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J Dent Res 84(10):913-918, 2005
© 2005 International and American Associations for Dental Research

RESEARCH REPORT
Biological

Effects of Positive Pressure in Odontogenic Keratocysts

S. Oka1, Y. Kubota1,*, T. Yamashiro1, S. Ogata1, T. Ninomiya1, S. Ito2, and K. Shirasuna1

1 Department of Oral and Maxillofacial Surgery and 2 Department of Dental Anesthesiology, Graduate School of Dental Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan;

* corresponding author, yasu{at}dent.kyushu-u.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Intracystic fluid pressure is thought to be involved in odontogenic cyst growth. In this study, we investigated the effects of positive pressure on the expression of interleukin-1{alpha} (IL-1{alpha}), matrix metalloproteinases (MMPs), and prostaglandin E2 (PGE2) in odontogenic keratocysts to determine whether this pressure stimulates inflammatory cytokine production and signaling of osteoclastogenic events. Positive pressure enhanced the expression of IL-1{alpha} mRNA and protein in odontogenic keratocyst epithelial cells, and increased the secretion of MMP-1, MMP-2, MMP-3, and PGE2 in a co-culture of odontogenic keratocyst fibroblasts and the epithelial cells. The pressure-induced secretions were inhibited by an interleukin-1 receptor antagonist. Recombinant human interleukin-1{alpha} (rhIL-1{alpha}) increased the secretion of MMP-1, MMP-2, MMP-3, and PGE2 in the fibroblasts. Furthermore, in the fibroblasts, rhIL-1{alpha} enhanced the expression of macrophage colony-stimulating factor (M-CSF) mRNA, and rhIL-1{alpha}-induced PGE2 increased the expression of nuclear factor {kappa}B ligand (RANKL) mRNA. Thus, positive pressure may play a crucial role in odontogenic keratocyst growth via stimulating the expression of IL-1{alpha} in epithelial cells.

KEY WORDS: positive pressure • interleukin-1{alpha} • odontogenic keratocysts.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mechanical stress is thought to play a crucial role in the regulation of bone metabolism (Kanzaki et al., 2002; Morinobu et al., 2003; Tsuji et al., 2004). Recently, we have found that the intracystic fluid pressure of odontogenic jaw cysts is over 80 mm Hg in an early growth stage (Kubota et al., 2004). The decompression of the fluid pressure leads to the gradual reduction of the bone resorption by the cysts (Marker et al., 1996). Positive pressure-dependent regulatory mechanisms, therefore, may be involved in odontogenic cyst growth in the jaws.

Interleukin-1{alpha} (IL-1{alpha}), which is one of the inflammatory and multifunctional cytokines, stimulates bone resorption by producing collagenases, including matrix metalloproteinases (MMPs) (Tewari et al., 1996; Kusano et al., 1998; Kubota et al., 2002). Furthermore, IL-1{alpha} stimulates osteoclast-like cell formation (Lader and Flanagan, 1998; Tani-Ishii et al., 1999). Osteoclastogenesis is regulated by many factors, such as receptor activation of nuclear factor {kappa}B ligand (RANKL), macrophage colony-stimulating factor (M-CSF), and a pseudo-receptor for RANKL [osteoprotegerin (OPG)] (Simonet et al., 1997; Lacey et al., 1998; Nakagawa et al., 1998; Yasuda et al., 1998). Recently, we demonstrated that IL-1{alpha} is highly expressed in the epithelial cells of odontogenic keratocyst tissue sections, and the expression is dramatically decreased after decompression (Ninomiya et al., 2002). Therefore, it is hypothesized that intracystic positive pressure might regulate the expression of IL-1{alpha} in odontogenic keratocyst epithelail cells, and play a crucial role in the growth of the cysts.

The goals of this study were to examine the effects of positive pressure on the expression of IL-1{alpha} in odontogenic keratocyst epithelial cells, and establish whether IL-1{alpha} stimulated the expression of MMP-1, MMP-2, MMP-3, PGE2, and RANKL in odontogenic keratocyst fibroblasts.


