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Originally published online as doi:10.1189/jlb.0304198 on July 7, 2004

Published online before print July 7, 2004
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(Journal of Leukocyte Biology. 2004;76:820-826.)
© 2004 by Society for Leukocyte Biology

Human milk oligosaccharides reduce platelet-neutrophil complex formation leading to a decrease in neutrophil ß 2 integrin expression

Lars Bode*,1, Silvia Rudloff*, Clemens Kunz*, Stephan Strobel{dagger} and Nigel Klein{ddagger}

* Institute of Nutritional Science, Justus-Liebig-University Giessen, Germany; and
{dagger} Immunobiology Unit and
{ddagger} Infectious Diseases and Microbiology Unit, Institute of Child Health, University College London, United Kingdom

1Correspondence at current address: The Burnham Institute, Glycobiology and Carbohydrate Chemistry Program, 10901 N. Torrey Pines Rd., La Jolla, CA 92037. E-mail: Lbode{at}burnham.org


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Human milk is thought by many authorities to be preferable to formula as a source of nutrients for infants. Some of the benefits may stem from its high concentration of unbound oligosaccharides (5-10 g/L). These sugars have structural similarities to selectin ligands, known to mediate important cell–cell interactions in the immune system. Platelet-neutrophil complexes (PNC) exist in healthy individuals but have been implicated in disease states. Formation of these complexes requires selectins and as such, could be influenced by human milk oligosaccharides (HMO). Here, we investigate this possibility by examining the effect of HMO on the formation of PNC and activation of associated neutrophils. We collected blood from 10 healthy volunteers, activated platelets with adenosine 5'-diphosphate, and added HMO, oligosaccharide standards, or phosphate-buffered saline as a control. We determined the influence of HMO on PNC formation and adjacent neutrophil activation with fluorescein-activated cell sorter analysis after labeling with antibodies for the platelet marker CD42a and the neutrophil activation marker CD11b. Within physiologically achievable concentrations (6.25-125 µg/mL), an acidic HMO fraction reduced PNC formation up to 20%, which was similar to the effect seen with high concentrations of sialyl-Lewis x. Associated neutrophils showed a dose-dependent decrease in ß 2 integrin expression, up to 30%, at high but physiological concentrations. The neutral HMO fraction had no effect. These results support the hypothesis that acidic HMO serve as anti-inflammatory components of human milk and thus, contribute to the lower incidence of inflammatory diseases such as necrotizing enterocolitis in breast-fed versus formula-fed infants.

Key Words: breast-feeding • inflammation • platelets • neutrophils • selectins • integrins • necrotizing enterocolitis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Human milk is a rich source of unbound oligosaccharides (5–10 g/L) [1 ]. In contrast, only trace amounts of these oligosaccharides are present in bovine milk and as a consequence, in infant milk formula [1 ]. There is good evidence that human milk oligosaccharides (HMO) reach the systemic circulation: HMO remain virtually undigested in the infant’s gastrointestinal tract [2 , 3 ], they can cross the intestinal epithelium by receptor-mediated transcytosis or paracellular pathways [4 ], and they are excreted with the urine of term [5 ] and preterm infants [6 ] fed with human milk. However, these oligosaccharides are not detectable in the urine of formula-fed infants [5 , 6 ].

HMO have been shown to have a number of biological properties that could be pertinent to human biology [1 ]. In particular, protein-carbohydrate interactions, such as those mediated by selectins [7 ], are amenable to modulation by HMO.

Selectins belong to a subclass of carbohydrate-binding proteins involved in cell adhesion in the immune system [8 ]. The physiological-binding determinant for selectin ligands is the tetrasaccharide sialyl-Lewis x (sLex), which consists of lactosamine [galactose (Gal)ß1-4N-acetyl-glucosamine (GlcNAc)], modified with an {alpha}2-3-linked N-acetyl-neuraminic acid (NeuAc; sialic acid) at the Gal and an {alpha}1-3-linked fucose (Fuc) at the GlcNAc [9 10 11 ]. Several oligosaccharides, which carry these particular binding determinants [12 ], could be detected in human milk, suggesting that these HMO structures might act as soluble selectin-ligand analogs.

