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The Journal of Immunology, 1998, 161: 4549-4554.
Copyright © 1998 by The American Association of Immunologists

Evidence for an Important Interaction Between a Complement-Derived CD21 Ligand on Follicular Dendritic Cells and CD21 on B Cells in the Initiation of IgG Responses1

Dahui Qin*, Jiuhua Wu*, Michael C. Carroll{ddagger}, Gregory F. Burton*, Andras K. Szakal{dagger} and John G. Tew2,*

Departments of * Microbiology and Immunology and {dagger} Anatomy, Division of Immunobiology, Medical College of Virginia, Virginia Commonwealth University, Richmond, VA 23298; and {ddagger} Department of Pathology, Harvard Medical School, Boston, MA 02115


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The addition of Ags to mononuclear leukocyte cultures typically elicits modest Ab responses, implying that cosignals beyond those provided by T cells and macrophages may be needed. Recently, we reported that Ab responses could be dramatically enhanced (10–1000-fold) by the addition of follicular dendritic cells (FDC), suggesting that FDC may provide an important costimulatory signal. This result prompted a study of molecules involved in FDC-mediated enhancement of Ab responses stimulated by specific Ag with memory T and B cells or nonspecifically by the addition of LPS. In this study, we report evidence supporting the concept that FDC bear a ligand that engages complement receptor II (CR2 or CD21) on B cells and provides a critical cosignal for both Ag-specific and polyclonal responses. A blockade of the CR2 ligand on FDC by the use of soluble CR2 or a blockade of CR2 on B cells by use of CR2 knockout mice (or B cells with CR2 blocked) reduced Ab responses from the µg/ml to the ng/ml range (10–1000-fold reductions). FDC from C3 knockout mice, which cannot generate the CR2-binding fragments (iC3b, C3d, and C3dg), were unable to provide costimulatory activity, suggesting the CR2 ligand on FDC consists of C3 fragments. FDC trap complement-activating Ag-Ab complexes, and it appears that FDC present B cells with both specific Ag to engage B cell receptors and a CR2 ligand to engage B cell-CR2. In short, optimal induction of specific Ab responses appears to require the combination of specific Ag and costimulatory molecules from both T cells and FDC.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
In addition to the signals delivered by specific Ag, costimulatory signals are known to be important for induction of efficient immune responses (1, 2). Although CD40L and T cell-derived lymphokines are known to provide important cosignals, the addition of Ag to cultures containing both B cells and T cells generally elicits only modest responses when compared with responses in vivo (3), suggesting that other cosignals beyond those provided by the T cells may be important. Identification of such cosignals may be crucial to our understanding of immune responses and may provide new options for modulating Ab responses. Apart from CD40, it is known that CD21 on B cells can serve as a coreceptor that can dramatically enhance Ab responses (4, 5, 6, 7). However, the locations where CD21L-CD21 interactions may occur and the physiology involved in presenting the ligand(s) that engage B cell-CD21 are not clear.

Germinal centers are well-known "hot spots" where B memory cells and Ab-forming cells (AFC)3 are generated (8, 9, 10). Follicular dendritic cells (FDC) are an important component of the germinal center microenvironment, where they form an extensive network with their long, slender dendrites in the light zones (11, 12). The FDC dendrites forming these networks bear immune complexes with C3 fragments (iC3b, C3d, and C3dg) attached to the immune complexes and adjacent cell membranes via ester linkages (13, 14). These C3 fragments are known CR2 ligands (CR2L) and are bound to FDC surfaces that interface with B cells in the area (13, 14). Furthermore, recent data indicates that immune complex bearing FDC provide a potent costimulatory signal needed to optimize T cell-dependent Ab responses (15). These relationships prompted the hypothesis that these complement-derived CR2L on FDC bind the CR2 coreceptor on B cells, leading to optimal Ab responses in the presence of Th cells. The results reported here demonstrated that when the interactions of FDC-CR2L and B cell-CR2 were interrupted either by blocking CR2 on B cells or by blocking CR2L on FDC, the FDC-enhanced Ab responses were substantially suppressed, indicating that the interaction between FDC-CR2L and B cell-CR2 is critical for an optimal Ab response. This CR2L on the FDC may provide an important target for modulating Ab responses and altering the outcome of a variety of diseases where Ab plays an important role.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

Female BALB/cByJ, C57BL mice (H-2b), 6 to 8 wk of age, were purchased from The Jackson Laboratory (Bar Harbor, ME). C3 knockout, CR2 knockout, and control mice with (H-2b) background were provided by Dr. M. C. Carroll (4, 16). The mice were housed in standard plastic shoe-box cages with filter tops. Food and water were supplied ad libitum, and the mice were used between 8 to 20 wk of age.