   MATERIALS & METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture
Odontogenic keratocyst tissues were obtained from patients admitted to Kyushu University Dental Hospital under institutionally approved protocols after informed consent was obtained. Fibroblasts and epithelial cells were isolated from the biopsied odontogenic keratocyst tissues by the explant outgrowth method, as described previously (Kubota et al., 2000). Fibroblasts were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Sigma Chemical Co., St. Louis, MO, USA) with 10% heat-inactivated fetal calf serum (FCS) and antibiotics (100 IU/mL penicillin and 100 µg/mL streptomycin) under a 95% air and 5% CO2 atmosphere at 37°C, and then used for experiments from the third to seventh passages. Epithelial cells were cultured in serum-free low-calcium KGM (Invitrocyte Inc., Seattle, WA, USA) containing human keratinocyte growth supplement and antibiotics under a 95% air and 5% CO2 atmosphere at 37°C.

Treatment with Positive Pressure or rhIL-1{alpha}
Fibroblasts (2 x 104 cells/cm2) and epithelial cells (4 x 104 cells/cm2) were seeded onto plastic dishes. In co-culture experiments, fibroblasts (2 x 104 cells/cm2) were seeded onto epithelial cells (4 x 104 cells/cm2). The cells were pre-incubated with serum-free DMEM for 24 hrs, and then incubated with fresh serum-free DMEM in the presence or absence of recombinant human IL-1{alpha} (rhIL-1{alpha}) in an acrylic chamber, where the pressure was continuously regulated at either 80 mm Hg or atmospheric pressure (0 mm Hg) of humidified 5% CO2 at 37°C. In some experiments, a recombinant human IL-1 receptor antagonist (IL-1ra; R & D Systems Inc., Minneapolis, MN, USA) was added 15 min before the application of positive pressure. The rhIL-1{alpha} was supplied courtesy of Dainippon Pharmacy Co. (Osaka, Japan). The mouse thymocyte co-mitogenic activity (LAF) of the rhIL-1{alpha} is 2.01 x 10-7 U/mg.

Gelatin Zymography
Gelatin zymography was performed on 10% SDS-polyacrylamide gels impregnated with 2 mg/mL gelatin as described previously (Kubota et al., 2000 , 2001, 2002). The supernatant of the conditioned medium was subjected to electrophoresis under non-reducing conditions, and the gels were washed with 2.5% Triton-X 100 for over 1 hr, and then incubated in 200 mM NaCl, 5 mM CaCl2, 30 mM Tris-HCl (pH 7.6), and 0.02% NaN3 at 37°C for 18 hrs. The lysis of gelatin was visualized under long-wave UV light, and gelatinolytic activities and activity ratios (62-kDa MMP-2 activities/72- and 62-kDa MMP-2 activities) were calculated from the density of each band with the use of a computer system (Kubota et al, 2002). Finally, the gel was stained with 0.2% Coomassie Brilliant Blue R250, and de-stained.

Western Immunoblotting
Samples were run on 10% SDS-polyacrylamide gels, and transferred onto nitrocellulose paper as described previously (Kubota et al., 2000, 2001, 2002). The nitrocellulose paper was incubated with 5% bovine serum albumin in TBST [150 mM NaCl, 10 mM Tris-HCl (pH 8.0), 0.05% Tween-20, and 0.02% NaN3] for 1 hr, incubated with a 1:100 dilution of polyclonal antibody against MMP-1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), MMP-2 (Calbiochem, Cambridge, MA, USA), or MMP-3 (Oncogene Research Products, Boston, MA, USA), and then developed by a horseradish peroxidase ABC kit. The amounts of MMP-1 and MMP-3 were quantitated from densitometric scans of immunostained nitrocellulose blots.

Measurements of IL-1{alpha} and PGE2 Concentrations
The concentrations of human IL-1{alpha} (Amersham International plc, Buckinghamshire, UK) and prostaglandin E2 (PGE2, Amersham Biosciences, Piscataway, NJ, USA) were measured by enzyme-linked immunosorbent assay (ELISA) according to the manufacturers’ instructions. Absorbance was measured at 450 nm with a microplate reader (Colona Electric, Ibaragi, Japan).