Selectins are critical for the formation of platelet-neutrophil complexes (PNC) [13 , 14 ]. These heterogeneous cell aggregates represent a large subpopulation of neutrophils with a greater capacity for phagocytosis and an increased production of reactive oxygen species (ROS) [15 ]. In resting blood, up to 25% of the neutrophils are associated with platelets [16 ]. Upon platelet activation, e.g., with histamine, thrombin, or adenosine 5'-diphosphate (ADP), the number of PNC is enhanced significantly [16 ]. The initial step of PNC formation is mediated by P-selectin on activated platelets and P-selectin glycoprotein ligand 1 (PSGL-1) on neutrophils [14 ]. This interaction induces signaling pathways leading to an increased expression of adhesion molecules, including the ß 2 integrin CD11b/CD18 [17 ], which can be blocked by P-selectin antibodies [15 , 16 ]. The heterodimer CD11b/CD18 tightens platelet-neutrophil binding [18 , 19 ] and might also contribute to leukocyte cross-linking [20 ]. Furthermore, an increased adhesion molecule expression enhances neutrophil transmigration through activated endothelium at sites of inflammation [15 , 20 21 22 ].

In view of the potential of highly activated neutrophils to cause damage in several diseases, the objective of the present study was to investigate the influence of HMO on selectin-mediated PNC formation and PNC-associated neutrophil activation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
HMO preparation
Oligosaccharides were isolated from human milk as described previously (ref. [23 ] and manuscript submitted by L. Bode et al.). Briefly, after centrifugation, the lipid layer was removed, and the proteins were precipitated from the aqueous phase with ice-cold ethanol. Lactose was removed by gel filtration on Sephadex G-25 (Pharmacia Biotech, Uppsala, Sweden). The total oligosaccharide fraction was further separated into an acidic fraction (aHMO), consisting of compounds with NeuAc residues, and a neutral fraction (nHMO) without NeuAc residues, using high-pressure liquid chromatography (HPLC) anion-exchange chromatography on a Resource Q column (Pharmacia Biotech) at the following conditions: 100% eluent A (H2O) from 0 to 7.5 min, a linear gradient to 55% eluent B (0.6 M NaCl) for 42.5 min, a linear gradient to 100% eluent B for 2 min, and a constant flow with 100% eluent B for 8 min. A flow rate of 2 mL/min was used. The eluting fractions were monitored at 214 nm. Afterwards, the pooled HPLC fractions were desalted by gel filtration on Sephadex G-25 (Pharmacia Biotech).

Oligosaccharide standards
The oligosaccharide standards, sLex[NeuAc{alpha}2-3Galß1-4(Fuc{alpha}1-3)GlcNAc] and galactotriose (TriGal; Gal{alpha}1-3Galß1-4Gal) were obtained from Dextra Laboratories (Reading, UK).

Oligosaccharide analysis
Oligosaccharide analysis was performed by high pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) on a CarboPac PA1 column (Dionex, Sunnyvale, CA) using the same conditions as described previously [23 ]. Furthermore, the aHMO and nHMO fractions were analyzed by nano-electrospray mass spectrometry (ESI-MS) using a quadrupole time-of-flight mass spectrometer (Micromass, Manchester, UK) in positive ion mode [ESI(+)] for nHMO and negative ion mode [ESI(–)] for aHMO. A Z-spray atmospheric pressure ionization source was used with the source temperature set to 80°C and a desolvation gas (N2) flow rate of 75 L/h. Oligosaccharide preparations were dissolved in 0.2% trifluoroacetic acid/methanol 1:1 (by vol) to a final concentration of roughly 100 pmol/µL and were applied to nanospray capillaries. The capillary tip was set to a potential of 1.1 kV, and the cone voltage was 40 V. Acquisition and analysis of the data were performed with the MassLynx Windows NT PC data system. Sodium iodide was used as mass standard for calibration, and the mass accuracy of all measurements was within 0.1 atomic mass unit.

