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NDT Advance Access originally published online on December 1, 2006
Nephrology Dialysis Transplantation 2007 22(3):756-762; doi:10.1093/ndt/gfl715
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© The Author [2006]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Leptin augments myofibroblastic conversion and fibrogenic activity of human peritoneal mesothelial cells: A functional implication for peritoneal fibrosis

An-Hang Yang1,4, Seng-Wong Huang5, Jin-Yang Chen2,6, Jan-Kou Lin3,5 and Chun-Yi Chen1

1Division of Ultrastructural and Molecular Pathology, Department of Pathology, 2Division of Nephrology, Department of Medicine and 3Division of Colorectal surgery, Department of Surgery, Taipei Veterans General Hospital, Taipei, Taiwan, 4Department of Pathology, 5Department of Surgery and 6Department of Medicine, National Yang Ming University, Taipei, Taiwan

Correspondence and offprint requests to: Dr An-Hang Yang, Division of Ultrastructural and Molecular Pathology, Department of Pathology, Taipei Veterans General Hospital, Taipei 112, Taiwan. Email: ahyang{at}vghtpe.gov.tw



   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Background. Myofibroblastic conversion of mesothelial cells is proposed to play an important role in pathological changes following serosal membrane injury.

Methods. Human peritoneal mesothelial cells (HPMCs) were isolated and maintained in culture. The gene expression was assessed by RT–PCR. Activation of signal transduction was determined by western blot and densitometry. Morphological changes were observed by phase-contrast and electron microscopy.

Results. In vitro study showed that TGF-ß1-induced myofibroblastic growth of HPMCs was significantly enhanced in the presence of leptin. Augmented expression of {alpha}-smooth muscle actin, fibronectin and type I collagen mRNA in HPMCs induced by leptin were TGF-ß1-dependent, suggesting that leptin promoted peritoneal fibrogenesis through synergistic activation of the TGF-ß1 signaling system. Leptin and TGF-ß1 synergistically augmented activation of signalling components of mitogen-activated protein kinase (MAPK), STAT3 and Smad but did not modulate the expression of LEPR-B.

Conclusion. Leptin may act as a profibrogenic TGF-ß1 activated cytokine in peritoneal bioenvironment associated with TGF-ß1 activated pathogenic processes.

Keywords: dialysis; fibrosis; leptin; mesothelial cell; myofibroblast; TGF-ß1



   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The transition of mesothelial cells to myofibroblasts was observed in the human serosal membrane under various inflammatory conditions induced by peritoneal dialysis [1, 2]. Interesting enough is to find that dialysate leptin concentration is positively correlated with plasma leptin, which is usually elevated in patients receiving peritoneal dialysis [3, 4]. Recently, leptin has been reported to exert fibrogenic effects on liver and kidney tissues. The rat Kupffer cells and hepatic sinusoidal endothelial cells (SECs), but not hepatic stellate cells (HSCs), expressed the functional isoform of LEPR (Ob-Rb) and upregulated TGF-ß1 in response to leptin stimulation [5]. The colocalization of leptin and {alpha}-smooth muscle actin ({alpha}-SMA) in activated HSCs implied the capability of leptin to promote fibrogenic effects in a paracrine fashion through myofibroblastic transdifferentiation [6]. In the kidney, leptin not only stimulated glomerular endothelial cells to produce TGF-ß1 and type IV collagen, but also enhanced mesangial cells to synthesis synthesize TGF-ß receptors (TGFßR) and type I collagen [7, 8] The mesangial cells might respond to leptin stimulation through a short form of leptin receptor (Ob-Ra) and phosphatidylinositol-3 kinase (PI-3K)-dependent signal pathway [8].

Our previous study has found that TGF-ß1 constantly activated myofibroblastic conversion of human peritoneal mesothelial cells (HPMCs) in vitro [9]. Since leptin can enhance fibrogenic activity of HSCs and mesangial cells via a process of myofibroblastic transition, we speculate that a similar process might occur in mesothelial cells if they are stimulated to undergo myofibroblastic conversion in a leptin-rich environment.