Reverse-transcriptase/Polymerase Chain-reaction (RT-PCR)
Total RNA was extracted by means of Trizol reagent according to the manufacturer’s protocol (Gibco/BRL, Gaithersburg, MD, USA). First-strand cDNA was synthesized from 3 µg total RNA, and PCR amplification was performed with the use of 25 µL of cDNA reaction mixture, as described previously (Kubota et al., 2002). The specific primers for IL-1{alpha} (Maas-Szabowski et al., 1999), RANKL (Hofbauer et al., 1999), M-CSF (Kanzaki et al., 2002), OPG (Quinn et al., 2000), and ß-actin (Yamamura et al., 1992) were used (TableGo). The cDNA amplification was carried out with a DNA thermocycler (ATTO, Tokyo, Japan). PCR products were resolved by electrophoresis on 1.8% agarose gels, and detected by ethidium bromide staining. The images of the gels were captured digitally, and the relative amounts of IL-1{alpha}, RANKL, M-CSF, and OPG mRNAs were calculated by normalization with the amount of ß-actin mRNA (Yokoi et al., 1993; Kubota et al., 2002).


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  		Table. Specific Primers and Annealing Temperatures for PCR Amplification
 
Measurement of Cellular Injury and Cell Viability
Injury to the plasma membrane was evaluated by fluo-3 and fluoresceinated dextran. For measurement of the fluorescent intensity of fluo-3, cells attached to a glass coverslip were incubated with 5 µM acetoxymethyl ester of fluo-3 (fluo-3 AM) for 30 min at room temperature (Fukugasako et al., 2003). The cells were mounted on the bottom of a chamber (500-µL capacity), and a constant positive pressure was applied in the chamber. The change in the fluorescent intensity of fluo-3 was determined with a confocal laser scanning microscope (IX70, Olympus). We measured fluoresceinated dextran uptake by adding 5 mg/mL fluoresceinated dextran (Sigma Chemical Co., St. Louis, MO, USA) to the medium before applying positive pressure (Lee et al., 1997). The fluorescent intensity in the cells was monitored with a fluorescent microscope (TE300/TE-FM, Nikon). Cell viability was calculated as the number of trypan-blue-excluded cells/number of total cells (Lee et al., 1997).

Statistical Analysis
Data are expressed as mean ± SD. The Mann-Whitney U-test was used for statistical analyses, and p values < 0.05 were considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of Positive Pressure on the Expression of IL-1{alpha}
Odontogenic keratocyst epithelial cells expressed IL-1{alpha} mRNA spontaneously, and the expression of IL-1{alpha} mRNA was enhanced by 80 mm Hg of positive pressure to 1.8 ± 0.5 times that of the control within 15 min (n = 5) (Fig. 1AGo). Furthermore, the concentration of IL-1{alpha} in the medium was significantly increased (about 1.5 times the control) by the application of positive pressure for 24 hrs (Fig. 1BGo). Odontogenic keratocyst fibroblasts did not secrete detectable amounts of IL-1{alpha} (data not shown). When positive pressure from 0 to 120 mm Hg was applied to the epithelial cells for 15 min, no significant change in the fluorescent intensity of fluo-3 was detected in the presence of 1 mM extracellular Ca2+ (Fig. 1CGo). In addition, no fluoresceinated dextran uptake into the cells was detected (data not shown). When 80 mm Hg of positive pressure was applied to the epithelial cells for 24 hrs, the percentage of cell viability (93.4 ± 2.5%, n = 5) was not significantly decreased (91.7 ± 3.5%, n = 5).