Endotoxin reduction and measurement
All samples were processed on affinity chromatography columns with immobilized polymixin B to remove endotoxins [lipopolysaccharides (LPS); ref. 24 ]. The amount of endotoxins present in the samples was determined by quantitative chromogenic limulus amebocyte lysate test (QCL-1000, BioWhittaker, Walkersville, MD). An endotoxin standard (LPS from Escherichia coli serotype 0111:B4, Sigma Chemical Co., St. Louis, MO) was used for control experiments.

Antibodies
Monoclonal antibodies (mAb) were purchased as follows: CD11b:fluorescein isothiocyanate (FITC), from Serotec (Oxford, UK); isotype-control antibody anti-mouse immunoglobulin G1 (IgG1):FITC, from Dako (High Wycombe, UK); CD42a:peridinin chlorophyll protein (PerCP) and isotype-control antibody anti-mouse IgG1:PerCP, from Becton Dickinson (Oxford, UK).

Whole blood stimulation
Blood was drawn via a 21G butterfly needle from nonsmoking, healthy volunteers who had not been on any medication for at least 2 weeks. The first 2 ml blood was discarded, and the required volume was collected into sodium citrate to a final concentration of 0.38%. Immediately after sampling, whole blood was stimulated with ADP in a final concentration of 10 µM and coincubated with different oligosaccharides (or phosphate-buffered saline as a control) at different concentrations and at different incubation times.

Measurement of platelet-neutrophil complexes
PNC were analyzed as described previously [16 ]. Briefly, 50 µl whole blood was incubated with a combination of directly conjugated mAb for CD42a (PerCP), as a specific platelet marker, and for CD11b (FITC), as a neutrophil activation marker. A combination of corresponding isotype-control antibodies was used to determine nonspecific binding. The samples were left at room temperature for 5 min. Samples were then incubated with 250 µl FACSLyse for 5 min to lyse red blood cells. Finally, 250 µl FACSFix was added.

Flow cytometry was performed on a Becton Dickinson FACSCalibur and CellQuest 3.1 within 1 h of sample preparation. Neutrophils were gated according to their forward and sideward characteristics. Data were collected using FITC fluorescence at 515 nm and PerCP at >650 nm. Results were compared with istotype-matched antibody staining and considered positive if the fluorescence intensity exceeded that of 98% of the control antibodies. Events staining positive for CD42a and CD11b were considered to represent PNC. Each sample was analyzed at the highest flow rate. Data for 5000 events were collected. The median fluorescence intensity (MFI) for CD42a:PerCP and CD11b:FITC was recorded and compared with the respective MFIs measured in ADP-stimulated blood without further treatment using paired, sample t-tests (Microsoft Excel 97). In all cases, P< 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Oligosaccharide preparation
Total HMO were separated into aHMO, containing structures with one or more NeuAc residues, and nHMO without NeuAc residues. The HPAEC-PAD chromatograms and the nano-ESI-MS of both fractions were identical with those fractions used in previous studies (L. Bode et al., manuscript submitted). These HMO fractions were added to whole human blood ex vivo to analyze their effects on PNC formation and neutrophil ß 2 integrin expression.

PNC formation: structure dependency
PNC formation in whole blood was significantly induced after incubation with 10 µM ADP (Fig. 1 ). To quantify the effects of different oligosaccharides on PNC formation, the percentage of PNC in ADP-stimulated blood without oligosaccharide treatment was defined as 100% (Fig. 2 ). When ADP-stimulated blood was coincubated with soluble sLex in a concentration of 125 µg/mL for 15 min, PNC formation was reduced to 85.3 ± 8.8% (P<0.01). TriGal, used as a negative control, had no effect on PNC formation. The coincubation with the aHMO fraction (125 µg/mL) reduced PNC formation to 79.7 ± 7.4% (P<0.001). The same concentration of the nHMO fraction decreased PNC formation slightly to 96.3 ± 4.2% (P<0.05). The inhibitory effect of the aHMO fraction was significantly higher than seen with the nHMO fraction (P<0.001; Figs. 1 and 2 ). All experiments were performed on blood from 10 different donors.