Immunization

Mice were primed by injecting 100 to 200 µg of OVA or human serum albumin (HSA) (catalogue numbers A5503 and A3782, Sigma, St. Louis, MO) precipitated with aluminum potassium sulfate (catalogue number A7167, Sigma) in the nape of the neck as described previously (15, 17). Secondary immunizations were done 2 wk later by i.p. injection and s.c. into the front legs and hind feet (20 µg aluminum potassium sulfate Ag/site).

Purification of soluble CR2

The culture supernatant fluid of (CR2)2-IgG1, a chimeric molecule of CR2 and mouse IgG1, cell line (7) (T-Cell Sciences, Cambridge, MA) was pooled and the proteins in the supernatant fluid were precipitated using saturated ammonium sulfate. After dialysis, (CR2)2-IgG1 was purified on a 5-iodo-4-hydroxy-3-nitrophenacetyl (NIP)-caproate Sepharose affinity column. The concentration of (CR2)2-IgG1 was quantitated using Coomassie Protein Assay Reagent stain (Pierce, Rockford, IL) and ChromPure mouse whole IgG was used as the standard (catalogue number 015-000-003, Jackson ImmunoResearch Laboratories, West Grove, PA). The purified (CR2)2-IgG1 was filtered using centrifuge tube filter 0.22 mm cellulose acetate (catalogue number 8160, Costar, Cambridge, MA) and stored at 4°C.

FDC isolation

FDC were isolated from the draining lymph nodes (popliteal, brachial, axillary, inguinal, periaortic, and mesenteric) using procedures as described previously (17, 18, 19, 20) except that higher levels of irradiation were used (15). FDC preparations were incubated with soluble CR2 (5 mg/ml or different doses when doses responses were pursued; see Results) or isotype control (mouse IgG1) for 30 min at 4°C. Then, the treated FDC and the soluble CR2 in the incubation were transferred to lymphocyte cultures. FDC from C3 knockout mice were isolated in the same way, but without soluble CR2 treatment.

Lymphocyte preparation

Memory lymphocytes were obtained from draining lymph nodes of immunized BALB/c mice a month or more after the final Ag challenge (19). The lymphocyte preparations containing B cells were incubated with anti-CR1,2 or anti-CR1 or isotype control (rat IgG2b or rat IgG2a) mAb (5 mg/ml) for 30 min at 4°C. Then, the treated lymphocytes and the Abs in the incubation were transferred to cultures with FDC.

Cell cultures

Enriched FDC preparations (1 x 105 cells) were added to 3 x 105 B and T cells in 96-well tissue culture plates (catalogue number 3595, Costar) containing 200 µl complete culture medium per well. The culture medium used in all studies consisted of DMEM supplemented with 10% FCS, 20 mM HEPES, 2 mM glutamine, 50 µg/ml gentamicin, and MEM-nonessential amino acids. The cell cultures were incubated at 37°C in a 5% CO2 incubator for 14 days. At day 7, culture media were replaced. Abs produced from day 7 to day 14 were measured using ELISA.

ELISA

Ag-specific IgG or total IgG were measured by means of a solid-phase ELISA as described by Helm et al. (21). The Immuno 4 ELISA plates were coated with OVA (0.1 mg/ml), HSA (0.1 mg/ml), or goat anti-mouse IgG (10 mg/ml) (catalogue number 115-005-003, Jackson ImmunoResearch Laboratories). The culture supernatant fluid harvested from day 7 to 14 was used to measure Ab levels. Murine IgG specifically bound to the coated plates were detected using biotinylated goat anti-mouse IgG (Southern Biotechnology Associates, Birmingham, AL) and alkaline phosphatase-labeled streptavidin (Kirkegaard & Perry Laboratories, Gaithersburg, MD). Mouse anti-OVA or mouse anti-HSA, or ChromPure mouse IgG (catalogue number 015-000-003, Jackson ImmunoResearch Laboratories) were used as standard curves. The anti-OVA and anti-HSA serum was collected from hyperimmunized BALB/c mice, and the Ab level in the serum was determined using quantitative precipitin analysis (22).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Inhibitory effect of soluble CR2 on in vitro IgG production