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  		Figure 1. Effects of positive pressure on the expression of IL-1{alpha} and on the plasma membrane permeability. (A) Odontogenic keratocyst epithelial cells were incubated in serum-free DMEM for 15 min at 37°C under atmospheric pressure or 80 mm Hg of positive pressure. Total cellular RNA was extracted, and RT-PCR amplifications were performed at 30 cycles for IL-1{alpha}, and 27 cycles for ß-actin, as described in MATERIALS & METHODS. (B) Odontogenic keratocyst epithelial cells were cultured in serum-free DMEM for 24 hrs at 37°C under atmospheric pressure or 80 mm Hg of positive pressure. The concentration of IL-1{alpha} in the conditioned media was measured by ELISA, as described in MATERIALS & METHODS. Vertical bars indicate mean ± SD (n = 4). *Significant difference between atmospheric pressure and positive pressure at p < 0.05. (C) Odontogenic keratocyst epithelial cells were incubated with 5 µM fluo-3 AM for 30 min at room temperature, as described in MATERIALS & METHODS. The fluorescent intensity for fluo-3 was monitored before (a) and after (b) application of 80 mm Hg positive pressure to the cells. Bar represents 40 µm.

 
Effects of Positive Pressure and of rhIL-1{alpha} on the Expression of MMP-1, -2, and -3, and PGE2
Odontogenic keratocyst fibroblasts secreted proMMP-1, proMMP-3, and 72-kDa gelatinase spontaneously (Fig. 2AGo, lane 1). The 72-kDa gelatinase was identified as proMMP-2 by immunoblotting (data not shown). Eighty mm Hg of positive pressure did not affect the secretion of these MMPs from the fibroblasts (Fig. 2AGo, lane 2). Odontogenic keratocyst epithelial cells did not secrete MMP-1, -3, and -2 spontaneously, while 80 mm Hg of positive pressure induced the secretion of proMMP-3 (Fig. 2AGo, lanes 3, 4). When the fibroblasts were seeded onto the epithelial cells, the spontaneous secretion of the proMMP-1, -3, and -2 was enhanced [2.33 ± 0.09 (n = 3), 4.27 ± 0.37 (n = 3), and 2.01 ± 0.05 (n = 3) fold, respectively]. The secretions of the proMMPs were further enhanced by the application of positive pressure [1.81 ± 0.05 (n = 3), 1.26 ± 0.07 (n = 3), and 1.42 ± 0.08 (n = 3) fold, respectively]. Activation of proMMP-2 was also enhanced when the fibroblasts were seeded onto the epithelial cells, and positive pressure resulted in a further increase in the activity ratio, from 0.24 ± 0.05 (n = 3) to 0.51 ± 0.06 (n = 3) (Fig. 2AGo, lanes 5, 6). These enhancements by positive pressure were inhibited by IL-1ra (Fig. 2AGo, lane 7). Odontogenic keratocyst fibroblasts (0.13 ± 0.02 ng/mL, n = 4) and epithelial cells (0.04 ± 0.02 ng/mL, n = 4) secreted a small amount of PGE2 spontaneously, and 80 mm Hg of positive pressure did not affect the secretion in either cell type. Spontaneous secretion of PGE2 was increased (1.03 ± 0.11 ng/mL, n = 4) by the co-cultivation of the epithelial cells and the fibroblasts, and positive pressure further enhanced the secretion significantly (3.86 ± 0.26 ng/mL, n = 4). This enhancement was inhibited by IL-1ra (Fig. 2BGo).



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  		Figure 2. Effects of positive pressure and rhIL-1{alpha} on the secretion of MMP-1, MMP-2, MMP-3, and PGE2. (A,B) Odontogenic keratocyst fibroblasts (2 x 104 cells/cm2) (a) and odontogenic keratocyst epithelial cells (4 x 104 cells/cm2) (b) were seeded alone or co-cultured (c), and then incubated in serum-free DMEM for 24 hrs at 37°C under atmospheric pressure or 80 mm Hg of positive pressure. IL-1ra was added 15 min before the application of positive pressure. (C,D) Odontogenic keratocyst fibroblasts (a) and odontogenic keratocyst epithelial cells (b) were incubated in serum-free DMEM for 24 hrs at 37°C in the absence or presence of rhIL-1{alpha}. (A,C) The culture media (200 µL) were concentrated and subjected to Western immunoblotting for MMP-1 and MMP-3, and the 30-µL culture media were subjected to gelatin zymography. (B,D) The concentration of PGE2 in the culture media was measured by ELISA as described in MATERIALS & METHODS. Vertical bars indicate mean ± SD (n = 4). *Significant difference at p < 0.05.

