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(Journal of Leukocyte Biology. 2001;70:578-584.)
© 2001 by Society for Leukocyte Biology

Spontaneous formation of germinal centers in autoimmune mice

Irina G. Luzina*, Sergei P. Atamas*, Catherine E. Storrer*, Ludmila C. daSilva*, Garnett Kelsoe3, John C. Papadimitriou4 and Barry S. Handwerger*

* Department of Medicine, and
§ Department of Pathology, University of Maryland School of Medicine, Baltimore, Maryland;
{dagger} Medical Service, Department of Veterans Affairs Medical Center, Baltimore, Maryland; and
{ddagger} Department of Immunology, Duke University Medical Center, Durham, North Carolina

Correspondence: Irina G. Luzina, M.D., Ph.D., Division of Rheumatology and Clinical Immunology, University of Maryland School of Medicine, 8-34 MSTF, 10 South Pine Street, Baltimore, MD 21201-1192. E-mail: iluzina{at}umaryland.edu


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ABSTRACT
 
The mechanisms of autoantibody production are not well understood. Germinal centers (GC) may be important sites of immune disregulation in autoimmune diseases. In this study, we document the presence of spontaneous GC formation in the spleens of several autoimmune mouse strains that spontaneously develop autoimmune Type I diabetes and a lupus-like disease. In contrast, mouse strains that do not develop lupus did not exhibit spontaneous formation of GC. In all of the autoimmune strains studied, GC were present at 1–2 months of age, a time that closely parallels the appearance of autoantibodies. Like the GC that develop after purposeful immunization, GC in autoimmune mice contained B220+, PNA+, and GL-7+ B cells, and FDC-M1+ follicular dendritic cells. In addition, spontaneously formed GC in autoimmunity and those caused by immunization were abrogated in a similar way by a short-term treatment with anti-CD40 ligand antibody. These data indicate that spontaneously forming GC in autoimmunity are similar to those appearing after purposeful immunization.

Key Words: CD40 • lupus • diabetes • autoimmunity


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INTRODUCTION
 
Autoimmune diseases result from altered regulation of self-reactivity. The exact mechanisms underlying the immunoregulatory abnormalities that lead to autoimmunity are unknown. Germinal centers (GC) are sites of immunoglobulin (Ig) class switching [1 2 ], Ig gene V-region somatic hypermutation [3 4 5 6 ], and B-cell tolerization [7 8 9 ]. Because GC are most likely the sites where mutated IgG autoantibodies arise, GC may be important sites of immune disregulation in autoimmune diseases.

GC formation in mice that spontaneously develop autoimmunity, in the absence of purposeful immunization, has not been studied extensively. GC have been observed histologically in the spleens and lymph nodes of (NZBxNZW)F1 (NZB/W) mice [10 ] and C3H/lpr and C3H/gld mice treated with anti-CD8 monoclonal antibody (mAb) [11 ]. However, these studies did not document whether the observed GC were phenotypically normal nor did they determine whether the mice were free of adventitious infections that may have stimulated the GC reaction. In this study, we demonstrate that in the absence of purposeful immunization and adventitious infection, GC are present in the spleens of several strains of mice that spontaneously develop a systemic lupus erythematosus-like disease or autoimmune Type I diabetes mellitus. GC are present as early as 1–2 months of age, a time that correlates with the onset of autoantibody production in these mice. Cells present in spontaneously developing GC are phenotypically similar with respect to cell-surface markers to cells in the GC that develop in response to immunization. In addition, we demonstrate that maintenance of GC in autoimmune mice is dependent on CD40-CD40 ligand (CD40L) interaction.