To determine whether interaction of FDC-CR2L and B cell-CR2 is important for Ab responses, a previously described in vitro model was used (15). Briefly, FDC were isolated from OVA-immunized mouse lymph nodes 3 days after Ag challenge when the FDC are known to bear OVA and to be active in releasing iccosomes (15). Typically, when memory B and T cells are cultured with soluble OVA, the produced concentrations of anti-OVA IgG are no more than 200 ng/ml. However, in the presence of OVA-bearing FDC, µg/ml levels of anti-OVA IgG are generated by the memory cells (15). To determine whether CD21L on FDC plays a role in enhancing Ab responses, (CR2)2-IgG1 was added to block any CR2L on FDC. As the dose of soluble CR2 increased, the anti-OVA IgG response was reduced to less than 10% of the uninhibited isotype control (Fig. 1Go).



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  		FIGURE 1. Inhibitory effect of soluble CR2 on anti-OVA IgG production in vitro. OVA-primed lymphocytes (LyOVA) were isolated from OVA-immune mouse lymph nodes. OVA-bearing FDC (FDCOVA) were isolated from OVA-immune mouse lymph nodes 3 days after the challenge. Lymphocytes were cultured in a 96-well culture plate (3 x 105/well) with FDCOVA (5 x 104/well). Different doses of soluble CR2 ((CR2)2-IgG1) or isotype control were added to LyOVA + FDCOVA cultures, respectively. The culture supernatant fluids were harvested on day 7, and new culture medium was added. Anti-OVA IgG in the supernatant fluid was measured with ELISA on day 14. The samples in each group were done in triplicate, and the result is representative of three experiments of this type.

 
The ability of (CR2)2-IgG1 to inhibit specific immune responses was further tested using preformed immune complex as an immunogen (Fig. 2Go). In this case, B and T cells were isolated from HSA-immunized mice but the FDC were isolated from nonimmunized mice, where the Ags in the immune complexes on the FDC would be limited to environmental Ags. HSA-anti-HSA complexes were added as the source of Ag (10 ng/well). Note that the immune complexes are poorly immunogenic in the absence of FDC. However, the addition of FDC dramatically enhanced the ability of preformed immune complexes to induce an Ab response in vitro as has been observed by Wu et al. (unpublished results). Notice that the addition of soluble CR2 resulted in a marked reduction in the ability of FDC to provide costimulatory activity and enhance anti-HSA production induced by preformed immune complexes (Fig. 2Go). Interestingly, a similar result was obtained when LPS was used to induce a polyclonal IgG response (Fig. 3Go).



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  		FIGURE 2. Effect of blocking FDC-CR2L on anti-HSA Ab responses. The HSA-primed lymphocytes (LyHSA) were isolated from HSA-immune mouse lymph nodes. LyHSA (3 x 105/well) were cultured with FDC (5 x 104/well) in the presence of preformed HSA-anti-HSA (HSA-IC, 10 ng/ml). The CR2L on FDC were blocked using soluble CR2:(CR2)2-IgG1, and mouse IgG1 was used as control. Culture supernatant fluids were replaced at day 7. Anti-HSA IgG accumulated in supernatant fluids from day 7 to day 14 were measured using ELISA. The samples in each group were done in triplicate, and the result is representative of three experiments of this type.

 


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  		FIGURE 3. Effect of blocking FDC-CR2L on Ab responses induced by LPS. Lymphocytes (Ly) were cultured (3 x 105/well) with FDC (5 x 104/well) in the presence of LPS (10 mg/ml). The CR2L on FDC were blocked using soluble CR2:(CR2)2-IgG1, and mouse IgG1 was used as control. Culture supernatant fluids were replaced on day 7, and total mouse IgG accumulated in supernatant fluids from day 7 to day 14 were measured using ELISA. The samples in each group were done in triplicate, and the result is representative of three experiments of this type.

