emeryville, CA), and 500 mL was transferred into tissue culture bags (LifeCell-500; Baxter Fenwall Division, Deerfield, IL). When the cells reached a density of more than 1 x 10⁶/mL, they were transferred into larger bags (LifeCell-3000) with 2000 mL of fresh medium that contained 60 U/mL IL-2.

When the density reached 2 x 10⁶ cells/mL (approximately 6 to 8 days later), the cells were harvested and re-exposed to immobilized anti-CD3 on precoated T-150 tissue culture flasks. After 18 hours, the cells were again cultured in X-Vivo-15 medium that contained IL-2, as previously described. The initial concentration was 3 x 10⁵ cells/mL. As the cell density reached 1 x 10⁶ cells/mL, additional medium was added. After 6 to 8 days of second expansion, the final culture volumes could reach up to 25 to 40 L.

Adoptive Transfer and IL-2 Therapy
Activated T cells for adoptive transfer were harvested with the Fenwal Cell Harvester (Baxter Health Care Corp, Deerfield, IL). After being washed in normal saline, the cells were resuspended in 700 mL normal saline that contained 5% human serum albumin and 420,000 U of IL-2 in a transfer pack (4R2032; Baxter). Cells were infused through a subclavian central venous catheter over a 30-minute interval in the Clinical Research Center of the University of Michigan Medical Center. Intravenous IL-2 administration began immediately after completion of cell infusion, at a dose and schedule of 360,000 U/kg every 8 hours for 5 days. While receiving IL-2, all patients received indomethacin 50 mg orally every 8 hours, acetaminophen 650 mg orally every 6 hours, and ranitidine 150 mg orally every 12 hours to reduce febrile and gastric side effects.

Toxicity was assessed using the National Cancer Institute Common Toxicity Scale. Hypotension was managed by 500-mL fluid boluses (maximum of two) followed by institution of an ephedrine drip. IL-2 therapy was discontinued for observance of any neurotoxicity or cardiac toxicity and for grade 4 toxicity. A 50% dose reduction of IL-2 was instituted for grade 3 metabolic toxicities.

Subjects were evaluated with repeat radiologic examinations every 4 weeks for 2 months. All subjects deemed to have stable or regressing disease by the end of that interval were treated with a repeat adoptive transfer of activated lymph node (LN) cells plus IL-2. Activated LN cells for repeat treatments were generated from cryopreserved VPLN cells obtained initially. A complete response (CR) was defined as complete regression of all evaluable disease without the appearance of new lesions. A partial response (PR) was defined as a reduction of all measurable disease by 50% of the sum products of the two greatest perpendicular diameters without the appearance of new lesions. A minor response was defined as a greater than 25% but less than 50% reduction in all measurable lesions.

Phenotypic Analysis of Lymphoid Cells
The phenotype of fresh and activated VPLN cells was detected by direct staining of cells with phycoerythrin (PE)– or fluorescein isothiocyanate (FITC)–labeled mouse antihuman antibodies. Fluorescence was detected by FACScan flow cytometer (Becton Dickinson Immunocytometry Systems, Mountain View, CA), and data analysis was performed using Lysis software (Becton Dickinson). Control staining was performed using PE- or FITC-labeled mouse monoclonal immunoglobulin isotype control. All the antibodies were purchased from BD PharMingen (San Diego, CA). One million cells were stained with 15 µL of appropriate antibody and incubated on ice for 45 minutes. The sample was then washed twice with ice-cold PBS that contained 0.05% Tween-20 and fixed for 30 minutes in 1% paraformaldehyde/1xPBS at 4°C. The cells were resuspended in 0.5 mL of 1xPBS with 5% fetal bovine serum (FBS). Analysis of cells was performed within 3 days after fixation.

