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Originally published online as doi:10.1189/jlb.0902474 on July 22, 2003

Published online before print July 22, 2003
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(Journal of Leukocyte Biology. 2003;74:952-958.)
© 2003 by Society for Leukocyte Biology

Different antigens trigger different Th1/Th2 reactions in neonatal mononuclear cells (MNCs) relating to T-bet/GATA-3 expression

Hong-Ren Yu, Jen-Chieh Chang, Rong-Fu Chen, Hau Chuang, Kuo-ching Hong, Lin Wang and Kuender D. Yang1

Division of Clinical Immunology & Allergy, Chang Gung Children’s Hospital and Graduate Institute of Clinical Medical Sciences, Chang Gung University, Kaohsing, Taiwan, R.O.C.

1Correspondence: Office of the Superintendents, Chang Gung Children’s Hospital, 123, Ta-Pei Road, Niao-Sung Hsiang, Kaohsiung Hsien 833, Taiwan. E-mail: yangkd{at}adm.cgmh.org.tw


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Neonates are known to have poor cellular immunity, especially poor Th1 response. We investigated how neonatal mononuclear cells raised different Th1/Th2 reactions in response to different antigens. Employing Dermatophagoides pteronyssinus (Der p) extract and varicella zoster virus (VZV) as antigens, we assessed Th1/Th2 reactions as demonstrated by IL-4/IFN{gamma} production and mRNA expression, and transcriptional factors T-bet/GATA-3 mRNA expression in mononuclear cells from human umbilical cord blood (CBMC). Results showed that VZV induced a dramatic increase of IFN{gamma} production by adult peripheral blood mononuclear cells (PBMC), whereas VZV did not drive CBMC to release significant IFN{gamma} production (1614.7±362.0 vs. 49.0±29.3,p<0.005). However, Der p induced higher IFN{gamma} production by CBMC than VZV (298.1±171.8 vs. 49.0±29.3, P=0.047). In contrast, VZV did not induce significant IL-4 production either by CBMC or by PBMC. Der p induced a comparative IL-4 production by CBMC and PBMC (2.58±0.84 vs. 2.04±0.37, p>0.05). A real-time RT-PCR analysis of IL-4 and IFN{gamma} mRNA expression showed that VZV induced a significantly higher IFN{gamma}, but not IL-4, mRNA expression in PBMC than CBMC. Der p did not induce significant difference of IFN{gamma} or IL-4 mRNA expression in PBMC and CBMC. VZV enhanced Th1-related transcription factor T-bet mRNA expression, in association with later down-regulation of Th2-related GATA-3 mRNA expression in PBMC. However, VZV did not up-regulate T-bet or down-regulate GATA-3 expression significantly in CBMC. In contrast, Der p induced an early GATA-3 expression and later T-bet expression in CBMC. These results suggest that different antigens trigger various Th1/Th2 reactions in PBMC and CBMC resulting from kinetic changes of T-bet/GATA-3 expression.

Key Words: Neonate • Th1/Th2 • Dermatophagoides pteronyssinus (Der p) • varicella • IFN{gamma} • IL-4 • transcription factor


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Helper-T (Th) cells play an important role in cellular and humoral immunity. Th cells can be subdivided into Th1 and Th2, according to the cytokines they produce when activated. Th1 cells produce IL-2, IFN{gamma}, and TNFß that promote cytotoxic T cell reaction and delayed type hypersensitivity and Th2 cells produce IL-4, IL5, IL-6, IL-10, and IL-13, which assist B cell responses and allergic reaction [1 , 2 ]. Human host defense to virus mainly depends on T-cell cytotoxicity of virus-infected cells. In addition, NK cells and macrophages are also important since secretion of cytokines from NK cells, macrophages can augment T-cell cytotoxicity [1 ].