 
Comparison of wild-type and C3 knockout FDC

To determine whether a complement-derived CR2L is involved in delivering cosignal rather than other potential ligands that might be on FDC (23), C3 knockout mice were used as a source of FDC. These mice lack C3 (16) and consequently cannot generate iC3b, C3d, or C3dg. We reasoned that the FDC isolated from the C3 knockout mice would not bear CR2L generated from C3 fragments and would not be able to enhance humoral immune responses as well as wild-type FDC. To test this prediction, FDC were isolated from lymph nodes of C3 knockout mice and were used in lymphocyte cultures. The results showed FDC from these C3 knockout mice were much less efficient in enhancing LPS-induced IgG responses (Fig. 4Go).



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  		FIGURE 4. Comparison of costimulatory activity between FDC from C3 knockout mice and wild-type FDC. C3 knockout FDC (FDCC3-/-) were isolated from the lymph nodes of C3 knockout mice, and wild-type FDC (FDCC3+/+) were isolated from the control mice lymph nodes. Both FDCC3-/- and FDCC3+/+ were cultured in a 96-well culture plate (5 x 104/well) with wild-type lymphocytes (3 x 105/well), respectively, in the presence of LPS (10 mg/ml). The culture supernatant fluids were harvested on day 7, and new culture media were added. Total IgG in the supernatant fluid on day 14 was measured with ELISA.

 
Effect of blocking CR2 on B cells

We reasoned that C3 fragments on FDC enhance Ab responses by engaging CR2 on B cells and we predicted that the FDC-derived cosignals should be blocked if CR2 on the B cell were blocked. To test this prediction, mAbs for CR2 (7G6, binds both CR1 and CR2), and CR1 (8C12) (6) were used to mask CR2 and/or CR1 on B cells in our in vitro system, and Ab responses were monitored. The anti-CR1 and -CR2 (7G6) suppressed 80 to 90% of the Ab responses induced by HSA (Fig. 5GoA), OVA (Fig. 5GoB), or LPS (Fig. 5GoC). The anti-CR1 (8C12) suppressed about 20 to 50% of the Ab responses (Fig. 5GoC), but the isotype controls for these Abs were without inhibitory effect.



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  		FIGURE 5. Effects of Abs reactive with CR1 and CR2 on specific IgG responses and polyclonal IgG responses. Abs, {alpha}-CR1,2 (7G6), {alpha}-CR1 (8C12), or isotype controls (used at 10 mg/ml), were incubated with B cells in the cold for 30 min to mask CR1 and CR2 or CR1 only before the B cells were set up in culture. Culture media were replaced on day 7, and Ag-specific IgG or total IgG accumulated in supernatant fluids from day 7 to day 14 were measured using the appropriate ELISA. A, HSA-specific memory T and B lymphocytes (LyHSA) were cultured with HSA-bearing FDC (FDCHSA), which were isolated from HSA-immunized mice 3 days after Ag challenge. Note the dramatic inhibitory effect of {alpha}-CR1,2 on the anti-HSA response. B, OVA-specific memory lymphocytes (LyOVA) were incubated with FDC from normal animals in the presence of preformed OVA-anti-OVA (OVA-IC, 10 ng/ml), and once again {alpha}-CR1,2 was markedly inhibitory. However, note that {alpha}-CR1 also had some inhibitory activity. The inhibitory effects of {alpha}-CR1,2 and {alpha}-CR1 are also apparent in C, where lymphocytes were stimulated with LPS (10 mg/ml) rather than specific Ag, and total IgG is being measured. In all cases, {alpha}-CR1,2 was a potent inhibitor, {alpha}-CR1 was a more modest inhibitor, and the isotype controls were without inhibitory effect.

 
CR2 knockout B cells and FDC mediated costimulation

To confirm the need for B cell CR2, CR2-null B cells were studied in cultures with wild-type FDC. While FDC markedly enhanced LPS-induced Ab responses in wild-type B cells, the Ab responses obtained from B cells from CR2-null mice were only about 10% of normal (about 3,000 ng/ml compared with 30,000 ng/ml), and this response was achieved only at the highest dose of LPS (Fig. 6Go). Notice that the Ab response induced by LPS alone in the CR2-null B cells was slightly higher than that obtained in wild-type B cells, indicating that the ability of the CR2-null B cells to respond to LPS was not depressed; this is consistent with previous work (4). In short, it appeared that the ability of the CR2-null B cells to receive the costimulatory signals from the FDC was aberrant.