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MATERIALS AND METHODS
 
Animals
NOD mice, a strain that spontaneously develops autoimmune, Type 1 diabetes mellitus, and several strains of mice that spontaneously develop an autoimmune, lupus-like disease—i.e., female PN, NZB, NZB/W, MRL/lpr, MRL/++, and B6/lpr, and male BXSB mice—were studied. Breeding pairs of PN mice were obtained from Dr. Sara E. Walker (University of Missouri, Columbia, MO). Subsequently, a separate inbred colony has been maintained at the University of Maryland School of Medicine (Baltimore, MD). Male and female BXSB mice, and female NOD, NZB, MRL/lpr, B6/lpr, MRL/++, DBA/2, and C57BL/6 mice were purchased from Jackson Laboratory (Bar Harbor, ME). NZB/W mice were obtained from Dr. Bevra Hahn (University of California, Los Angeles, CA). The University of Maryland Animal Facility is AALAC-approved. The mice were serologically monitored on a regular basis; all mice were free of bacterial, viral, and mycoplasma infections.

Tissues
Mice were killed by CO2 asphyxiation; their spleens were removed and embedded in Tissue-Tec OCT compound (VWR, Bridgeport, NJ) and flash-frozen in 2-methyl butane cooled with liquid N2. Blocks of frozen tissues were stored at -70°C until sectioning. Sections (6 µm) were cut on a cryostat (International Equipment Co., Needham Heights, MA), allowed to air-dry for 10 min, fixed in ice-cold acetone for 10 min, air dried, and stored at -20°C until use.

Immunohistological staining of splenic sections
Serial sections were stained with horseradish peroxidase (HRP)-conjugated peanut agglutinin (PNA; Sigma Chemical Co., St. Louis, MO), which stains germinal center B cells and biotinylated anti-CD4 mAb (PharMingen, San Diego, CA), biotinylated anti-B220 mAb, biotinylated anti-GL7 mAb (which also stains germinal-center B cells), or FDC-M1 mAb (all from PharMingen), which reacts with follicular dendritic cells (FDC) in GC [12 ]. Bound FDC-M1 mAb was detected using biotinylated monoclonal anti-rat Ig antibody (Southern Biotechnology Associates, Birmingham, AL); bound biotinylated antibodies were visualized using alkaline phosphatase-conjugated streptavidin (streptavidin-AP; PharMingen), followed by enzymatic detection with napthol-AS-MX-phosphate (Sigma), Fast Blue BB salt (Sigma), and levamisole in 0.1 M Tris-HCl, pH 8.5. HRP-conjugated antibodies were visualized using an AEC substrate kit for peroxidase (Vector Laboratories, Burlingame, CA). Endogenous peroxidase activity was blocked by a 5-min incubation in 0.3% H2O2 before staining. Nonspecific avidin binding was blocked by an avidin/biotin blocking reagent (Vector Laboratories). Stained sections were washed in phosphate-buffered saline (PBS) and mounted (Crystal Mount, Biomedia Corp., Foster City, CA).

5-bromo-2'-deoxyuridine (BrdU) assay for detection of proliferating cells
Proliferating cells were identified by incorporation of BrdU as described [13 ]. Briefly, young (3-months old) and old (12-months old) PN mice were injected intraperitoneally (i.p.) with 2 mg BrdU (Sigma). Two hours later, spleens were removed, and 6 µm cryostat sections were prepared, embedded in OCT compound, and frozen in liquid nitrogen. To detect the cells that had incorporated BrdU into their DNA, sections were incubated with HRP-conjugated PNA and biotinylated anti-CD4 mAb. After color development, the sections were incubated in 1 N HCl for 1 h to expose and partially degrade the DNA. BrdU, incorporated into newly synthesized DNA, was then detected by sequential incubation of the tissue with unlabeled anti-BrdU mAb (Sigma), biotin-conjugated, goat anti-mouse IgG (PharMingen) as second antibody, and streptavidin-AP, followed by color development with Fast Red TR salt (Sigma).

Short-term, anti-CD40L treatment
Three-month-old, female C57BL/6 mice were immunized with a single i.p. injection of 100 µg alum-precipitated (4-hydroxy-3-nitrophenyl), acetyl-chicken {gamma}-globulin (NP-CGG) [3 ]. Beginning 6 days after immunization, immunized C57BL/6 mice and non-immunized 3-month- and 12-month-old PN mice were injected intravenously (i.v.; 250 µg/injection on days 6, 8, and 10) [8 ] with anti-CD40L mAb (PharMingen) or control hamster Ig (Pierce, Rockford, IL). Two days after the third injection (day 12), the mice were sacrificed, and their spleens were removed for immunohistological evaluation.