Cytokine Release Assay
Release of IFN and IL-10 by activated VPLN cells in response to tumor stimulation was evaluated. VPLN cells were activated with immobilized anti-CD3 for 2 days and expanded in 60 U/mL of IL-2 for 5 days. After activation, 2 x 10⁶ activated VPLN cells were cocultured with 0.5 x 10⁶ irradiated autologous tumor cells. Cultured tumor cells were used in all assays. These were used after three to five passages in tissue culture flasks containing complete media. Cultured tumor cells were irradiated with 6,000 cGy by a ¹³⁷Cs source (500 cGy/min; Gamma Cell Irradiator; Atomic Energy of Canada, Kanata, Canada). The assay was carried out in 2 mL of complete medium (CM) containing 4 U/mL IL-2 in 24-well culture plates. After 48 hours, culture supernatants were collected and centrifuged at 1,500 rpm for 5 minutes to separate any cells. Culture supernatants were stored at -20°C for subsequent cytokine measurement using commercially available enzyme-linked immunosorbent assay kits (BD PharMingen). For IFN, a standard curve starting at 20,000 pg/mL with serial twofold dilutions was performed. For IL-10, a standard curve starting at 10,000 pg/mL was established in a similar fashion. Experimental values were computed after subtracting background measurements by T cells or tumor cells alone.

Statistical Analysis
A Wilcoxon rank sum test was used to compare the median ratios of IFN:IL-10 for responders and nonresponders. Results were further confirmed with a two-sample t test. The Kaplan-Meier method was used to generate overall survival curves for responders and nonresponders. Differences in overall survival were assessed with a log-rank test. A P value less than .05 was deemed to represent statistical significance.

	RESULTS

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Patient Characteristics
A total of 39 subjects were entered during a study period starting in December 1995. There were 28 male and 11 female patients, with a mean age of 55 years (range, 41 to 78 years). Prior IL-2 therapy had failed in three of the subjects. Eight of the 39 subjects had had nephrectomies before entering the study. These individuals had tumor harvested from nodal recurrences,⁴ subcutaneous metastases,² chest wall metastases,¹ or renal bed recurrence.¹ The remaining patients underwent nephrectomies for tumor harvest. All patients except one recovered from their tumor harvest and received autologous tumor vaccines approximately 3 to 4 weeks after surgery. The individual who did not receive tumor vaccination developed severe hypercalcemia and dehydration with a decreased performance status that required removal from the study. Three other subjects who had received tumor vaccination were removed from the study before cell infusion because of rapidly progressive disease. One subject who had a nephrectomy and subsequent tumor vaccination was found to have complete regression of her pulmonary metastases at the time of cell infusion and did not receive activated lymphocytes. This left a total of 34 subjects who completed cell therapy as per protocol.

VPLN Cells Before and After Activation
Administration of the vaccine in both thighs of each patient resulted in a mean (± SEM) number of VPLN cells/patient of 1.1 (± 0.2) x 10⁹ cells. For each cell infusion, approximately 5 x 10⁸ VPLN cells were placed in culture for activation. After an average (± SEM) of 19 (± 0.6) days in culture, there was a mean (± SEM) 104-fold (± 28) expansion of cells.

Lymphocyte markers were examined by flow analysis before and after anti-CD3/IL-2 activation. As shown in Fig 1, approximately 70% of the freshly harvested VPLNs were made up of CD3⁺ T cells. Among those freshly harvested T cells, approximately 57% were CD4⁺ and 11% were CD8⁺. After activation, virtually 100% of the cells were CD3⁺, of which 77% were CD8⁺ and 18% were CD4⁺. This mimics our prior reported experience using the same activation protocol, where we also documented negligible expression of the natural killer (NK) marker CD56 before and after activation.¹⁰

View larger version (42K): [in this window] [in a new window]   Fig 1. Flow analysis of vaccine-primed lymph node cells before and after activation by the anti-CD3/interleukin-2 culture method. Data represent the mean (± SEM) percentage positively stained cells.

View larger version (44K): [in this window] [in a new window]   Fig 2. Computed tomography scans of complete response in lung. Pretreatment scans in upper panel reveal multiple bilateral pulmonary metastases. Posttreatment scans are shown in lower panel.

View larger version (66K): [in this window] [in a new window]   Fig 3. Complete response in scapula. Pretreatment computed tomography scan on left demonstrates a lytic metastatic lesion. Posttreatment scan on right shows complete resolution with new bone formation.

View larger version (16K): [in this window] [in a new window]   Fig 4. Overall survival of responders and nonresponders. The difference between the group was significant at P < .0001.