   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Acknowledgements
 References
 
Culture conditions
We prepared HPMCs as previously described [12]. The confluent HPMCs were transferred to lower serum-containing medium (2% FBS) for 48 h prior to treatment. The induction of myofibroblastic conversion was initiated by continuously exposing HPMC to recombinant human TGF-ß1 (3 ng/ml, R&D Systems, Minneapolis, MN, USA) for 6 days. Other parallel experimental groups included leptin treatment alone (2, 20 and 200 ng/ml; R&D Systems, Minneapolis, MN, USA) and combined leptin and TGF-ß1 treatment. The control groups were parallel duplication of experimental groups in terms of cell batch, basic growth condition and culture period.

Western blot analyses
Western blot analyses of RIPA lysates were performed according to standard procedures using antibodies following manufacturers' suggestions. Activation of p44/42 MAPK (Erk1/Erk2), STAT3 and Smad2/3 were determined by measuring the phosphorylation state of respective proteins. Before termination of culture, cells received final treatment of leptin, TGF-ß1 or combined leptin and TGF-ß1 for 15 min to assess activation of p44/42 MAPK, for 30 min to assess activation of STAT3 or for 60 min to assess activation of Smad2/3. The monolayers were then solubilized by the direct addition of protease inhibitor cocktail containing RIPA lysis buffer (Upstate, Charlottesville, Virginia, USA) and cell debris were removed by centrifugation at 4°C. Protein concentration was determined using Bio-Rad Protein Assay (BIO-RAD, Hercules, CA, USA). The extracts (50 µg/lane) were further subjected to SDS–PAGE (4% stacking/8% resolving gel) in reducing conditions, followed by transfer to a nitrocellulose membrane (PolyScreen® PVDF Transfer Membrane, PerkinElmer Life Sciences, CT, USA) and probed with primary antibodies against phospho-Smad2 (Ser465/Ser467), phospho-p44/42 MAPK (Thr202/Tyr204) (Cell Signaling Technology, Beverly, MA, USA) and phospho-STAT3 (Tyr705) (Upstate, NY, USA). The same cell extract was used to detect LEPR in separate western blotting with mouse monoclonal antibody against human long-form LEPR (Chemicon, CA, USA). After incubation with the appropriate horseradish peroxidase-conjugated secondary antibodies, the specific labelling of protein was demonstrated by chemiluminescence (Western Lightning Plus, PerkinElmer Life Sciences, CT, USA) recorded on high-speed film (Kodak Biomax Light Film, Rochester, NY, USA). Membranes were subsequently stripped with western blot stripping buffer (Pierce, Rockford, IL, USA), and reprobed with antibodies for total Smad2/3 (Cell Signaling Technology, Beverly, MA, USA), total p44/42 MAPK (Cell Signaling Technology, Beverly, MA, USA), total STAT3 (Upstate, NY, USA) and {alpha}-tubulin (Amersham Life Science, UK). Relative band density was determined from light scans of the resulting films using densitometric analysis software (AlphaEaseFC 4.0, Alpha Innotech, USA).