Morphological evaluation of germinal centers
Germinal centers in spleen sections were evaluated microscopically for size and number per spleen section. Individual GC were graded for size, by one individual (IL), on a 0 to 4+ scale. Size was determined by comparison to photographic images of reference GC. Electronic spot recognition and counting (ChemImager 4000, Alpha Innotech, San Leandro, CA) of reference GC images demonstrated that a grade of 0 = no PNA+ cells, 1+ = 1–100 PNA+ cells, 2+ = 101–250 PNA+ cells, 3+ = 251–400 PNA+ cells, and 4+ = >400 PNA+ cells. The number/spleen section of GC of each size was determined microscopically.

Statistical analysis of data
Three to five mice per age group were evaluated for all strains, except PN mice, where three to seven mice per group were studied. Groups were compared statistically using the Mann-Whitney U test (Statistica software, StatSoft, Tulsa, OK).


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RESULTS
 
Kinetics of spontaneous GC formation in autoimmune mice
With age, PN mice spontaneously develop a systemic, autoimmune, lupus-like disease, characterized by the production of multiple autoantibodies and the presence of immune complex-mediated glomerulonephritis and a severe systemic vasculitis [14 15 16 ]. GC were not present in the splenic follicles of 3-month- or 12-month-old DBA/2 mice (Fig. 1A and B) but were histologically obvious in age- and sex-matched PN mice (Fig. 1C and 1D) . In both strains, the splenic red and white pulps were well organized. Expansions of the white pulp in PN, but not DBA/2, mice indicated follicular proliferation and active GC responses. Indeed, prominent GC were observed in the splenic follicles of PN mice in an age-dependent manner (Fig. 2 ). Newborn PN mice exhibited characteristic periarteriolar lymphoid sheath (PALS) structures when labeled by an antibody specific for CD4 and B-cell follicles containing no or few B cells that bound PNA (Fig. 2A) . With time (Fig. 2B 2C 2D 2E 2F) , PNA+ splenic B cells in PN mice became organized into large GC, causing expansion of the splenic follicles. As is typical of GC induced by immunization, the spontaneous GC present in older PN mice were generally in contact with the T-cell-rich PALS and surrounded by a follicular mantle of small B lymphocytes (e.g., Fig. 2D 2E 2F ).



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  		Figure 1. H&E staining of spleens from 3-month- and 12-month-old PN mice and age- and sex-matched DBA/2 control mice. (A) Three-month-old DBA/2, (B) 12-month-old DBA/2, (C) 3-month-old PN, and (D) 12-month-old PN. Magnification x100.



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  		Figure 2. Immunohistochemical staining of germinal centers in the spleens of PN mice of varying ages: (A) newborn, (B) 1 month, (C) 3 months, (D) 7 months, (E) 9 months, and (F) 12 months. The PNA+ GC B cells are stained red; CD4+ T cells are stained blue. These data are representative of five to seven mice studied at each of the indicated ages. Magnification x100.

Immunohistological methods were used to evaluate further the GC that spontaneously occur in PN mice. GC were not seen in the spleens of newborn PN mice; however, by 1 month of age, GC were present in the spleens of non-immunized, female PN mice (Fig. 2) . The size and the number of GC/spleen section did not increase significantly between 1 and 9 months of age (Fig. 3 ); however, 12-month-old PN mice exhibited significantly more GC/spleen section and significantly larger GC than 1-month- to 9-month-old PN mice.



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  		Figure 3. Kinetics of spontaneous GC formation in different strains of autoimmune mice. Data are presented as median number of germinal centers of different sizes (0 to 4+) per spleen section; bars indicate minimal and maximal values observed for a given group. Three to five mice at each age for each strain were studied, except for PN mice, where seven to nine animals were studied.

