* INRS-Institut Armand-Frappier/Santé Humaine, Université du Québec, Canada; and
Department of Internal Medicine, University Hospital Zürich, Switzerland
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: signal transduction inhibitors • cytology • flow cytometry • gelsolin
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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12 h in the circulation). Cell
turnover must therefore be under strict control. In this sense,
neutrophils are known to spontaneously undergo apoptosis without
addition of special reagents in the milieu. Normally, when freshly
isolated human neutrophils are incubated for 24 h (37°C, 5%
CO2) in RPMI-1640 supplemented with fetal calf serum or
autologous serum (ranging from 5–10%), about 30–60% of cells will
be in apoptosis, depending on the donor [1
2
3
4
]. Because
they have been established as terminally differentiated non-dividing
cells, neutrophils were erroneously characterized as cells with little
activity, namely with respect to their capacity to synthesize proteins.
However, it is becoming increasingly clear that these cells are more
capable of protein synthesis than previously thought
[3
4
5
6
7
8
9
10
]. Various cytokines are among proteins that are
synthesized by neutrophils and this indicates that these cells have the
potential to perform biological activities in both afferent and
efferent arms of the immune response. Among these cytokines, some are
potent modulators of neutrophil apoptosis such as interleukin-8 (IL-8)
and tumor necrosis factor 
(TNF-) [5
,
6
, 9
, 11
]. Now it is generally
accepted that neutrophils play roles in host response that extend well
beyond their capacities for phagocytosis and releasing cytotoxic
compounds [12
]. The importance of studying and
discovering molecules that can modulate the neutrophil apoptotic rate
resides in the fact that clearance of apoptotic neutrophils by cells
such as macrophages leads to the resolution of inflammation
[13
14
15
]. In addition to this, deregulation of normal
cell turnover via modulation of apoptosis may lead to cancer or
autoimmunity. Viscum album agglutinin-I (VAA-I) is a galactoside-specific plant lectin that possesses a molecular mass of 63 kDa [16 , 17 ]. It consists of two distinct subunits, the A chain (29 kDa) and the B chain (34 kDa). The A chain confers the protein synthesis inhibitory property to the VAA-I molecule by acting as a ribosome-inactivating agent. This is due to RNA-glycosidase activity that inhibits N-glycosilation of a single adenine within a universally conserved GAGA sequence on the 28S rRNA (at position 4324 in rat liver cells) [18 , 19 ]. The B chain allows the VAA-I molecule to bind to terminal galactoside residues on the membrane of various cells. VAA-I belongs to the family of type II ribosome-inactivating proteins including abrin, modeccin, and ricin. VAA-I was recently found to act as an immunomodulator [16 17 18 19 20 21 22 23 24 ]. This lectin is known to induce pro-inflammatory cytokine expression at both gene and protein levels in human peripheral blood mononuclear cells [17 ] and to induce apoptosis in human lymphocytes, monocytes, monocytic THP-1 cells, and murine thymocytes [16 ]. The recombinant form of the lectin (rVAA) was recently found to augment the secretion of IL-12 and to potentiate the cytokine-induced natural killer (NK) cell activation [22 ].
It was previously demonstrated, by flow cytometry, that fluorescein isothiocyanate (FITC)-conjugated VAA-I binds to monocytes and granulocytes with higher affinity than on lymphocytes [17 ]. Studies dealing with VAA-I/neutrophil interactions are limited and data reveal that VAA-I induces neutrophil aggregation, O2- and H2O2 production, and phagocytosis [25 26 27 ]. Because of the potential therapeutic use of VAA-I, at least in cancer [23 , 24 ], and its great capacity to modulate the immune response, it is important to better understand the interaction of VAA-I and human neutrophils for therapeutic purposes. Knowing that these cells are at the basis of an inflammatory reaction and that they can produce various distinct proteins, it is also important to better understand how VAA-I can alter both de novo protein synthesis and the neutrophil apoptotic rate.