RNA isolation and RT-PCR analyses
Total RNA was extracted with TRlzol (Invitrogen, CA, USA). Contaminated genomic DNA was removed with RNAse-free DNAse (Ambion, Austin, TX, USA) following manufacturer's protocol. First-strand cDNA was synthesized from total RNA with SuperscriptTM First-strand cDNA synthesis system for RT-PCR (Invitrogen, CA, USA) using Oligo(dT) as primers. The expression of LEPR isoforms in HPMC was assessed by RT-PCR analysis using the following primers [13]: LEPR-A (Hub219.3) sense 5'-ATTCAATTGGTGCTTCTGTT-3', antisense 5'-CATTGGGTTCATCTGTAGTG-3'; LEPR-B (OBRb, long-form, accession no. U43168 [GenBank] ) sense 5'-CAGAAGCCAGAAACGTTTGAG-3', antisense 5'-AGCCCTTGTTCTTCACCAGT-3'; LEPR-C (Hub219.1, accession no. U52912 [GenBank] ) sense 5'-TTGGAAGCCCCTGATGAAA-3', antisense 5'-AGCAGATAAACAAGTGAACAAAG-3'; LEPR-D (Hub219.2, accession no. U52913 [GenBank] ) sense 5'-TTGGAAGCCCCTGATGAAA-3', antisense 5'-AGGTGCGCACGAGGTAGGA-3'. GAPDH (sense 5'-ATCAAGAAGGTGGTGAAGCAGG-3', antisense 5'-GCAACTGTGAGGAGGGGAGATT-3') were used as control. Amplification was carried out in a thermal cycler (Tpersonal 48, Biometra, Gijttingen, Germany) by 30 s denaturation at 94°C, 30 s annealing at 66°C and 40 s extension at 72°C. Quantitative evaluation of collagen I, collagen III, {alpha}-smooth muscle actin ({alpha}-SMA), fibronectin and connective tissue growth factor (CTGF) mRNA transcripts by real-time RT–PCR were performed using the following primers: collagen I sense (accession no. BC036531 [GenBank] ) 5'-CCTGCGTGTACCCCACTCA-3', antisense 5'-ACCAGACATGCCTCTTGTCCTT-3'; collagen III (accession no. BC028178 [GenBank] ) sense 5'-TCTTGGTCAGTCCTATGCGGATA-3', antisense 5'-GGATCCTGAGTCACAGACACATATTT-3'; {alpha}-SMA (accession no. BC017554 [GenBank] ) sense 5'-TCCTCCCTTGAGAAGAGTTACGA -3', antisense 5'-GGCAGCGGAAACGTTCATT -3'; fibronectin (accession no. X02761 [GenBank] ) sense 5'-TCGCCATCAGTAGAAGGTAGCA -3', antisense 5'-TATACTGAACACCAGGTTGCAAGTC -3'); CTGF (accession no. M92934 [GenBank] ) sense 5'-TGCACCGCCAAAGATGGT -3', antisense 5'-GACTCTCCGCTCCGGCACAC-3'. All primers used in real-time RT-PCR reactions were designed using Primers Express V2.0 (Applied Biosystems, Foster City, CA, USA) and checked for homology in BLAST. Real-time RT-PCRs were carried out in triplicate with both 18S rRNA internal controls and no-template controls using a SYBR Green PCR kit (Applied Biosystems, Foster City, CA, USA) on an ABI Prism 7500 sequence detector (Applied Biosystems, Foster City, CA, USA). The quantitative method involved obtaining the CT values for the PCR products, normalizing to 18S rRNA housekeeping gene and deriving the fold increase compared with control, unstimulated cells.

Electron microscopy
The HPMCs growing on Transwell membranes (Transwell-Clear 3452, Costar) were initially fixed in 2.5% glutaraldehyde in phosphate buffer. The cells were subsequently processed through osmication, dehydration and embedding as a routine. The membranes embedded in Spurr resin (Electron Microscopy Science, Ft. Washington, PA, USA) were sectioned vertically and thin sections were viewed and digitally recorded using a transmission electron microscope (JEM-1230, JEOL, Japan) equipped with multiscan CCD camera (791 MSC, Gatan, USA).

Statistical analyses
Real-time RT–PCR data were analysed using repeated one-way analysis of variance followed by Newman–Keuls tests for each pair for multiple comparisons and by linear trend tests for the dose effect of leptin. Differences were considered significant if P < 0.05. All analyses were performed using the GraphPad Prism version 3.00 for Windows, (GraphPad Software, San Diego, CA, USA).



   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Acknowledgements
 References
 
The effects of leptin on HPMC growth
To examine the effects of leptin on the growth of HPMCs, we used both TGF-ß1 stimulated and TGF-ß1 free culture systems. Under TGF-ß1 free culture conditions, HPMCs constantly maintained epithelial cell morphology with characteristic cobblestone-like growth pattern, no matter whether they were treated with leptin or not (Figure 1A–D). However, simultaneous administration of leptin (2–200 ng/ml) and TGF-ß1 augmented myofibroblastic conversion of HPMCs by enhancing crisscrossed spindle morphology, multilayering growth and formation of multifocal cellular hillocks (Figure 1E–H). While leptin alone failed to alter cellular structures of HPMCs (Figure 1I), the simultaneous treatment of HPMCs with leptin (2–200 ng/ml) and TGF-ß1 (0.3 ng/ml) increased cytoplasmic actin bundles and extracellular collagenous matrix (Figure 1J). The ultrastructural examination of cellular hillocks showed focal piling up of spindled HPMCs with interspersed deposition of collagenous matrix (Figure 1J).