Several other mouse strains that spontaneously develop a lupus-like disease, including female NZB, NZB/W, MRL/lpr, MRL/++, and B6/lpr, and male BXSB mice, also exhibited spontaneous GC formation in their spleens (Fig. 3) . NOD mice that spontaneously develop autoimmune Type 1 diabetes mellitus also exhibit spontaneous GC formation in spleen. In contrast, non-immunized DBA/2 mice and female BXSB mice, which do not develop lupus, had few or no GC in their spleens. Between 2 months and 6–8 months of age, the size and number of GC/spleen section did not increase with age in female NZB, NZB/W, MRL/++, B6/lpr, or NOD mice. GC were present in the spleens of 2-month-old, female MRL/lpr mice but then decreased with age; at 6 months of age, GC were no longer detectable in the spleens of MRL/lpr mice (Fig. 4 ).



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  		Figure 4. Phenotype of cells in GC of 12-month-old PN mice. In all panels, PNA+ B cells are red. (A) BrdU+ cells are pink, and CD4+ cells are blue; (B) GL7+ B cells are blue; (C) FDC-M1+ cells are blue; and (D) B220+ cells are blue. These data are representative of seven mice studied. Magnification x200.

Phenotype of GC cells in autoimmune mice
Like the GC that develop after purposeful immunization, the GC of 3-month- (unpublished results) and 12-month-old (Fig. 4) PN mice were predominantly composed of PNA+, B220+, and GL7+ B cells. Approximately 5% of the cells in the GC were FDC that reacted with the FDC-M1 antibody, and approximately another 5% were CD4+ T cells, which were also prominently seen in the areas surrounding GC.

Anti-CD40L treatment induces the loss of germinal centers in PN mice
Cellular interactions mediated by CD40L and CD40 play fundamental roles in T-cell-dependent antibody production,B-cell proliferation and differentiation, expression of B-cell activation markers, isotype switching, and the generation of memory B cells [17 18 ]. Signaling through CD40 prevents apoptosis of germinal center B cells [17 18 19 20 21 ]; in vivo administration of the anti-CD40L mAb, MR-1, inhibits CD40-CD40L interaction and induces the loss of GC that has formed in recently immunized mice [8 22 ]. To determine if the GC present in the spleens of autoimmune-prone mice depend on CD40-CD40L interaction, we injected non-immunized 3-month- and 12-month-old, female PN mice and NP-CGG-immunized C57BL/6 mice with anti-CD40L mAb or control hamster Ig. Two days after the last injection, mice were sacrificed, and their spleens were removed for histological evaluation (Fig. 5 ). Large, well-formed GC were present in the spleens of 12-month-old, female PN mice (Fig. 5A) and NP-CGG-immunized, C57BL/6 controls (Fig. 5E) . Administration of anti-CD40L mAb caused the total loss of GC from the spleens of PN mice (Fig. 5B) and NP-CGG-immunized, C57BL/6 mice (Fig. 5E) . In contrast, injection of control hamster Ig had no effect on GC formation in PN (Fig. 5C) or immunized control mice (Fig. 5G) . GC were not present in the spleens of non-immunized C57BL/6 controls (Fig. 5D) . Anti-CD40L mAb and control hamster Ig had similar effects on GC in the spleens of 3-month-old PN mice (unpublished results).



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  		Figure 5. GC formation in the spleens of mice treated in vivo with anti-CD40L mAb or control hamster Ig. GC cells were visualized by staining with HRP-conjugated PNA (red) and biotinylated, anti-CD4 mAb followed by streptavidin-AP (blue). (A–C) Twelve-month-old PN mice untreated (A), after in vivo treatment with anti-CD40L mAb (B), and after in vivo treatment with control hamster Ig (C). (D) Control, non-immunized, untreated C57BL/6 mice. (E–G) NP-CGG-immunized C57BL/6 mice untreated (E), after in vivo treatment with anti-CD40L mAb (F), and after in vivo treatment with control hamster Ig (G). These data are representative of three mice studied per experimental group. Magnification x100.