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  		FIGURE 6. FDC-costimulated LPS induce IgG response in wild-type B cells but not in CR2 knockout B cells. Lymphocytes were isolated from CR2 knockout mice (LyCR2-/-) or control mice lymph nodes (LyCR2+/+). FDC were isolated from normal mouse lymph nodes. Both LyCR2-/- and LyCR2+/+ were cultured in a 96-well culture plate (3 x 105/well) with or without FDC (5 x 104/well), respectively. Different doses of LPS were added to cultures. The culture supernatant fluids were harvested on day 7, and new culture media were added. Total IgG in the supernatant fluid on day 14 was measured with ELISA. The samples in each group were done in triplicate, and the result is representative of three experiments of this type.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Immunogens are converted into immune complexes as soon as they encounter natural Ab or Ab induced in the primary response, and these Ag-Ab complexes are trapped by FDC (24). When Ab is present from prior immunization(s), Ag-Ab complexes form almost instantaneously and are trapped by Ag-transporting cells (ATC) and are transported to the lymph node follicles within minutes (25, 26). As the ATC begin entering the follicles, the draining lymph nodes undergo a series of dynamic changes including "germinal center dissociation" (27, 28). These rapid changes after immunization contrast with the rather static state of the follicle before immunization. Changes include the observation that the germinal centers become edematous, the peanut agglutinin (PNA)-positive B cells that make up the majority of cells in the germinal center leave, and PNA-positive cells can be found leaving the area in the thoracic duct lymph (9, 28, 29). At the same time, ATC can be seen entering the area with lymphocytes in tight association (25). These ATC-associated lymphocytes are thought to be memory B cells from the surrounding area that are transported into the developing germinal center. These B cells entering with the ATC are thought to be precursors to the AFC that emerge from the germinal centers in the period 3 to 5 days after immunization, as well as to the new generation of memory B cells that emerge during the next week or two (9, 28). We reason that the FDC-associated CD21L is critical to the generation of the AFC in the germinal center, which then migrate to the bone marrow where the vast majority of Ab is made during a secondary Ab response (8, 9). A few micrograms of Ag is sufficient to elicit a potent secondary response in spite of the fact that the vast majority of Ag is catabolized by phagocytic cells (25, 26). This suggests that a mechanism exists in vivo to utilize small quantities of Ag-Ab complexes in a highly efficient fashion. The results of the present study support the concept that CR2L on FDC delivers a potent costimulatory signal by engaging CR2 on B cells and this may be an in vivo mechanism to utilize small quantities of Ag-Ab complexes in a highly efficient fashion for Ab responses in the presence of Th cells.

Recent studies using fusion proteins of C3d and hen egg lysozyme demonstrate a 1,000- to 10,000-fold increase in efficiency of the response when the immunogen includes C3d, and this C3d effect is dependent upon the presence of a high density of C3d. In these studies, it was possible to get Ab responses using only low picomolar concentrations of Ag (30). Interestingly, the level of Ag retained on the FDC is very low (in the picomolar range) and only a few picograms of FDC-Ag is capable of eliciting potent immune responses (15). In some experiments, we have been able to get good Ab production using less than a picogram of FDC-associated Ag (our unpublished observations). This ability of FDC to stimulate high Ab responses may relate to the fact that FDC are heavily decorated with C3 fragments (13). Thus, the efficient presentation of Ag by the FDC may be explained by the combination of both Ag and the CR2L costimulator. This is consistent with the increase in efficiency found by Carter and Fearon (31) when they were able to reduce the requirement for B cell receptor (BCR) stimulation by two orders of magnitude by simultaneous stimulation of the CD19/CD21/TAPA-1 complex. To address the question if CR2L on FDC is an important source of the signals, a soluble (CR2)2-IgG1, which is a chimeric molecule of CR2 and mouse IgG1 and can mask CR2L (7), was introduced into our in vitro Ab response system to mask CR2L on FDC. The results showed that when CR2L on FDC were blocked by soluble (CR2)2-IgG1, the FDC-derived enhancement effect was diminished, indicating that CR2L on FDC is important for Ab responses. This was confirmed using FDC from C3 knockout mice. No C3 was generated in these mice and no CR2L from C3 fragments was on FDC. The results showed that FDC from these mice do not have a comparable enhancement effect, which confirms that CR2L on FDC is important for Ab responses and also indicates that C3 fragments are a major source of CR2L on FDC.