In this study, we report that VAA-I can up-regulate or down-regulate de novo protein synthesis of human neutrophils depending on the concentration used and that its ability to inhibit de novo protein synthesis correlates well with its ability to induce neutrophil apoptosis. Moreover, we bring evidence that VAA-I-induced-neutrophil apoptosis is a caspase-dependent mechanism.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Isolation of VAA-I
The plant lectin VAA-I derived from V. album was
isolated and purified as previously published, and endotoxin
contamination was measured by Limulus amebocyte lysate
(LAL) assay and was <5 pg/mL [21
]. The purity
was verified by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE; data not shown). As other agents used in the
study, the lectin was present in the culture for the indicated period
of incubation. Stimulation of cells was performed in 96-well plates,
without agitation, at 37°C, 5%CO2, for the de
novo protein synthesis assay and in 24-well plates for apoptosis
[3
, 4
, 9
, 10
];
whereas they were incubated in 5 mL polystyrene tubes in a water bath
(37°C), under light agitation, for the tyrosine phosphorylation assay
[9
].
Neutrophil isolation
Cells were isolated from venous blood of healthy volunteers by
centrifugation over Ficoll-Hypaque as described previously
[3
, 4
, 9
, 10
,
28
]. Blood donations were obtained from informed and
consenting individuals according to institutionally approved
procedures. Cell viability (>99%) was monitored by Trypan blue
exclusion, and the purity (>98%) was verified by cytology from
cytospin preparations followed by Diff-Quick staining [3
,
4
, 9
]. Treatment of neutrophils with VAA-I
(0.1 through 1000 ng/mL in RPMI-1640 supplemented with 10% autologous
serum) did not induce cell necrosis in contrast to methylmercury after
24 h of treatment (Fig. 1
).
 View larger version (88K): [in a new window] |
Figure 1. VAA-I and neutrophil cytotoxicity. Freshly isolated human cells were
incubated in the presence of increasing concentrations of VAA-I
(0.1–1000 ng/mL) or with methylmercury chloride (CH3HgCl),
a pesticide known to induce cell necrosis, and cell viability was
monitored for up to 24 h as described in Materials and Methods.
(A) cells incubated with 1000 ng/mL VAA-I for 24 h; (B) cells
incubated with 10-4 M CH3HgCl for 24 h.
Note that CH3HgCl induces necrosis as the trypan blue dye
penetrates into cells but not VAA-I. Results are representative of at
least 10 experiments conducted with different blood donors.
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Assessment of neutrophil apoptosis by cytology and by flow
cytometry
Freshly isolated human neutrophils (100 µL of a 10 x
106 cells/mL suspension in RPMI-1640 supplemented with 10%
autologous serum) were incubated for indicated times in the presence or
absence of VAA-I or appropriate controls as indicated in the figure
legends, and apoptosis was evaluated by cytology and flow cytometry.
Cytocentrifuge preparations of neutrophils were performed with a
Cyto-tek® centrifuge (Miles Scientific) essentially as
previously described and were stained with a Diff-Quick staining kit
(Baxter, FL), according to the manufacturer’s instructions
[3
, 4
, 9
, 10
].
Cells were examined by light microscopy at x400 final magnification,
and apoptotic neutrophils were defined as cells containing one or more
characteristic darkly stained pyknotic nuclei. An ocular containing a
10 x 10-square grill was used to count at least five different
fields (>100 cells) for assessment of apoptotic cells. Results were
expressed as percentage of apoptotic cells. For the flow cytometric
procedure, apoptosis was investigated using FITC-Annexin-V/PI labeling
as previously published [29
, 30
]. Ten
thousand cells were analyzed by FACS Calibur (Becton-Dickinson, San
Jose, CA) using CellQuest program [17
]. The number of
late apoptotic and necrotic cells were subtracted in order to evaluate
the percentage of apoptotic cells.
Tyrosine phosphorylation
Neutrophils (100 µL of 40 x 106 cells/mL in
RPMI-1640) were incubated for 1, 5, 15, or 30 min at 37°C with buffer
alone, GM-CSF (65 ng/mL), or increasing concentration of VAA-I (1–1000
ng/mL) in a final volume of 120 µL. Reactions were stopped by adding
120 µL of a 2x Laemmli sample buffer as we have detailed elsewhere
[9
]. Aliquots corresponding to 1 x 106
cells were loaded onto 10% SDS-PAGE and transferred from gel to PVDF
membranes (Millipore, Bedford, MA) according to Towbin et al.