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DISCUSSION
 
The GC reaction is a characteristic feature of T-cell-dependent, humoral immune responses. GC are necessary for the generation of memory B cells [23 24 25 ] and sites of Ig gene V-region hypermutation [3 4 5 6 ]. A few days after exposure to antigen, antigen-specific T cells and B cells migrate to the FDC-rich areas of lymphoid follicles to form GC. In GC, B cells proliferate rapidly and undergo isotype switching, somatic mutation, and selection for high affinities for the immunizing antigen [26 27 28 29 30 ]. GC also appear to have the ability to purge by apoptosis B cells that acquire reactivity to antigen(s) not present on the FDC surface [7 8 9 ]. Thus, antigen-driven activation of GC B lymphocytes results in death without survival signals mediated by the FDC and/or CD4+ T cells present in the GC environment. Survival signals are known to be mediated by CD40L present on T-helper cells [31 ] and C3d-decorated antigen held on the surface of FDC [32 ]. Thus, V(D)J mutations cannot widen the specificity of GC B cells to antigens that are not recognized by GC T cells or are not free in the circulation. These requirements normally act to prevent affinity maturation from creating self-reactivity.

Two laboratories have previously demonstrated the presence of GC in the spleens and lymph nodes of NZB/W mice [10 ] and anti-CD8, mAb-treated C3H/lpr and C3H/gld mice [11 ]. However, these studies did not document that the mice were free of adventitious infections that may have stimulated the GC reaction nor did they determine whether the observed GC were phenotypically normal. We demonstrate that in the absence of purposeful immunization and adventitious infection, strains of mice that typically develop an autoimmune, lupus-like disease or Type I diabetes mellitus exhibit spontaneous GC formation in the spleen. As determined by immunohistology, the splenic GC cells in autoimmune mice are similar to those that develop in normal mice after immunization. In both cases, GC are composed predominantly of B220+ and GL7+ B cells but also contain some CD4+ T cells and FDC-M1+. In addition, spontaneous and induced GC contain distinct light and dark zones, centroblasts, and tingible body macrophages and contain similar numbers of proliferating cells. Like the GC that develop in normal mice following immunization with a foreign antigen [8 ], GC in PN mice can be completely eliminated by in vivo treatment with anti-CD40L mAb.

Spontaneous GC develop by 1–2 months of age in the spleens of PN, NZB, NZB/W, BXSB, MRL/++, MRL/lpr, B6/lpr, and NOD mice. In PN mice, the size and numbers of GC/spleen section did not increase between 1 and 9 months of age (Fig. 3) ; by 12 months of age, PN mice were significantly larger, and more GC were present than 1-month- to 9-month-old PN mice. In the other autoimmune mice studied, except MRL/lpr, the GC size and numbers were also stable between 2 months and 7–8 months of age. It should be noted, however, that at 7–8 months of age, male BXSB mice and female NZB/W and MRL/lpr mice have a severe, systemic, lupus-like autoimmune disease similar in severity to that of 12-month-old PN mice; 50% mortality occurs at 5.0 months in male BXSB, 8.5 months in NZB/W, 6.0 months in MRL/lpr mice, and 12 months in female PN mice [33 ]. Thus, the increase in the size of GC and number of GC/spleen section that occurs late in disease in PN mice is not universally seen in other strains of lupus mice that have developed a similar degree of disease severity. The reason for this disparity is unknown. Carroll and his collaborators [32 ], however, have demonstrated that deficiencies in the early components of complement or complement receptors 1 and 2 (CD35 and CD21, respectively) reduce the GC response. As their autoimmune disease progresses, some strains of lupus mice (i.e., BXSB, NZB/W, and MRL/lpr) may generate sufficient levels of immune complexes to create complement deficiencies by consumption, which limit GC size and numbers; other lupus strains, like PN, do not.

In contrast to all of the other autoimmune strains of mice examined, the size of GC and number of GC/spleen section significantly decreased with age in MRL/lpr mice: GC were readily detectable in the spleens of 2-month-old mice but absent from the spleens of 6-month to 8-month-old mice. It is likely that the loss, with age, of GC from the spleens of MRL/lpr mice is related to the alteration in splenic architecture that results from the massive accumulation of double-negative (DN) T cells in the spleens of these mice. This possibility is supported by the observation that chronic anti-CD8 mAb treatment of C3H/lpr mice is associated with a marked reduction in DN T cells, the presence of prominent splenic GC [11 ], and our observation of prominent GC formation in 6-month-old B6/lpr mice that have less accumulation of DN T cells in spleen than 6-month- to 8-month-old MRL/lpr mice.