 
In odontogenic keratocyst fibroblasts, rhIL-1{alpha} enhanced the secretion of proMMP-1, proMMP-2, and proMMP-3 (Fig. 2CGo). The fibroblasts secreted a small amount of PGE2 spontaneously, and this secretion was also increased significantly by 0.1 nM rhIL-1{alpha} (Fig. 2DGo). In contrast, in odontogenic keratocyst epithelial cells, IL-1{alpha} induced the secretion of proMMP-3, but did not induce the secretion of MMP-1, MMP-2, or PGE2 (Figs. 2CGo, 2DGo).

Effects of rhIL-1{alpha} on the Expression of RANKL, M-CSF, and OPG mRNAs
Although the expression of RANKL mRNA was not detected in the control condition in odontogenic keratocyst fibroblasts, rhIL-1{alpha} (over 1 nM) did induce its expression (Fig. 3AGo). When the amount of RANKL mRNA was normalized with the amount of ß-actin mRNA, the relative value of RANKL mRNA was increased from 0 to 0.52 ± 0.21 (n = 5) and 1.04 ± 0.38 (n = 5) by 1 nM and 10 nM rhIL-1{alpha}, respectively. The expression of RANKL mRNA was also induced by 10 µM PGE2, and the rhIL-1{alpha}-induced expression of RANKL mRNA was completely inhibited by 1 µM indomethacin. M-CSF mRNA was expressed weakly in the control, and was enhanced by rhIL-1{alpha}. However, indomethacin failed to inhibit the rhIL-1{alpha}-induced expression of M-CSF mRNA. Furthermore, PGE2 did not enhance the expression of M-CSF mRNA. Strong expression of OPG mRNA was detected in the control, but neither rhIL-1{alpha} nor PGE2 affected its expression (Fig. 3BGo).



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  		Figure 3. Effects of rhIL-1{alpha} and PGE2 on the expression of RANKL, M-CSF, and OPG mRNAs in odontogenic keratocyst fibroblasts. (A) Effects of rhIL-1{alpha} on the expression of RANKL mRNA in odontogenic keratocyst fibroblasts. Odontogenic keratocyst fibroblasts were incubated in serum-free DMEM for 12 hrs at 37°C in the absence or presence of rhIL-1{alpha}. RT-PCR amplifications were performed at 35 cycles for RANKL and 27 cycles for ß-actin, respectively, as described in MATERIALS & METHODS. (B) Effects of PGE2 on the expression of RANKL, M-CSF, and OPG mRNAs in odontogenic keratocyst fibroblasts. Odontogenic keratocyst fibroblasts were incubated in serum-free DMEM for 12 hrs at 37°C in the absence (lane 1) or presence of 10 nM rhIL-1{alpha} (lanes 2 and 3) or 10 µM PGE2 (lane 4). Indomethacin (1 µM) was added 15 min before the application of 10 nM rhIL-1{alpha} (lane 3). RT-PCR amplifications were performed at 35 cycles for RANKL, 30 cycles for M-CSF, 25 cycles for OPG, and 27 cycles for ß-actin, respectively, as described in MATERIALS & METHODS.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we have demonstrated one possible pathological mechanism for positive pressure-induced bone resorption in odontogenic keratocysts. Positive pressure increased the expression of IL-1{alpha} mRNA and protein in odontogenic keratocyst epithelial cells. This finding is supported by the results of our previous study, in which we showed that strong signal intensities for IL-1{alpha} mRNA and protein were detected in the epithelial cells of odontogenic keratocyst tissue sections, and were significantly decreased after decompression (Ninomiya et al., 2002). It has been reported that mechanical deformation leads to the rapid release of IL-1{alpha} and IL-1ra through transient disruption of the plasma membrane (Lee et al., 1997). Positive pressure, however, did not increase the fluorescent intensity of fluo-3 and dextran in odontogenic keratocyst epithelial cells. Neither cell viability nor cell numbers (data not shown) of the epithelial cells were changed by the pressure. Thus, under our conditions, the positive-pressure-induced secretion of IL-1{alpha} may not be due to an elevation of plasma membrane permeability or the increase of cell numbers.