[31
]. Nonspecific sites were blocked with 2% gelatin in
TBS-Tween (25 mM Tris-HCl, pH 7.8, 190 mM NaCl, 0.15% Tween-20) for
1 h at 37°C. The monoclonal anti-phosphotyrosine UB 05-321
(1:5000) was then incubated with membranes for 1 h at 37°C
followed by washes, and incubated with a horseradish peroxidase-labeled
sheep anti-mouse IgG (1:15,000, Bio/Can) for 1 h at 37°C in
fresh blocking solution. Membranes were washed three times with
TBS-Tween, and phosphotyrosine bands were revealed with the enhanced
chemiluminescence (ECL) Western blotting detection system (Amersham,
Pharmacia Biotech). Protein loading was verified by staining the
membranes with Coomassie blue at the end of the experiments.
Fragmentation of gelsolin
The degradation of gelsolin into an 41-kDa fragment was
recently found to be dependent on caspase-3 activity during neutrophil
apoptosis [32
]. We next verify whether VAA-I can induce
such fragmentation in VAA-I-induced neutrophil apoptosis. Neutrophils
(106 cells/mL in 24-well plate) were incubated with or
without 500 ng/mL VAA-I for 24 h and then harvested for the
preparation of cell lysates in Laemmli sample buffer. In other wells,
cells were pre-incubated 1 h with the diluent or 50 µM z-VAD-FMK
before an additional 23 h of incubation (total period of 24 h). Aliquots corresponding to 1 x 106 cells were
loaded onto 10% SDS-PAGE and transferred from gel to PVDF membranes as
for the tyrosine phosphorylation assay. Nonspecific sites were blocked
with 1% bovine serum albumin (BSA) in TBS-Tween (25 mM Tris-HCl, pH
7.8, 190 mM NaCl, 0.15% Tween-20) for 1 h at 37°C. Membranes
were incubated with monoclonal anti-human gelsolin (Sigma, clone
GS-2C4) in a final dilution of 1:500 for 1 h at 37°C followed by
washes, and incubated with a horseradish peroxidase-labeled sheep
anti-mouse IgG (1:15,000; Bio/Can) for 1 h at 37°C in fresh
blocking solution. The other steps were performed exactly as described
for the tyrosine phosphorylation assay.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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 View larger version (72K): [in a new window] |
Figure 2. Modulation of de novo protein synthesis by VAA-I. Freshly
isolated human neutrophils were metabolically labeled as described in
Materials and Methods and incubated for 22 h in the presence or
absence of stimuli. Intracellular and extracellular fractions were
prepared and then run by SDS-PAGE. (A) cell lysates prepared from cells
treated with 1, 100, or 1000 ng/mL VAA-I (lanes 1, 2, and 3,
respectively), 65 ng/mL GM-CSF (lane 4), 10 µg/mL cycloheximide (lane
5), or buffer (lane 6). (B) samples were prepared from the external
milieu of cells treated with buffer (C, control), 65 ng/mL GM-CSF (GM),
10 µg/mL cycloheximide (CHX), or 1000 ng/mL VAA-I. Results are one
representative experiment out of at least 15 different experiments.
Note that, in panel A, de novo synthesis of different
polypeptides is increased by treatment with low concentrations of VAA-I
(lanes 1 and 2) and GM-CSF (lane 4) when compared with untreated cells
(small arrows in lane 6). Intensity of the bands (arrows) was
quantified with the Bio-Rad Multi-AnalystTM/PC Version 1.1
program. Values are from top to bottom. Lane 1: 1.98, 2.51, 2.71, 2.71,
2.54, 3.53, 1.99. Lane 2: 1.00, 1.43, 1.48, 1.37, 1.59, 2.51, 1.46.
Lane 3: 0.73, 0.83, 0.79, 0.62, 0.81, 1.30, 0.55. Lane 4: 1.02, 1.87,
1.52, 1.63, 2.09, 2.63, 0.97.
|
 View larger version (23K): [in a new window] |
Figure 3. Induction of neutrophil apoptosis by high concentration of VAA-I.