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  		Figure 1. Fluorescein-activated cell sorter (FACS) analysis of PNC formation. Events stained positive for CD11b and CD42a were considered to be PNCs (upper right quadrants, values indicate % PNC). The black lines represent the 98% thresholds for the respective isotype-control antibodies. PNC formation was analyzed in unstimulated (A) and ADP-stimulated blood (B–F) and after the incubation with different oligosaccharides (C–F).

 


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  		Figure 2. PNC formation: structure dependency. The bars represent the effects of different oligosaccharide standards or HMO fractions on PNC formation in ADP-stimulated blood indicated as mean and standard deviation. PNC formation in ADP-stimulated blood without prior oligosaccharide treatment was defined as 100% (open bar). All samples were used at 125 µg/mL. *, P< 0.05; **, P< 0.01; ***, P< 0.001; the presence of common letters next to the data bars indicates that these groups are not statistically different from each other.

 
ADP-stimulated blood was coincubated with the aHMO fraction (125 µg/mL) for 5, 15, 30, 45, and 60 min, and PNC formation was reduced to 85.9 ± 6.3% (P<0.01), 79.7 ± 7.4 (P<0.001), 80.7 ± 6.9% (P<0.001), 81.4 ± 8.1% (P<0.001), and 82.1 ± 7.5% (P<0.001), respectively. Consequently, we used a 15-min incubation time in all following experiments, as longer incubation times had no further inhibitory effect on PNC formation.

PNC formation: concentration dependency
To investigate whether the aHMO fraction inhibited PNC formation in a concentration-dependent manner, the aHMO concentration in the blood was gradually reduced to 6.25 µg/mL. After adding the aHMO fraction in a concentration of 87.5, 50, 25, and 12.5 µg/mL, PNC formation in ADP-stimulated blood was reduced to 82.2 ± 7.0% (P<0.001), 86.4 ± 7.1% (P<0.001), 90.2 ± 4.8% (P<0.001), and 92.7 ± 3.7% (P<0.001), respectively. However, at the lowest concentration of 6.25 µg/mL, PNC formation was not affected anymore (Fig. 3 ).



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  		Figure 3. PNC formation: concentration dependency. The bars represent the effects of different aHMO concentrations on PNC formation in ADP-stimulated blood, indicated as mean and standard deviation. PNC formation in ADP-stimulated blood without prior oligosaccharide treatment was defined as 100% (open bar). ***, P < 0.001; the presence of common letters next to the data bars indicates that these groups are not statistically different from each other.

 
Neutrophil ß 2 integrin expression: structure dependency
In previous studies, P-selectin-mediated PNC formation was associated with an increased expression of the ß 2 integrin, CD11b/CD18. Therefore, we investigated whether the inhibition of PNC formation with oligosaccharides also affected neutrophil CD11b/CD18 expression. CD11b/CD18 was present on resting neutrophils but increased further following the addition of 10 µM ADP. The CD11b/CD18 expression fell to 75.1 ± 20.5% of the ADP-stimulated blood after the addition of soluble sLex in a concentration of 125 µg/mL (P<0.05; ADP-stimulated blood alone was referred to as 100%). CD11b/CD18 expression on neutrophils was not affected by the addition of TriGal. When 125 µg/mL aHMO was added to ADP-stimulated blood, CD11b/CD18 expression fell significantly to 69.5 ± 15.7% (P<0.001). In contrast, the nHMO fraction had no effect (Figs. 4 and 5 ).



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  		Figure 4. FACS analysis of CD11b expression on neutrophil. CD11b expression was compared between unstimulated (black line) and ADP-stimulated blood (shaded pattern; A). The addition of the aHMO (B) and nHMO fraction (C) to ADP-stimulated blood was analyzed (black lines) and compared with ADP-stimulated blood without further treatment (shaded pattern).

 


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  		Figure 5. CD11b expression: structure dependency. The bars represent the effects of different oligosaccharide standards or HMO fractions on CD11b expression in ADP-stimulated blood, indicated as mean and standard deviation. PNC formation in ADP-stimulated blood without prior oligosaccharide treatment was defined as 100% (open bar). All samples were used at 125 µg/mL. *, P< 0.05; ***, P< 0.001; the presence of common letters next to the data bars indicates that these groups are not statistically different from each other.