Neonates are known to have poor or immature immunity to infections. Neonates, however, have normal or higher CD4/CD8 ratios [3 , 4 ], although they do have lower cytotoxic T cells and NK cells [3 4 5 ]. These studies partly explain why neonates have poor cellular immunity. Accumulated evidence showed that peripheral blood mononuclear cells (PBMC) from adults could produce a large amount of IFN{gamma} in response to varicella zoster virus (VZV) antigen [6 7 8 ], suggesting that Th1 response with IFN{gamma} production by mononuclear cells may be responsible for the host defense to VZV [9 ]. It is well known that neonates develop Th2-dominant responses after mitogen stimulation [10 , 11 ]. It is, however, not known whether mononuclear cells in neonates always raise Th2 reaction greater than Th1 in response to VZV and other antigens. Since different antigens may activate different intracellular signaling pathways for inducing different spectra of cytokine production [12 13 14 ]. To explore how neonates raise different Th1/Th2 profiles in response to different antigens and understand the molecular mechanism of Th1/Th2 polarization in neonates, we investigated Th1/Th2 responses as demonstrated by cytokine production, and its mRNA and transcription factor expression in human cord blood mononuclear cells (CBMC) stimulated by Dermatophagoides pteronyssinus (Der p) extract and VZV antigen.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Subjects and antigens studied
Human umbilical cord blood was collected at the time of elective Caesarean sections or normal spontaneous delivery of healthy mothers after informed consult was obtained. The blood was then collected into heparinized tubes (10 U/ml) by cordocentesis. Heparinized adult peripheral blood was also obtained from healthy adult volunteers who were 23–35 years old as a control. Every one or two cord blood samples were studied simultaneously with one adult blood as a control. Sixteen cord blood and 10 adult peripheral blood samples were enrolled in this study.

Reagents
Allermed Laboratories, Inc. (San Diego, CA) kindly provided the Der p extract. Varieclla zoster virus (VZV) vaccine (Valrix; SmithKline Beecham Biologicals, Rixensart, Belgium) contained a virus titer of more than 103.3 plaque-forming units (pfu) per dose was used as VZV antigen.

Preparation of mononuclear cells
Peripheral blood mononuclear cells (PBMC) and umbilical cord blood mononuclear cells (CBMC) were isolated by dextran (4.5%) sedimentation of RBC at a ratio of 1:5 to separate leukocytes from red blood cells. Leukocytes were separated into mononuclear cells (MNCs) and polymorphonuclear cells by density gradient centrifugation (Ficoll-Paque; Amersham Pharmacia, Biotech AB, Uppsala, Sweden). PBMC and CBMC were counted and suspended to 2 x 106/ml in RPMI-1640 medium supplemented with 10% heat-inactivated fetal calf serum for studies [15 ].

In vitro immune response to different antigens
PBMC and CBMC (2 x106 cells/ml) were cocultured with and without Der p (50 AU/ml) or VZV at 1:20 (v/v) in 48-well culture plates with a total of 0.5 ml. The reactions were incubated in a humidified atmosphere of 5% CO2 at 37°C. Supernatants and cell pellets harvested after culture for 10 h and 6 days were subject to assessment of IFN{gamma}/IL-4 production and mRNA expression, respectively, as described below:

Measurement of cytokine production
Supernatants collected from parallel cultures of nonstimulated and antigen-stimulated CBMC and PBMC were aliquoted and stored at –80°C until studies were completed. In a pilot study with three replicate experiments, IL-4 production peaked 10 h after Der p stimulation (Fig. 1A ), while IL-4 level in culture supernatant decreased rapidly after culture for 24 h. A high-sensitivity IL-4 ELISA was used to detect the low range of IL-4 levels in the reaction. As shown in Fig. 1B , the reliability can be seen in this correlated standard curve. In contrast, IFN{gamma} production peaked in 6 days after VZV stimulation (Fig. 1C) . The early detection of IL-4 within 24 h [16 ] and the later detection of IFN{gamma} [17 ] have also been described previously. Therefore, we collected the supernatant from the culture for 10 h and 6 days for measurement and comparison of the IL-4/IFN{gamma} production by using a commercial ELISA kit (R&D System Inc., Minneapolis MN) [18 ].



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  		Figure 1. The IL-4/IFN{gamma} production by PBMC after stimulation by Der p/VZV antigens for different time periods. PBMC were incubated with (•) and without ({circ}) Der p antigen (50 AU/ml) or with ({blacktriangleup}) and without ({triangleup}) VZV at 1:20 (v/v) for 10 h, and 1 to 6 days. The IL-4 production after Der p antigen stimulation was only detectable in 10 h (A). The standard curve of IL-4 ELISA was shown (B). In contrast, the IFN{gamma} production progressively increased over 6 days after VZV antigen stimulation (C).