Positive pressure increased secretions of proMMP-1, proMMP-2, proMMP-3, and PGE2 in a co-culture of odontogenic keratocyst fibroblasts and epithelial cells. These events were inhibited by IL-1ra. In odontogenic keratocyst fibroblasts, secretions of these proMMPs and PGE2 were not enhanced by positive pressure, but were enhanced by rhIL-1{alpha}. Furthermore, neither positive pressure nor rhIL-1{alpha} induced the secretions of the proMMP-1, proMMP-2, and PGE2 in odontogenic keratocyst epithelial cells (data not shown). Only a small amount of proMMP-3 was induced by positive pressure or rhIL-1{alpha} in the epithelial cells. Thus, our findings suggest that, in the co-cultivation experiments, the positive pressure may enhance the secretion of the proMMPs and PGE2 from the fibroblasts by inducing the secretion of IL-1{alpha} from the epithelial cells. When positive pressure was applied to odontogenic keratocyst epithelial cells, the concentration of IL-1{alpha} in the conditioned media was approximately 3 pM. This concentration is too low to induce the secretions of proMMPs and PGE2 from odontogenic keratocyst fibroblasts. However, the local concentration of IL-1{alpha} secreted from the epithelial cells around the fibroblasts might be higher than the average values of the conditioned media, because the fibroblasts were seeded directly onto the epithelial cells. Activation of proMMP-2 was also induced in the co-culture by positive pressure. IL-1{alpha} stimulated the activation of proMMP-2 when fibroblasts were cultured on a type I collagen-coated dish (Kubota et al., 2002). It has been reported that both soluble collagen I and procollagen I mRNA transcript levels are increased by keratinocyte-fibroblast interaction (Lim et al., 2002). Therefore, the activation of proMMP-2 in co-cultivation might be induced by positive-pressure-induced IL-1{alpha}.

Osteoclastogenesis is regulated by many signals, such as RANKL/RANK and M-CSF/M-CSF receptor (c-Fms) (Yasuda et al., 1998; Arai et al., 1999). c-Fms is expressed in osteoclast precursors at an early stage of osteoclastogenesis. M-CSF activates c-Fms, and enhances RANK expression in osteoclast precursors (Arai et al., 1999). Binding RANKL to RANK on the osteoclast precursors stimulates osteoclast differentiation. The interesting finding in this study is that rhIL-1{alpha} enhanced the expression of RANKL and M-CSF mRNAs in odontogenic keratocyst fibroblasts, suggesting that odontogenic keratocyst fibroblasts may support osteoclast differentiation in the presence of IL-1{alpha}. The enhanced expression of RANKL mRNA was completely inhibited by indomethacin, and PGE2 enhanced its expression in the fibroblasts. Since rhIL-1{alpha} increased the secretion of PGE2 from the fibroblasts, this IL-1{alpha}-induced enhancement of RANKL mRNA expression may be induced not by the direct action of IL-1{alpha} on the cells, but by indirect action via PGE2. In contrast, the expression of M-CSF mRNA was not inhibited by indomethacin, and PGE2 did not enhance its expression. Thus, in odontogenic keratocyst fibroblasts, the mechanisms for the IL-1{alpha}-induced expression of M-CSF mRNA may be different from those for the IL-1{alpha}-induced expression of RANKL mRNA.

In summary, the results from this study demonstrate that positive pressure up-regulates the expression of IL-1{alpha} in epithelial cells, and that the secreted IL-1{alpha} enhances the expression of RANKL and M-CSF mRNAs, and the secretion of proMMP-1, proMMP-2, proMMP-3, and PGE2 in co-cultured fibroblasts. Thus, there may be a strong relationship among intracystic pressure, the expression of IL-1{alpha}, and bone resorption. These findings may explain one possible mechanism for the intracystic positive-pressure-dependent growth of odontogenic keratocysts in the jaw.


   ACKNOWLEDGMENTS
 
This work was supported by a Grant in Aid from the Ministry of Education of Japan (No. 09672054 and No. 15592117).

Received June 28, 2004; Last revision June 21, 2005; Accepted June 22, 2005


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 DISCUSSION
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