GC in autoimmune mice are likely to be the sites where mutated, self-reactive autoantibodies are generated. Consistent with this possibility is the observation that autoantibody production is apparent in female PN, NZB, NZB/W, MRL/lpr, MRL/++, and NOD mice and male BXSB mice by as early as 1–2 months of age [15 34 35 36 37 ], a time that correlates with the initial appearance of GC in the spleens of these mice.

CD40-CD40L interactions appear to be playing a critical role in the immunopathogenesis of murine lupus. The serum levels of nephritogenic, IgG antibodies to DNA, and nucleosomes (histone/DNA complexes) are markedly decreased in anti-CD40L-treated SNF1 mice. Brief treatment of prenephritic SNF1 mice at 3 months of age with anti-CD40L mAb reduces the incidence of severe nephritis at 12 months of age from 90% to 40% [38 ]. In addition, long-term treatment of SNF1 mice with already-established lupus nephritis prolongs survival and reduces the incidence of severe glomerulonephritis [39 ]. Similarly, in vivo treatment with anti-CD40L mAb inhibits the development of glomerulonephritis and prolongs survival in NZB/W mice [40 ]. Blockade of CD40-CD40L interaction also inhibits autoantibody production in mice with graft-versus-host disease-induced lupus [21 ]. CD40-CD40L interaction also has been shown to be important in several experimentally induced models of organ-specific autoimmune disease, including collagen-induced arthritis [41 ] and experimental allergic encephalomyelitis [42 ]. Our data document that short-term treatment of PN mice with anti-CD40L mAb completely eliminates pre-existing GC, demonstrating that CD40-CD40L interactions are critical for the maintenance of GC in PN mice. Furthermore, because pathogenic autoantibodies in autoimmune disease (especially lupus) are usually of the IgG isotype, and GC are critical sites where isotype switching and somatic hypermutation of V region genes occur, our data suggest that an important mechanism of action of anti-CD40L mAb in the treatment of autoimmunity may be the elimination of pre-existing GC that contain autoreactive B cells, which results in the inhibition of IgG autoantibody production.

In conclusion, we have demonstrated that beginning as early as 1–2 months of age, multiple strains of mice that spontaneously develop autoimmune disease—i.e., lupus or autoimmune Type I diabetes mellitus—spontaneously develop GC in spleen that are phenotypically similar to those that develop in normal mice following immunization with a foreign antigen. The continued presence of GC in the spleens of autoimmune mice is dependent on CD40-CD40L interactions: In vivo treatment of autoimmune mice with anti-CD40L mAb completely eliminates GC from the spleen. These findings indicate that the GC that arise spontaneously in autoimmune mouse strains share a common histological structure, cellular compartments, and physiology with GC induced by immunization. These similarities may indicate that an aberrant GC reaction is not a cause of humoral autoimmunity but a marker of its progression. For example, if nuclear antigens and apoptotic cell debris cannot be cleared effectively, these normally cryptic antigens might reach immunogenic levels and initiate antibody responses. In this case, immunological ignorance, not tolerance, would be broached, and the resulting GC response would be entirely physiologic. Conversely, there is good evidence for the ability of the GC microenvironment to ensure the maintenance of B-cell and T-cell tolerance [7 8 9 43 ]. Thus, if this capacity of the GC were impaired—as it may be in mice deficient for various apoptotic pathways—the GC reaction could generate autoimmunity as well as expand and refine it.


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ACKNOWLEDGEMENTS
 
This work was supported by the Maryland Chapter of the Arthritis Foundation (I. G. L.), U.S. Scleroderma Foundation (S. P. A.), National Institutes of Health (grants AI27885 and AI24335 to G. K.), and U. S. Department of Veteran Affairs (Merit Review to B. S. H.). The authors thank Jordan M. Denner, RBP, Chief, Medical Production Service, Department of Veterans Affairs Medical Center, Baltimore Maryland, for his expert assistance in the preparation of photoimages.

Received December 27, 2000; accepted May 3, 2001.


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