 
Flow cytometric analysis of intracellular IFN{gamma} staining
PBMC and CBMC (2x106 cells/ml) were cocultured with and without VZV at 1:20 (v/v) in 48-well culture plates with a total of 0.5 ml for 3 days. After culture, the cells were first stained with PE-conjugated antihuman CD4, CD8, or CD56 antibody (2 µg/0.1ml) for 30 min, followed by cell fixation with IntraPrep Reagent 1 (Beckman Culter, Marseille, France) for 15 min and cell membrane permeabilization with IntraPrep Reagent 2 (Beckman Culter, Marseille, France) for 5 min before another staining of intracellular IFN{gamma} by FITC-anti-IFN{gamma} antibody (0.5 µg/0.1ml) (Beckman Culter, Marseille, France) for 30 min. Cells were analyzed for the intracellular IFN{gamma} by two-color flow cytometry (Becton-Dickinson, Franklin Lakes, NJ).

Isolation of total RNA from PBMC and CBMC
Total RNA from the PBMC and CBMC (1x106 cells) cultured with and without antigen for 10 h and 6 days were harvested after collection of supernatants for measurement of cytokine production. The PBMC and CBMC were suspended in 250 µl of PBS before adding 750 µl of Tri-reagent solution (Sigma Chemical Company, St. Louis, MO) to separate RNA from DNA and protein as our previous description [19 ]. The RNA was precipitated with 75% ethanol (Merck KgaA, Darmstadt, Germany), resuspended in 20 µl of 0.1% DEPC-treated water, and stored at -70°C until analysis.

Measurement of IL-4/IFN{gamma} and T-bet/GATA-3 mRNA expression by real-time quantitative RT-PCR
The real-time quantitative RT-PCR was carried out in an ABI 7700 quantitative PCR machine system (Perkin Elmer, Norwalk, CT), using TaqMan technology, as we described previously [18 ]. A fluorescent threshold was manually set across all samples in the experiment starting with the exponential phase of the fluorescent signal increase. As the threshold is set at 10 times the standard deviation of the mean baseline fluorescence emission calculated from the first three to 15 PCR cycles, the fractional number of RT-PCR cycles demonstrating fluorescence exceeding the threshold values was designed the cycle threshold (Ct). Primer sequences for the internal control (ß-actin) were 5'-GGC CAA CCG CGA GAA GAT-3' (380-397) for the forward primer, 5'-CGT CAC CGG AGT CCA TCA C-3' (495-513) for the reverse primer, and 5'-TGC TAT CCC TGT ACG CCT CTG GCC-3' (457-480) for the probe sequence. For detecting IL-4 mRNA, the forward primer was 5'-CCA CGG ACA CAA GTG CGA TA-3', the reverse primer was 5'-AGC CCT GCA GAA GGT TTC CT-3', and the probe sequence for IL-4 was 5'-AAA ACT TTG AAC AGC CTC ACA GAG CAG AAG ACT-3'. For detecting IFN{gamma} mRNA, the forward primer was 5'-TGC AGA GCC AAA TTG TCT CCT-3', the reverse primer was 5'-GCT TTG CGT TGG ACA TTC AA-3', and the probe sequence was 5'-CCA AAA GAG TGT GGA GAC CAT CAA GGA AGA-3'. Recently, molecular mechanisms of Th1/Th2 differentiation have been related to reciprocal T-bet/GATA-3 expression [20 21 22 ]. Therefore, we designed RT-PCR primers for quantitative detecting T-bet/GATA-3 mRNA expression. The forward primer for detecting T-bet mRNA was 5'-AACACAGGAGCGCACTGGAT-3', the reverse primer was 5'-TGCTCTCCTGGCTGCAGAC-3', and the probe sequence for T-bet was 5'-TGACCCAGATGATTGTGCTCCAGTCC-3'. For detecting GATA-3 mRNA, the forward primer was 5'-ACCGGCTTCGGATGCAA-3', the reverse primer was 5'-TGCTCTCCTGGCTGCAGAC-3', and the probe sequence for GATA-3 was 5'-TCCAGCACAGGCAGGGAGTGTGTG-3'. The RT-PCR cycling parameters were set as follows: the initial RT reaction at 50°C, 2 min; 60°C, 30 min; and 95°C, 5 min; followed by 40 cycles of PCR reactions at 94°C, 20 s, and 60°C, 1 min. The results were detected in a real-time fashion and recorded on a plot showing fluorescence vs. time. Reaction products were also visualized on ethidium bromide-stained 2.5% agarose (Pierce, Rockford, IL) gel with a 100-bp ladder (Pharmacia Biotech, Piscataway, NJ) as a reference. The ß-actin, IL-4, IFN{gamma}, T-bet, and GATA-3 cDNA fragments were detected at 124, 151, 193, 164, and 207 bp, respectively. The quantity of the T-bet and GATA-3 mRNA expression was therefore calculated assuming 100% efficient PCR where each CT value of the reactions was normalized to ß-actin mRNA expression, as shown by the equation