 
Neutrophil ß 2 integrin expression: concentration dependency
The influence of aHMO concentration on CD11b/CD18 expression was investigated. At concentrations of 87.5, 50, and 25 µg/mL, the CD11b/CD18 expression on neutrophils was reduced to 70.5 ± 14.9% (P<0.001), 79.8 ± 10.6% (P<0.001), and 76.6 ± 21.3% (P<0.01), respectively. At lower concentrations of 12.5 and 6.25 µg/mL, the aHMO fraction had no effect (Fig. 6 ).



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  		Figure 6. CD11b expression: concentration dependency. The bars represent the effects of different aHMO concentrations on CD11b expression in ADP-stimulated blood, indicated as mean and standard deviation. PNC formation in ADP-stimulated blood without prior oligosaccharide treatment was set as 100% (open bar). **, P < 0.01; ***, P < 0.001; the presence of common letters next to the data bars indicates that these groups are not statistically different from each other.

 
The effect of aHMO on PNC formation and neutrophil activation was not dependent on LPS
Endotoxins (LPS) accumulate in the HMO fractions as a result of their physicochemical similarities. The LPS content was markedly reduced from the HMO fractions by affinity chromatography on polymixin B. We determined the amount of remaining LPS in our nHMO and aHMO fractions and found it to be 815.5 ± 2.1 and 790.9 ± 121.4 pg/mg, respectively. As the HMO fractions were applied at a maximum concentration of 125 µg/mL blood, the amount of LPS added to the blood as part of the nHMO or aHMO fraction was 102.2 ± 0.2 and 98.9 ± 15.2 pg/ml, respectively. The LPS content in both fractions was not significantly different (P>0.79). The amount of LPS in oligosaccharide standards, sLex and TriGal, was below detection level. To verify that the LPS remainders in the samples had no effect on PNC formation or neutrophil activation, unstimulated and ADP-stimulated blood was incubated with LPS in a final concentration of 100 or 1000 pg/mL for 15 min. All experiments were performed on blood from three different donors. No effects on PNC formation could be detected after LPS exposure. Although neutrophil CD11b/CD18 expression was increased to 112.9 ± 7.4% in unstimulated blood after the incubation with LPS in a concentration of 1000 pg/mL (P<0.05), no effect was seen at 100 pg/mL, the levels found in the HMO fractions. Both LPS concentrations (100 and 1000 pg/mL) had no effect on the CD11b/CD18 expression in ADP-stimulated blood.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Neutrophils associated with platelets in PNC are highly activated and primed for adhesion, phagocytosis, and ROS production [15 ]. PNC formation is initiated by P-selectin, which binds to oligosaccharides on its ligand PSGL-1 [14 ]. HMO contain these oligosaccharide-binding determinants [12 ], and we hypothesized that they serve as soluble ligand analogs and block selectin binding to PSGL-1. Here, we report that an aHMO fraction indeed reduces PNC formation and neutrophil activation.

As previously reported [15 , 16 ], incubation of whole blood with ADP induces PNC formation and neutrophil activation. The addition of soluble sLex not only reduced PNC formation by 15% but also decreased CD11b expression by 25% (Figs. 2 and 5) . SLex is a known physiological-binding determinant for selectins [10 ], and soluble sLex inhibits selectin-ligand binding [25 ]. We conclude that soluble sLex competes with PSGL-1 for P-selectin binding and, as a consequence, reduces PNC formation. P-selectin binding to PSGL-1 also initiates signaling pathways, which enhance CD11b expression on the surface of neutrophils [17 , 19 , 26 27 28 ]. We assume that the presence of soluble sLex partially prevents P-selectin binding to PSGL-1 and, therefore, reduces the signal that leads to CD11b up-regulation. This finding is in accordance with the results of a previous study showing that CD11b up-regulation is reduced when P-selectin binding to PSGL-1 is blocked by P-selectin antibodies [15 , 16 ].