Figure 1
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  		Fig. 1. Phase-contrast and electron microscopy of HPMCs. (A) The control mesothelial cells without treatment of TGF-ß1 exhibited epithelial cell morphology with characteristic cobblestone-like growth pattern. (B–D) The mesothelial cells maintained epithelial morphology after continuous treatment of leptin (B: 2 ng/ml, C: 20 ng/ml, D: 200 ng/ml) for 6 days. (E) After continuous treatment of TGF-ß1 (0.3 ng/ml) for 6 days, the epithelial morphology of mesothelial cells completely converted to spindle fibroblast-like morphology with multilayered crisscross growth pattern. (F–H) The mesothelial cells showed exaggerated spindle cell morphology with focal hillocky aggregation of cells and extracellular matrix (arrows) after combined treatment of TGF-ß1 (0.3 ng/ml) and leptin (F: 2 ng/ml, G: 20 ng/ml, H: 200 ng/ml) for 6 days. (I) A representative electron micrographic montage of HPMC after continuous treatment of leptin (20 ng/ml) for 6 days. (J) A parallel control culture of unstimulated HPMC. The leptin-treated mesothelial cells maintained typical epithelial monolayer growth with scattered surface microvilli. (K) A representative electron micrographic montage of a hillock growth of HPMC after combined treatment of leptin (20 ng/ml) and TGF-ß1 (0.3 ng/ml) for 6 days. HPMCs showed characteristic myofibroblastic morphology with spindle multilayered growth, well-developed actin bundles decorated with dense bodies running through the cytoplasm (arrows), and abundant extracellular collagenous matrix between cells (double arrows). Scale bars: 500 µm for phase contrast images, 5 µm for electron microscopic images.

 
The effects of leptin on the expression of LEPR on HPMC
The constitutional expression of LEPR on HPMCs was demonstrated by RT-PCR and western blot analyses (Figure 2). HPMCs were found to express mRNA of both long isoform (LEPR-B) and short isoforms (LEPR-C, LEPR-D and LEPR-A) under all experimental conditions (Figure 2A). As further revealed by western blot analyses, the treatment of leptin and/or TGF-ß1 did not alter the amount of long isoform (LEPR-B) expression on HPMCs (Figure 2B).


Figure 2
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  		Fig. 2. The expression of LEPR on HPMCs treated with leptin, TGF-ß1, or both. (A) RT–PCR analysis of mRNA expression of LEPR long form (LEPR-B), LEPR short forms (LEPR-C, -D and -A) and internal control of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Lane marks: M (DNA ladder); Control (HPMCs without treatment of TGF-ß1/leptin); TG (HPMCs after continuous treatment of TGF-ß1 0.3 ng/ml for 6 days); L2/L20/L200: (HPMCs after continuous treatment of leptin 2/20/200 ng/ml in TGF-ß1-free medium for 6 days); L2TG/L20TG/L200TG (HPMCs after combined treatment of TGF-ß1 0.3 ng/ml and leptin 2/20/200 ng/ml for 6 days). (B) Western blot analysis of LEPR long-form expression on HPMCs. Lane marks represent the same conditions as those indicated in the panel (A). {alpha}-Tubulin served as protein loading control.

 
The modulation of signal transduction by leptin and TGF-ß1 on HPMC
The activation of signal transduction by leptin, TGF-ß1 or both was assessed by measuring the phosphorylation levels of p42/44 MAPK, STAT3 and Smad2 in HPMCs (Figure 3). Leptin alone at high dose (200 ng/ml) enhanced phosphorylation levels of p42/44 MAPK (Figure 3A and D) and STAT3 (Figure 3B and E), but not Smad2 (Figure 3C and F) as compared with untreated control groups. TGF-ß1 alone enhanced activation of Smad2 (Figure 3C and F) and p42/44 MAPK (Figure 3A and D) but not STAT3 (Figure 3B and E). Simultaneous treatment of leptin (2 and 20 ng/ml) and TGF-ß1 to HPMCs showed strong synergistic enhancement of p42/44 MAPK phosphorylation level, while high concentration of leptin (200 ng/ml) showed an additive effect with TGF-ß1 (Figure 3A and D). However, such treatment only exerted modest synergistic effects on STAT3 (Figure 3B and E) and Smad2 (Figure 3C and F) phosphorylation.