The Ct1(target) and Ct2(target) represent the Ct values for the target gene expression from stimulation group and non-stimulation group samples, respectively. Ct1 (actin) and Ct2 (actin) represent the CT values for the ß-actin gene expression in stimulation group and nonstimulation group samples, respectively.

Data analysis and statistics
Results were expressed as mean ± standard error of the mean. Differences in the cytokine levels and mRNA expression between different groups were analyzed by Student’s t test. Differences of intracellular IFN{gamma} staining were tested by Wilcoxon sign rank test. A P value of <0.05 was considered to be statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
VZV induction of higher Th1 cytokine production by PBMC than CBMC
Supernatants collected after culture with or without antigen stimulation for 10 h and 6 days were subject to measurement of IL-4 and IFN{gamma}, respectively. It was found that both PBMC and CBMC produced very low IL-4 and IFN{gamma} levels 10 h after stimulation with VZV. VZV, however, induced a large amount of IFN{gamma} by mononuclear cells 6 days after stimulation. The VZV-induced IFN{gamma} production by PBMC was significantly higher than that by CBMC after culture for 6 days (1614.7±362.0 vs. 49.0±29.3 pg/ml, p<0.005)(Fig. 2 ).



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  		Figure 2. The IFN{gamma} production by PBMC and CBMC after stimulation by different antigens for 6 days is shown. MNC from 16 cord blood samples and 10 adult peripheral blood samples were incubated with and without Der p extract (50 AU/ml) or VZV antigen (5x101.3 pfu/ml) for 6 days. Constitutive IFN{gamma} production was low by both PBMC and CBMC. The VZV-induced IFN{gamma} production by PBMC was significantly higher than that by CBMC (p<0.005). In contrast, the Der p-induced IFN{gamma} production by CBMC was comparable to that by PBMC.

 
Der p induction of both Th1 and Th2 cytokines production
Der p allergen induced higher IL-4 production by PBMC than that without antigen stimulation (2.04±0.37 pg/ml vs. undetectable, p<0.001) in early 10 h. CBMC stimulated by Der p allergen also produced higher IL-4 than those without antigen stimulation (2.58±0.84 vs. 0.04±0.03 pg/ml, P=0.021) (Fig. 3 ). This suggests that CBMC could raise a comparative Th2 reaction in response to Der p antigen as compared with PBMC. Production of IFN{gamma} by CBMC after Der p stimulation for 6 days was higher than that by PBMC (298.1±171.8 vs. 156.1±75.5 pg/ml) (Fig. 2) . This indicates that CBMC can raise a comparative Th1 reaction in response to Der p antigen when compared with PBMC. To make sure the increase of IFN{gamma} production is related to antigen-induced production, we used a dual-stain technique to detect intracellular IFN{gamma} levels in CD4+, CD8+ and CD56+ lymphocytes. It was found that VZV stimulation although not increased intracellular IFN{gamma}-expressing cells in CD4, CD8 and CD56 positive populations, the IFN{gamma} expressing intensity in these cells was significantly increased after stimulation (Table 1 ). This suggests that VZV did not increase IFN{gamma}-producing cells but augmented IFN{gamma} production by certain lymphocytes.