Immune Function Assays
Cytokine release assays were performed on activated VPLN cells as described in Patients and Methods. IFN was assessed as a type 1 cytokine and IL-10 as a type 2 cytokine. Tumor cell cultures were established for this purpose from freshly harvested or cryopreserved tumor cells; this was successfully accomplished approximately 70% of the time. Among the 34 subjects treated with activated VPLN cells, 24 had cultured tumor cells available to assess cytokine release in vitro: 17 were from the nonresponder group, and seven were from the responder group (Fig 5). For IFN, the mean (± SEM) values for nonresponders and responders were 3,914 (± 2,245) and 3,881 (± 2,182) pg/mL, respectively (P = .28). For IL-10, the mean (SEM) values for nonresponders and responders was 710 (± 292) and 260 (± 187), respectively (P = .12).

View larger version (13K): [in this window] [in a new window]   Fig 5. Mean (± SEM) values for interferon gamma and interleukin-10 release by vaccine-primed lymph node cells in response to autologous tumor in vitro. Comparison of responders versus nonresponders is depicted.

View larger version (15K): [in this window] [in a new window]   Fig 6. Ratio of interferon gamma to interleukin 10 cytokine release of vaccine-primed lymph node cells for responders and nonresponders. Each dot represents a patient. Median values are shown by the horizontal lines and were significantly different at P = .047.

	DISCUSSION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

We have focused our efforts on obtaining tumor-primed lymphoid cells from tumor-draining lymph nodes in animal models and, as a correlate in the clinical setting, from VPLNs. On the basis of our animal studies, we have found a greater frequency of tumor-reactive T cells within the draining nodal compartments as opposed to other nodal sites or the peripheral circulation.¹⁷ Our earlier clinical trial with anti-CD3-activated VPLN cells demonstrated the induction of relatively tumor-specific reactivity of the cells with tumor responses seen in RCC and melanoma.¹⁰ During this same period, Curti et al^18,19 reported on the nonspecific reactivity of anti-CD3 activated peripheral blood mononuclear cells from subjects with advanced cancers that had negligible antitumor efficacy in adoptive immunotherapy.

An important aspect of this study was the observation that the immune function of the VPLN cells correlated with tumor responses. On the basis of our animal models, we have previously shown that the release of IFN and granulocyte-macrophage colony-stimulating factor (GM-CSF) were important mediators in the tumor rejection response after adoptive transfer.¹⁷ The release of GM-CSF by tumor-primed lymphoid cells has paralleled the secretion of IFN in both our animal and clinical studies. Neutralization of these cytokines after cell transfer resulted in abrogation of the tumor response in these models. Furthermore, we found that the release of IL-10 by the transferred cells resulted in suppression of the tumor rejection response.²⁰ Neutralization of IL-10 enhanced the antitumor effects of the cell therapy. Because of these findings, we examined the ability of VPLN cells to release IFN (type 1 cytokine) and IL-10 (type 2 cytokine) in response to autologous tumor antigen. We have previously demonstrated that IFN secretion of VPLN cells in response to tumor antigen was restricted by major histocompatibility class I molecules.¹⁰ This would indicate that CD8⁺ T cells are responsible for this cytokine. In both nonresponders and responders, we found similar quantities of IFN released in response to tumor. By contrast, there was a trend toward lower levels of IL-10 being released by responding patients compared with nonresponders. When a ratio of IFN:IL-10 was calculated, we found a significant correlation to tumor response. Patients who responded had a higher ratio compared with nonresponders.