Figure 3
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  		Fig. 3. Western blot analysis of MAPK (panel A), STAT3 (panel B) and Smad2/3 (panel C) phosphorylation levels in HPMCs treated for 6 days with the conditions as lane marks indicated in Figure 2A. Densitometry quantification of activated MAPK (D), STAT3 (E) and Smad2/3 (F) are expressed as a ratio to total MAPK, STAT3 and Smad2/3, respectively. {alpha}-Tubulin served as protein loading control.

 
The quantitative assessments of myofibroblastic activity of HPMC
Real-time RT–PCR analyses showed that leptin alone failed to upregulate the expression of mRNA of {alpha}-smooth muscle actin, fibronectin, type I collagen, connective tissue growth factor and type III collagen in HPMCs (Figure 4). TGF-ß1 alone would significantly upregulate the expression of {alpha}-smooth muscle actin, fibronectin and type I collagen in HPMCs, but it failed to enhance the expression of connective growth factor and type III collagen. The simultaneous treatment of leptin and TGF-ß1 to HPMCs showed significant synergistic enhancement of the expression of {alpha}-smooth muscle actin, fibronectin and type I collagen (Figure 4A, B and D). The linear trend tests also revealed a positive dose effect of leptin on these three elements.


Figure 4
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  		Fig. 4. Real-time RT-PCR analysis of mRNA expression of {alpha}-smooth muscle actin (A), fibronectin (B), connective tissue growth factor (C), type I collagen (D) and type III collagen (E) in HPMCs treated for 6 days with the indicated conditions as lane marks described in Figure 2A. The relative changes are defined as the fold increase compared with control (unstimulated) HPMCs. Data are expressed as mean ± SE, n = 3. Asterisks indicate groups that show statistically significant intergroup difference by multiple comparisons.

 


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Acknowledgements
 References
 
As given earlier, a functional connection in terms of fibrogenic effects was found between leptin and TGF-ß1 in certain cell types [7–9]. Whether the inflammation plays a role in the local accumulation of leptin in the peritoneal cavity has not been studied extensively. The surveys of women with untreated pelvic endometriosis have revealed significantly higher peritoneal leptin levels in them than in normal controls [10, 11]. The raised leptin in peritoneal fluid in the early stage of endometriosis was speculated to be induced by inflammatory cytokines associated with the disease [11]. Similarly, the raised leptin in the peritoneal dialysate was speculated to be derived locally and systemically, and closely associated with the dialysis process [3, 4, 12]. On the basis of these findings, it can be hypothesized that either transient or prolonged accumulation of leptin in the peritoneal cavity may be potentially pathogenic to the peritoneal membrane, especially when mesothelial cells fully express both long and short forms of leptin receptors. However, our study failed to reveal that leptin alone could initiate the myofibroblastic conversion of HPMCs, although it did activate Erk MAPK and STAT3 signal pathways. In certain types of cells, including mesothelial cells, leptin could induce TGF-ß1 production [5, 7, 13]. The lack of activation of Smad2/3 by sole leptin stimulation in our experiment implies an ineffective autocrine loop of TGF-ß1 by leptin stimulation. On the other hand, the enhancement of Smad2/3 phosphorylation after treating HPMCs with both leptin and TGF-ß1 may attribute to the upregulation of TGFßR activity by leptin as was demonstrated in the glomerular mesangial cells [8]. So far, it is not clear how leptin modulates TGFßR. In a diabetic animal model, selective inhibition of p42/p44 MAPK significantly inhibited the bradykinin-induced increase in TGFßR2 suggesting a potential mechanistic pathway through which leptin may modulate these receptors in HPMCs [14]. Moreover, leptin might indirectly modulate R-Smad, I-Smad or other TGF-ß1 signalling components to enhance Smad2/3 phosphorylation since a previous study has demonstrated the presence of a positive crosstalk between Erk MAPK and Smad pathway in human mesangial cells [15]. The synergistic enhancement of Smad2/3 phosphorylation by leptin in the present study may contribute to the increased collagen I mRNA expression since it is well-known that regulation of collagen gene expression is a Smad3-dependent process [16]. Beside the Smad signalling system, the activation of Erk MAPK may be essential for collagen production since a previous study showed that induction of type I collagen promoter required a ras-dependent MAPK cascade in mesangial cells [17]. The lack of collagen III mRNA expression in response to TGF-ß1 stimulation represents an inherent property of HPMC, as has been demonstrated in our previous study [9].