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  		Figure 3. The IL-4 production by PBMC or CBMC after stimulated by Der p extract or VZV antigen for 10 h is shown. Constitutive IL-4 production was almost undetectable both in PBMC and CBMC. Both PBMC and CBMC that were stimulated with Der p antigen produced higher IL-4 than those stimulated with vehicle. ND = nondetectable.

 

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  		Table 1. Flow Cytometric Analysis of Intracellular IFN{gamma} Expression in CD4+, CD8+ and CD56+ Cells

 
Quantitative RT-PCR analysis of IFN{gamma}/IL-4 mRNA expression
Experiments were next performed to detect IL-4 and IFN{gamma} mRNA expression in PBMC and CBMC by fluorogenic quantitative RT-PCR. As shown by a real-time representative study in Fig. 3 , CBMC expressed the highest IL-4 mRNA expression after Der p stimulation. In contrast, VZV did not enhance the IL-4 mRNA expression in PBMC or CBMC. VZV, however, induced a higher IFN{gamma} mRNA expression in PBMC than CBMC. Der p was able to induce higher IFN{gamma} mRNA expression in CBMC than in PBMC. After 10 replicate experiments, it was found that PBMC showed significant IFN{gamma} mRNA expression under VZV stimulation than CBMC (p=0.042), and IL-4 mRNA was expressed significantly higher in Der p-stimulated CBMC than VZV-stimulated CBMC (p=0.049). Similarly, Der p also induced higher IL-4 mRNA expression in PBMC than VZV (p=0.019) (Fig. 4 ).



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  		Figure 4. Quantitative RT-PCR analysis of IL-4 and IFN{gamma} mRNA in PBMC and CBMC after different antigen stimulation. IL-4 and IFN-{gamma} mRNA expression by Der p-stimulated CBMC (C Der p) and PBMC (A Der p) as well as by VZV-stimulated CBMC (C VZV) and PBMC (A VZV) were presented. RT-PCR. On the basis of 10 replicate experiments, Der p was found to enhance IL-4 mRNA expression both in PBMC and CBMC, while VZV induced a significantly higher IFN{gamma} mRNA expression in PBMC than in CBMC.

 
Quantitative analysis of T-bet and GATA-3 mRNA expression in PBMC and CBMC
A reciprocal regulation of T-bet and GATA-3 mRNA expression has been shown to mediate Th1/Th2 reactions [20 21 22 ]. We next studied how Der p and VZV antigens differently polarize T-bet/GATA-3 mRNA expression. It was found that VZV induced a Th1-related transcriptional factor T-bet mRNA expression (Fig. 5A ) in association with later down-regulation of GATA-3 mRNA expression in PBMC (Fig. 5B) . VZV, however, did not up-regulate T-bet or down-regulate GATA-3 expression significantly in CBMC. In contrast, Der p induced an early GATA-3 expression both in PBMC and CBMC (Fig. 5A 5B) that is compatible with IL-4 production (Fig. 3) . Interestingly, Der p induced a later T-bet mRNA expression in day 6 in CBMC but not in PBMC (Fig. 5B) , which is compatible with the IFN{gamma} production (Fig. 2) . Taken together, these results suggest that different antigens trigger various Th1/Th2 reactions in PBMC and CBMC, relating to kinetic changes of T-bet/GATA-3 expression.