Freshly isolated human neutrophils (10 x106 cells/mL)
were incubated for 22 h in the absence (0) or presence of
increasing concentration of VAA-I (1–1000 ng/mL) and the number of
cells in apoptosis were assessed by flow cytometry using FITC-Annexin
V/PI staining (A) or by cytology from cytocentrifuge preparations (B)
as described in Materials and Methods. (A) results are means ±
SEM (n =3), *P < 0.05 vs. control (early apoptosis);
**P < 0.05 vs. control (late
apoptosis/necrotic) both by Student’s t test. Inset, about
42% of untreated cells are in early apoptosis vs. 7.8% for
VAA-I-induced neutrophils, whereas it can be observed that 18.4% and
92.1% of cells are in the late apoptotic/necrotic category,
respectively. (B) results are means ± SEM
(n 10), *P < 0.05 vs. control by
Student’s t test. Inset, top, untreated cells after 22 h of incubation. Only one cell is in apoptosis in this field (round
nucleus instead of polylobed). Inset: neutrophils treated for 22 h
with 1000 ng/mL VAA-I. Note that virtually all cells are in apoptosis
(pycnotic nuclei).
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 View larger version (29K): [in a new window] |
Figure 4. Assessment of neutrophil apoptosis during time-course experiments.
Cells were incubated for 4, 12, or 24 h with buffer (Ctrl), 65
ng/mL GM-CSF, or increasing concentrations of VAA-I (100, 500, or 1000
ng/mL) and apoptosis was assessed by cytology as described in Materials
and Methods. Results are means ± SEM
(n7), *P < 0.05 vs. control by Student’s
t test. Inset: apoptosis was assessed by flow cytometry
(FITC-Annexin V/PI staining). Results are means ± SEM
(n=3) and represented mainly viable cells (early and late
apoptosis) because <9% of cells were necrotic (trypan blue dye
exclusion). Note that signs of apoptosis can be detected more rapidly
by FITC Annexin-V binding but results are the same after 24 h;
almost 100% of cells are in apoptosis with the use of 1000 ng/mL.
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 View larger version (34K): [in a new window] |
Figure 5. VAA-I reverses the GM-CSF-induced suppression of neutrophil apoptosis
and the GM-CSF-induced de novo protein synthesis. Cells were
incubated for 24 h in the presence of buffer (Ctrl); 65 ng/mL
GM-CSF, 1000 ng/mL VAA-I; or a combination of GM-CSF (65 ng/mL) +
VAA-I (1000 ng/mL) and the number of apoptotic neutrophils was
evaluated by cytology as described in Materials and Methods. Results
are means ± SEM (n=5).
*P < 0.05 vs. control by Student’s
t test. Inset: neutrophils were metabolically labeled as in
legend of Figure 2
, and incubated for 24 h in the presence of
buffer (lane 1), 65 ng/mL GM-CSF (lane 2), 1000 ng/mL VAA-I (lane 3),
or a combination of GM-CSF and VAA-I (lane 4). Note that GM-CSF induced
de novo protein synthesis (lane 2) and that VAA-I can
reverse this response (lane 4).
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 View larger version (65K): [in a new window] |
Figure 6. Induction of neutrophil apoptosis by VAA-I is independent of tyrosine
phosphorylation events: VAA-I does not alter GM-CSF-induced tyrosine
phosphorylation. (A) cells were stimulated for 15 min with buffer (lane
1), 65 ng/mL GM-CSF (lane 2), VAA-I at 1, 10, or 1000 ng/mL (lanes 3,
4, 5, respectively), or a combination of GM-CSF + an increasing
concentration of 1, 10, 1000 ng/mL VAA-I (lanes 6, 7, and 8,
respectively). Immunoblotting was performed as described in Materials
and Methods. Top: tyrosine phosphorylation of intracellular proteins.