The mechanism for the synergistic effect of leptin and TGF-ß1 on the enhancement of MAPK activation in HPMCs is not clear. It is unlikely due to an autocrine influence of leptin since HPMCs were incapable of producing leptin in whatever experimental conditions were used in our study (data not shown). The finding that leptin and TGF-ß1 share a converged signalling transduction via ras-dependent pathway to activate Erk MAPK may account for the synergistic amplification of the signal in HPMCs [18]. However, the higher efficacy of synergistic enhancement of Erk MAPK by low concentrations of leptin (i.e. 20 ng/ml) with TGF-ß1 in HPMCs was an intriguing phenomenon since leptin per se of these amounts were unable to enhance Erk MAPK. There should be additional crosstalk mechanisms in terms of synergistic activation of Erk MAPK between leptin and TGF-ß1. The unresponsiveness of MAPK and STAT3 signalling systems to low concentration of leptin is speculated to be physiologically protective. The synergistic activation of these signal systems, especially at a low concentration of leptin, may pose a risk on the amplification of TGF-ß1 related pathogenic processes of peritoneal dialysis. The less synergistic enhancement of STAT3 by leptin and TGF-ß1 may be due to the lack of a converged signal pathway between them. However, synergistic crosstalk between them is still possible because a cooperative signalling with complex formation between Smads and STAT3 has been demonstrated during astrocyte differentiation [19].

Our previous study has demonstrated that TGF-ß1 could upregulate small guanosine triphosphate Rho, specifically RhoA, and downregulate Rho guanosine diphosphate inhibitor and myosin light chain kinase in HPMCs. These signal transductions might also contribute to actin–myosin assembly, fibronexus formation, and cell contraction during myofibroblastic conversion [9]. The strong synergistic enhancement of smooth muscle actin and fibronectin by leptin and TGF-ß1 in the present study further indicates that leptin can reinforce the similar signal transduction systems activated by TGF-ß1 during myofibroblastic conversion. This speculation is supported by a previous study to show that remodeling of actin cytoskeleton in response to leptin could be potentiated by constitutively active RhoA [20]. In addition, recent signal transduction research has revealed extensive crosstalk between RhoA and STAT3, implying a complex network in mediating the synergistic effects of leptin and TGF-ß1 on epithelial mesenchymal transition. In our study, both leptin and TGF-ß1 were unable to enhance HPMC to express CTGF. This phenomenon may be attributed to the concentration of leptin and TGF-ß1 used in our culture of HPMC. It has been reported that leptin concentration ranging from 0.2 to 1 µg/ml is required to enhance CTGF expression in renal interstitial fibroblasts [21]. Moreover, a higher concentration of TGF-ß1 (>0.1 ng/ml) was found to inhibit CTGF indirectly by enhancing vascular endothelial growth factor (VEGF) expression [22].

In summary, this study demonstrates that mesothelial cells express LEPR. Moreover, in vitro functional tests reveal significant synergistic action of leptin with TGF-ß1 to promote myofibroblastic conversion and fibrogenic activity. Our results have a clinical implication on the therapeutic management to reduce the fibrosis associated with peritoneal dialysis.



   Acknowledgements
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
References
 
The work was supported by Taipei-Veterans General Hospital Research Grant V95C1-033 and National Science Council Grant NSC 94-2320-B-075-009.

Conflict of interest statement. None declared.



   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 

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Received for publication: 22. 7.06
Accepted in revised form: 3.11.06


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