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  		Figure 5. Quantitative RT-PCR analysis of GATA-3 and T-bet mRNA in PBMC and CBMC after different antigen stimulation. Increases in GATA-3 and T-bet mRNA expression in PBMC (A) and CBMC (C) after Der p or VZV stimulation was analyzed by quantitative RT-PCR at 10 h and 6 days. Based on 10 replicate experiments, it was found that Der p enhanced GATA-3 mRNA expression both in PBMC and CBMC at 10 h, while VZV induced a significantly higher T-bet mRNA expression in PBMC than in CBMC (A). In contrast, Der p induced a higher T-bet expression in 6 days in CBMC than in PBMC; and VZV induced a higher T-bet but lower GATA-3 expression in PBMC than in CBMC (B).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Neonates are well known to have poor Th1 response than adults under mitogen stimulation [23 24 25 ]. This is supported by our study with VZV, showing that PBMC but not CBMC released a large amount of IFN{gamma} after stimulation. However, we showed that in response to the Der p antigen, MNCs from neonates did polarize a Th1 reaction similar to and even higher than that from normal adults. This suggests that neonatal Th1 response may not always be poorer than adult Th1, but can be dependent on different antigens. Certain studies have shown that lower production of IFN{gamma} by cord blood T cells is partly due to an augmented inhibitory effect of IL-4 on Th1 reactions [9 ]. This hypothesis is not supported by our results: VZV did not significantly induce IL-4 production but instead induced IFN{gamma} production by both PBMC and CBMC. In contrast, Der p induced comparative IL-4 production in the first day by PBMC and CBMC (Fig. 3) , which related to earlier GATA-3 expression. Later, comparative IFN{gamma} production (Fig. 2) by PBMC and CBMC was stimulated by Der p, which relates to T-bet expression. This suggests that different antigens drive various T-bet/GATA-3 expression for induction IFN{gamma}/IL-4 production. In fact, IFN{gamma} production is regulated by T-bet expression mediated by STAT-1 activation [26 , 27 ], and IL-4 production is regulated by GATA-3 expression mediated by STAT-6 [28 29 30 ]. Different antigens may trigger one or both STAT-1 and STAT-6 activation and result in different Th1/Th2 cytokine profiles. Thus, CBMC may not always reveal poor Th1 reaction; certain antigens can trigger Th1 reactions through different signal pathways.

Atopy is considered a disorder caused by the imbalance of Th1/Th2 function and is influenced by genetic and environmental factors. Evidence suggests that IL-4 is the most dominant factor in determining the Th2 polarization [31 , 32 ]. However, cord blood lymphocytes exhibit a specific pattern of immune function that differs in a number of ways from adult peripheral blood lymphocytes [33 ]. It was not possible to detect allergen-specific IL-4 production by CBMC using conventional sandwich ELISA [33 34 35 36 ]. To our knowledge, this is the first report to successfully measure IL-4 production by CBMC in response to different antigens. Why couldn’t others detect IL-4 directly from supernatant of antigen-stimulated CBMC? We think it may be due to three possible reasons: 1) The IL-4 levels produced by MNCs peak at a very early stage. In our study, we found the mean IL-4 level in culture supernatants from CBMC decreased rapidly to 0.39 ± 0.11 pg/ml after they were cultured for 24 h. This kinetic change was very different in different antigen stimulation [9 ]. Other reports usually detected IL-4 at 24 or 48 h later after culture [33 34 35 ], which seemed too late. 2) We used a low range of ELISA kit with a sensitivity threshold of 0.13 pg/ml. 3) People previously used different antigens to stimulate IL-4 production. The cytokine profiles after nonspecific stimulation may be not able represent allergen-specific response. In this study, IL-4 production by CBMC stimulated by Der p allergen appeared early at 10 h, while IFN{gamma} production appeared late, in 6 days. This was compatible with previous reports showing that the peak IL-4 production occurs before IFN{gamma} and declines rapidly [37 38 39 ]. The fact that CBMC produced comparative IL-4 levels at 10 h, as compared with PBMC upon stimulation with Der p allergen, suggests that newborns can raise Th2 reaction compared to adults. The mechanism may be related to lower T-bet induction in association with comparative GATA-3 expression after antigen stimulation.

In summary, we found that neonatal Th1 responses are not necessarily always poorer than adult ones, but rather are dependent on different antigen stimulation. Der p antigen can induce an early Th2 polarization and a late Th1 response in CBMC compared to PBMC. VZV antigen, however, induces a prominent Th1 response in PBMC but a poor Th1 response in CBMC. Different antigens trigger various Th1/Th2 reactions in PBMC and CBMC relating to kinetic change of T-bet/GATA-3 expression. Results from this study may point to the possibility of raising effective Th1 reaction and turning down the Th2 response in neonates through regulating reciprocal T-bet/GATA-3 expression.

Received September 29, 2002; revised May 15, 2003; accepted May 23, 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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