Bottom: Coomassie blue staining of the membrane to indicate equivalence
in loading. Results shown are representative of three experiments
conducted with three different blood donors. (B) Diff-quick staining of
cells treated with buffer + 50 µg/mL genistein (Ctrl +
genistein), 65 ng/mL GM-CSF alone (GM), GM-CSF + genistein
(GM + genistein), or with 1000 ng/mL VAA-I + genistein
(VAA-I + genistein). Photomicrographs are representative of three
experiments conducted separately. Small arrows, apoptotic neutrophils;
big arrowheads, normal neutrophils. Approximately 50, 0, 20, and 100%
of cells are in apoptosis from top to bottom.
|
3). Involvement of caspases during
VAA-I-induced neutrophil apoptosis was further demonstrated by the
fragmentation of the cytoskeletal gelsolin protein (Fig. 8
). Such fragmentation was largely inhibited by the z-VAD-FMK
inhibitor (Fig. 8)
but not the irrelevant Calphostin C inhibitor (data
not shown).
 View larger version (18K): [in a new window] |
Figure 7. Involvement of caspases in VAA-I-induced neutrophil apoptosis. Freshly
isolated human neutrophils were treated for 24 h with buffer
(CTRL), diluent (1% DMSO), caspase inhibitor z-VAD-FMK (50 µM;
z-VAD) alone, 500 ng/mL VAA-I alone, or 1 h with z-VAD and then
VAA-I for 23 h (total of 24 h, as other conditions) and
apoptotic neutrophils were quantified by cytology as described in
Materials and Methods. Results are means ± SEM
(n = 4). *P < 0.0005 by
Student’s t test.
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 View larger version (50K): [in a new window] |
Figure 8. Fragmentation of gelsolin in VAA-I-induced neutrophil apoptosis. Human
neutrophils were isolated and cultured as in legend of Figure 7
. The
fragmentation of the cytoskeletal gelsolin protein was visualized by
immunoblotting as described in Materials and Methods. C, control cells
incubated with diluent (-) or 50 µM z-VAD-FMK (+); VA, cells
incubated with 500 ng/mL VAA-I with diluent (-) or 50 µM z-VAD-FMK
(+). N, native gelsolin (90 kDa); p47, a 47-kDa peptide derived from a
chymotryptic cleavage of gelsolin recognized by the antibody. Note the
diminution of native gelsolin and an 50-kDa band induced by VAA-I in
lane 2, and the appearance of a more intense band (41 kDa) in the same
lane (small arrow). The degradation of native gelsolin was inhibited by
the z-VAD-FMK (compare lane 2 with lane 4). This is one representative
experiment out of four. Numbers on the left are molecular mass
standards.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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An increasing number of products are known to modulate neutrophil apoptosis [3 , 4 , 34 , 39 , 40 ]. Identifying such agents is of biological importance in various situations such as for patients receiving drug therapy for cancer treatments [41 ], AIDS patients [42 ], or during an inflammatory state [13 14 15 ]. Herein, we report that VAA-I is a potent agent that can induce neutrophil apoptosis. Using both flow cytometry (FITC-Annexin-V/PI labeling) and cytology (Diff-Quick staining of cytocentrifuge preparations) approaches, we have demonstrated that VAA-I can induce neutrophil apoptosis at high concentrations (>500 ng/mL). In addition, our results allow us to correlate the ability of VAA-I to induce neutrophil apoptosis and concomitantly inhibit de novo protein synthesis. This is in contrast to stimuli that delay neutrophil apoptosis and induce de novo protein synthesis such as GM-CSF [3, 10, this study], IL-4 [4 ], IL-15 [3 ], sodium butyrate [37 ], and dexamethasone [38 ]. This correlation is reinforced by the following observations. (1) Low concentrations of VAA-I induced de novo protein synthesis but do not alter the neutrophil apoptotic rate. (2) We have included in all of our experiments dealing on de novo protein synthesis one of the most widely used protein synthesis inhibitors as a control, CHX, at a concentration known to induce neutrophil apoptosis [37 ] and observed that it clearly inhibits de novo protein synthesis visualized by SDS-PAGE. (3) High concentrations of VAA-I were found to markedly reverse the ability of GM-CSF to both induce de novo protein synthesis and to delay neutrophil apoptosis without inducing cell necrosis. (4) Similar properties of VAA-I were previously observed with cells other than neutrophils, namely, human lymphocytes, monocytes, monocytic THP-1 cells, and murine thymocytes [16 ]. (5) To our knowledge, there is no clear evidence in the literature that argues against the correlation of induction of neutrophil apoptosis with inhibition of de novo protein synthesis. One unique study demonstrated that typical morphological features of apoptosis, in etoposide-induced rat pheochromocytoma PC12 cells, were not associated with oligonucleosomal DNA fragmentation or with de novo macromolecule synthesis [43 ]. However, the concentration of CHX used in pre-incubation study or during co-incubation was low because higher concentrations were found to be toxic for these cells. In addition, unlike the present study (where proteins are visualized), these authors did not perform SDS-PAGE from metabolically labeled cells, but rather added CHX with etoposide and enumerated the number of apoptotic cells [43 ].