We predict that methods to upregulate the ability of tumor-reactive T cells to release IFN and/or GM-CSF would enhance their therapeutic efficacy. In animal models, we have examined this question by activating tumor-draining lymph node (TDLN) cells with both anti-CD3 and anti-CD28 mAbs. Ligation of the CD28 receptor on T cells with the T-cell receptor represents a costimulatory signal that increases cytokine production compared with CD3 signaling alone.²¹ This has resulted in augmented cytokine release on antigen stimulation that included IFN-, GM-CSF, and IL-10. Despite the parallel enhancement of IL-10 release, the anti-CD3/anti-CD28 activated cells were found to be more potent than anti-CD3-activated cells in mediating tumor regression in animal models. One potential mechanism for the increased therapeutic efficacy of the doubly activated T cells is the generation of tumor-reactive CD4⁺ T cells after culture. In murine and human systems, we found the preferential generation of CD4⁺ T cells after anti-CD3/anti-CD28 activation of tumor-primed lymphoid cells.^21,22 On a per cell basis, these CD4⁺ T cells were more effective than CD8⁺ cells in mediating tumor regression in animal models. This is in contrast to anti-CD3 activation, which resulted in the generation of CD8⁺ effector T cells and not CD4⁺ T cells that could independently mediate tumor rejection.²³ The majority of cells infused in this study were CD8⁺. Curti et al²⁴ reported a clinical study of anti-CD3-stimulated CD4⁺ T cells given with IL-2 and cyclophosphamide that indicated that tumor responses were associated with the greatest CD4⁺ T cell expansion in vivo. These findings would indicate that the inclusion of tumor-reactive CD4⁺ cells generated via CD3/CD28 activation would result in a more effective cellular therapy.

In preliminary studies, we have also found that the ligation of CD3/CD28 by mAbs induced the expression of the 4–1BB costimulatory receptor on murine TDLN and human VPLN cells. 4–1BB is a member of the tumor necrosis factor (TNF) superfamily of receptors that is expressed on activated T cells and NK cells and on dendritic cells.^25–27 The activation of tumor-primed lymphoid cells with anti-CD3/anti-CD28/anti-4–1BB mAbs resulted in upregulated type 1 and downregulated type 2 cytokine release in response to tumor cells.²⁸ Furthermore, the antitumor reactivity of these cells was more effective in vivo than were anti-CD3/anti-CD28 activated cells.²⁹ Further directions in the area of cellular therapy will take advantage of culture conditions that will direct T cells toward specific phenotypes.

Another direction to follow to generate more potent effector T cells is to separate out the relevant tumor-reactive subpopulation from the harvested lymphoid compartment (ie, lymph node, tumor-infiltrating lymphocytes [TIL], peripheral blood) before in vitro expansion. In animal studies, we have found that the subpopulation of TDLN cells that was responsible for mediating tumor regression in vivo expressed the adhesion molecule P-selectin ligand (PselL) in high amounts.³⁰ PselL is expressed on lymphoid cells in variable amounts and is involved in lymphocyte-endothelial interactions during cell trafficking.^31–34 We have found that it also appears to be a marker of tumor activation. PselL^high-expressing TDLN cells secrete large quantities of IFN compared with PselL^low cells, which secrete negligible amounts. The latter have no antitumor reactivity on adoptive transfer in vivo. In contrast, PselL^high cells have significantly increased antitumor reactivity compared with the unselected, whole TDLN population. Using freshly harvested human VPLN cells from a patient with RCC, we isolated and activated PselL^high cells that resulted in increased IFN and decreased IL-10 release in response to autologous tumor compared with the unselected population (L. Stoolman, unpublished observation).

We have shown that the adoptive transfer of anti-CD3-activated VPLN cells in combination with moderate doses of IL-2 can result in durable responses in subjects with metastatic RCC. On the basis of current and prior studies, the cells that have been generated are predominantly CD8⁺ T cells with reactivity to autologous tumor cells in vitro.¹⁰ Responses correlated clinically with the cytokine release profile of the VPLN cells. Administration of VPLN cells, which demonstrated a higher IFN: IL-10 ratio in response to autologous tumor cells, was associated with a higher probability of a response. Future efforts should focus on developing protocols to increase the ability to generate T cells with this cytokine profile. We plan to examine alternative activation conditions as described above in our future clinical studies.

	ACKNOWLEDGMENTS

 
We thank the 7A nursing staff of the General Clinical Research Center for their care of the patients, Joann Goodson for data management, and Emily Knaggs and Jodi Hargreaves for their coordination of patient appointments and manuscript preparation.

	NOTES

 
Supported in part by National Institutes of Health grants CA69102 and MO1-RR00042 and by the Gillson Longenbaugh Foundation.

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Submitted August 12, 2002; accepted November 21, 2002.

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