The inhibitory effects were dependent on oligosaccharide structures that resemble the selectin-binding determinant. Neither PNC formation nor neutrophil activation was affected by TriGal, which is structurally unrelated to any known selectin ligand-binding determinants (Figs. 2 and 5) . It is mandatory for selectin binding that the oligosaccharides, soluble or as part of glycoconjugate ligands, carry a NeuAc residue [9 10 11 , 29 ]. The aHMO fraction, which consists of oligosaccharides with one or more NeuAc residues, reduced PNC formation by 20% and CD11b expression by 30%. Indeed, the aHMO fraction had an even more pronounced effect on PNC formation and neutrophil activation than the physiologic-binding determinant sLex. These differences between the aHMO fraction and sLex can be explained by the occurrence of multivalent binding sites on complex HMO, which supports greater selectin binding than monovalent sLex [30 31 32 ]. The nHMO fraction, which consists of oligosaccharides without NeuAc residues, had only a minor effect on PNC formation, which is consistent with the small amounts of acidic oligosaccharides detected by HPAEC-PAD and nano-ESI-MS. The nHMO fraction had no effect on CD11b expression. The residual LPS contained within the HMO fractions was found to be ineffectual in influencing PNC formation and CD11b/CD18 expression.

All experiments with the HMO fractions were performed at physiologically relevant concentrations. The most explicit evidences that HMO occur in the systemic circulation of breast-fed infants derive from data showing that ~1% of the ingested HMO appear in the urine of breast-fed infants but not in the urine of formula-fed infants [5 , 6 ]. These data are in accordance with other studies reporting that HMO are only minimally digested [2 , 3 ] and that ~1% is absorbed [4 ]. Although the fate of HMO after absorption and before urinary excretion yet remains a black box, we can conclude that approximately 1% of the daily intake appears in the systemic circulation. Calculations based on these studies [2 3 4 5 ] combined with data on the amount of HMO in human milk [1 ], the infant’s daily intake, and the infant’s blood volume of 80–100 mL per kg body weight show that the aHMO concentration in the blood of breast-fed infants can exceed 100 µg/mL. However, all these parameters can vary within a wide range and, thus, can vary the concentration in the blood. However, even with the highest aHMO concentration of 125 µg/mL, neither PNC formation nor neutrophil activation was reduced to baseline levels found in blood without ADP stimulation. These results are concordant with a previous study on leukocyte rolling and adhesion, another cell–cell interaction initially mediated by selectins. sLex and the aHMO fraction reduced leukocyte adhesion, whereas TriGal and the nHMO fraction had no influence. The inhibitory effects of the aHMO fraction were concentration-dependent and more pronounced than the effects of soluble sLex. Leukocyte adhesion could not be reduced to baseline levels by applying the aHMO fraction in physiological concentrations (L. Bode et al., manuscript submitted).

The physiological consequences of aHMO in neonates/infants are as yet unclear. A possible role could be in preventing/modulating inflammatory conditions known to affect neonates. In necrotizing enterocolitis (NEC), an inflammatory disease prevalent in neonatal intensive care (refs. [1 , 33 ] and manuscript submitted by L. Bode et al.), excessive leukocyte infiltration accelerates the progression of NEC [34 ], and it has been proposed that tissue damage may be caused largely by neutrophil-derived ROS. This leads to the breakdown of mucosal integrity and subsequent bacterial translocation [34 , 35 ]. ROS production is significantly higher in neutrophils associated with platelets but can be decreased by blocking P-selectin-mediated PNC formation [15 ]. In the present study, HMO also reduced PNC formation by serving as soluble selectin-ligand analogs. These results suggest that certain HMO might diminish the production of ROS and therefore reduce the deleterious effects of neutrophils in NEC. The inhibitory effects of HMO on leukocyte extravasation, activation, and PNC formation may help to explain why NEC is less common in breast-fed infants [36 , 37 ].

In conclusion, oligosaccharides from human milk may influence multiple levels of leukocyte activity including extravasation, neutrophil activation, and ROS production. The applicability of our in vitro results to neonatal physiology warrants further investigation.


   ACKNOWLEDGEMENTS
 
This work was supported by the German Research Foundation (DFG Grant #Ru529/4-3). The German Academic Exchange Service funded L. B.’s visit in London.

Received March 25, 2004; revised May 17, 2004; accepted June 4, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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