Results from this study allow us to establish that VAA-I does not induce tyrosine phosphorylation events in human neutrophils by itself and does not alter the GM-CSF-induced response. Involvement of tyrosine kinases to mediate biological actions was found to be of importance for several agonists [35 , 36 , 44 45 46 47 48 49 50 ]. Among them, GM-CSF and LPS were found to recruit tyrosine kinases for delaying neutrophil and eosinophil apoptosis [44 45 46 ]. Results from one recent study demonstrate that induction of human neutrophil apoptosis by the protein synthesis inhibitor CHX accelerate macrophage clearance of apoptotic neutrophils [51 ]. Whether or not VAA-I can act similarly remains unknown. In one study, it was found that treatment of cells with a specific inhibitor of Jak-2, tyrphostin B42, can reverse the ability of GM-CSF to delay eosinophil apoptosis, suggesting that this biological action of GM-CSF occurs via Jak-2 [45 ]. Herein, although pretreatments of human neutrophils by genistein can reverse the effect of GM-CSF, such treatments were ineffective in reversing the inducing effect of VAA-I. In addition, although we observed that tyrphostin B42 was able to reverse the delaying effect on neutrophil apoptosis, it was not able to reverse the VAA-I-induced neutrophil apoptosis (our unpublished observations). This suggests that VAA-I-induced neutrophil apoptosis is independent of tyrosine kinase activation and, in particular, it does not recruit Jak-2, an important molecule in neutrophil signaling [52 ]. Furthermore, in this study we bring evidence that VAA-I induces neutrophil apoptosis via a mechanism that involves caspases but not G proteins, protein kinase Cs, and phospholipase A2. Involvement of caspases was confirmed by inhibition of VAA-I-induced neutrophil apoptosis with the z-VAD-FMK inhibitor by both microscopic observations and by fragmentation of gelsolin visualized by immunoblotting. Our results are in agreement with the fact that caspases play an important role in apoptosis. Recently, it has been demonstrated that unlike anti-Fas-induced neutrophil apoptosis, specific inhibitors to caspase-3 and caspase-8 were unable to prevent spontaneous neutrophil apoptosis [53 ]. This is in agreement with our observation that the use of the z-VAD-FMK alone did not alter normal neutrophil apoptotic rate. However, it was found by others that caspase-3 is involved during spontaneous neutrophil apoptosis [54 ]. Although they speculate that the z-DEVD-FMK inhibitor is specific for caspase-3, it is specified by the manufacturer that it can inhibit as well caspases-6, -7, -8, and -10. The experimental conditions were different between the two studies, including the concentration of the inhibitor used (100 µM for the former and 20 µM in the other study). This testifies to the complexity of interpreting results. Herein, we demonstrate that among caspases-1, -3, -4, -7, the caspase-3 is at least involved. In addition, our results suggest that VAA-I can accelerate human neutrophil apoptosis without the synthesis of new factor(s). It seems that all the machinery (including the caspases) is already present in these cells and induction of apoptosis could be simply mediated by an "on/off" switch. This is in agreement with the fact that these cells are known to spontaneously undergo apoptosis without addition of external agents.
Taken together, our results demonstrate that VAA-I is a potent immunomodulator. The fact that VAA-I could act as a very potent inducer of neutrophil apoptosis offers an interesting potential therapeutic strategy in inflammatory disorders such as rheumatoid arthritis. Elaboration of a VAA-I immunotoxin that could be used to eliminate neutrophils by "killing" them by inducing apoptosis during an inflammatory response could be useful to attenuate/eliminate inflammation. This would be of great importance in general biology and medicine.
Received December 9, 1999; revised July 1, 2000; accepted July 5, 2000.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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