As reported here, the addition of FDC to LPS-stimulated B cells resulted in a dramatic increases in Ab production. This is probably not attributable to co-cross-linking of BCR and CD19 but likely relates to the delivery of two signals: one signal by the LPS and a second by the cross-linked CD19 on the B cell. Co-cross-linking of BCR and CD19 is a highly efficient mechanism for signaling B cells, but the LPS results indicate that the co-cross-linking is not obligatory. Our LPS results are consistent with work indicating that polymeric CR2 or microgram levels of CR2L attached to albumin can deliver a signal that markedly amplifies B cell responses induced by polyclonal activators (32, 33). We think it is important to appreciate that the dendritic processes of the FDC nearly surround the B cells (11, 25), and it is likely that large numbers of CR2 receptors are occupied by the FDC-CR2L. It is also probably true that large numbers of CR2 receptors are occupied when polymeric CR2L or microgram levels of CR2L attached to albumin are used to deliver a signal. It is also clear that LPS at high concentrations was able to stimulate B cells from CR2-null mice where the FDC-CR2L would not have been a factor. It was at the lower doses of LPS that the stimulation by CR2L on FDC was most important. This appears to be consistent with the reports indicating that high doses of Ag are also less dependent on CR2 stimulation (14).

We have noted in previous work that the presence of FDC promotes survival of lymphocytes in culture (20). It is known that FDC have the ability to protect B cells from apoptosis, including Fas-mediated apoptosis (34, 35, 36, 37), and this may help explain why the presence of FDC is associated with increased production of Ab. A recent paper indicates that B cells from CR2-null mice can enter the germinal centers but they do not persist (38), suggesting that the CR2 receptor may be critical for the FDC to block apoptotic signals. In short, it is likely that the ability of the FDC to protect B cells from apoptosis and improve lymphocyte survival does make a contribution to the increased Ab response observed in the presence of the FDC.

The results reported here confirm and extend in vivo studies showing that C3 and CR2 are critical for normal Ab responses (4, 7, 16, 39, 40, 41, 42). It appears that CD21L-CD21 interactions likely occur in germinal centers. A recent report by Vora et al. (43) makes it clear that FDC lacking Fc receptors do trap the Ag-Ab complexes and germinal center reactions are elicited. Clearly, FDC have complement receptors that can be used to trap the Ag. To meet the functional requirements defined here, the FDC would need to trap immune complexes and the immune complexes would need to activate the complement cascade and provide the CD21L. The critical issue is for the FDC to be able to present B cells with both specific Ag to engage BCR and a CR2L to engage B cell-CR2. A blockade of CR2L on FDC or BCR reduces in vitro Ab responses 10- to 1000-fold, which is compatible with the results in vivo (7, 44, 45).


   Acknowledgments
 
We thank T-Cell Sciences, Cambridge, MA, for providing soluble CR2 and R. Berry for setting up the NIP affinity column.


   Footnotes
 
1 This work was supported by National Institutes of Health Grant AI-17142. Back

2 Address correspondence and reprint requests to Dr. John G. Tew, Department of Microbiology/Immunology, P.O. Box 980678, MCV Station, Richmond, VA 23298-0678. Back

3 Abbreviations used in this paper: AFC, Ab forming cell; FDC, follicular dendritic cells; CR1, complement receptor I; CR2, complement receptor II; CR2L, ligand for complement receptor II; BCR, B cell receptor; HSA, human serum albumin; (CR2)2-IgG1, chimeric molecule of CR2 and mouse IgG1; 7G6, a blocking mAb for CR2 and CR1; 8C12, a blocking mAb for CR1. Back

Received for publication March 20, 1998. Accepted for publication June 24, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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