SEARCH

SEARCH BY CITATION

Keywords:

  • endothelial cells;
  • IL-6;
  • IL-8;
  • lipopolysaccharide;
  • MD-2;
  • NF-κB;
  • RANTES;
  • TLR4;
  • Toll-like receptor;
  • vitamin D

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

In addition to its well-known role in mineral and skeletal homeostasis, 1,25-dihydroxyvitamin D3[1,25-(OH)2, D3] regulates the differentiation, growth and function of a broad range of immune system cells, including monocytes, dendritic cells, T and B lymphocytes. Vascular endothelial cells play a major role in the innate immune activation during infections, sepsis and transplant rejection; however, currently there are no data on the effect of 1,25-(OH)2 D3 on microbial antigen-induced endothelial cell activation. Here we show that 1,25-(OH)2 D3 pretreatment of human microvessel endothelial cells (HMEC) inhibited the enteric Gram-negative bacterial lipopolysaccharide (LPS) activation of transcription factor NF-κB and interleukin (IL)-6, IL-8 and regulated upon activation normal T cell exposed and secreted (RANTES) release. The effect of 1,25-(OH)2 D3 was not due to increased cell death or inhibition of endothelial cell proliferation. 1,25-(OH)2 D3 pretreatment of HMEC did not block MyD88-independent LPS-induced interferon (IFN)-β promoter activation. 1,25-(OH)2 D3 pretreatment of HMEC did not modulate Toll-like receptor 4 (TLR4) or MD-2 expression. These data suggest that 1,25-(OH)2 D3 may play a role in LPS-induced immune activation of endothelial cells during Gram-negative bacterial infections, and a suggest a potential role for 1,25-(OH)2 D3 and its analogues as an adjuvant in the treatment of Gram-negative sepsis.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The active form of vitamin D, 1α,25-(OH)2 D3, is known to have immunomodulatory effects, which have been studied most extensively in myeloid and lymphocytoid cells (reviewed in [1]). 1,25-(OH)2 D3 treatment decreases interferon γ[2,3] and interleukin (IL)-12 production in T lymphocytes [4]. In monocytes, 1α,25-(OH)2 D3 treatment leads to their differentiation [5,6], induces CD14 expression and augments lipopolysaccharide (LPS) responses [7–9]. 1α,25-(OH)2 D3 and vitamin D analogues induce a persistent state of immaturity in dendritic cells both in vitro and in vivo[10,11]. Currently there are no data on the immunomodulatory effect of 1α,25-(OH)2 D3 on endothelial cells, which play an important role in the regulation of immune and inflammatory responses (reviewed in [12]).

Vascular endothelial cells (EC) are critical targets for LPS and many cytokines [13–17]. Activation of vascular endothelium by LPS results in EC production of various proinflammatory molecules, including leucocyte adhesion molecules, as well as soluble cytokines and chemokines [13–15], and it has been proposed that widespread vascular endothelial activation, dysfunction and eventually injury occurs in septic shock, ultimately resulting in multi-organ failure [18,19].

Here, we examined the effect of 1α,25-(OH)2 D3 pretreatment of human dermal microvessel endothelial cells (HMEC) on LPS-induced proinflammatory cytokine [interleukin (IL)-6 and IL-8] and chemokine [regulated upon activation normal T cell exposed and secreted (RANTES)] release as well as nuclear transcription factor (NF)-κB activation.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Cells and reagents

The immortalized HMEC obtained from Dr F. J. Candal (Centers for Disease Control and Prevention, Atlanta, GA, USA) [20] were cultured in MCDB-131 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM glutamine and 100 µg/ml penicillin and streptomycin in 24-well plates. The cells were used routinely between passages 10 and 14, as described previously [21]. The cells were plated 2·5 × 104 cells/100 µl. Tissue culture reagents were purchased from Life Technologies (Rockville, MD, USA). Highly purified, phenol-water-extracted Escherichia coli K235 LPS (< 0·008% protein), which was prepared according to the method of McIntyre et al. [22], was generously provided by Dr Stefanie Vogel (University of Maryland, Baltimore, MD, USA). 1α,25-(OH)2 D3 was purchased from Calbiochem (San Diego, CA, USA). All reagents were verified to be LPS-free by the Limulus amoebocyte lysate assay (< 0·03 endotoxin U/ml; Pyrotell; Associates of Cape Cod, Woods Hole, MA, USA).

Expression vectors and endothelial cell transfection

Endothelial leucocyte adhesion molecule (ELAM)-NF-κB-luciferase and pCMV-β-galactosidase vectors were used as described previously [21]. Interferon (IFN)-β luciferase cDNA was kindly obtained from Dr Moshe Arditi (Cedars-Sinai Medical Center, Los Angeles, CA, USA). HMEC were plated at a concentration of 50 000 cells/well in 24-well plates and cultured overnight in MCDB-131 containing 5% serum with or without 1α,25-(OH)2 D3. Cells were co-transfected the following day with reporter genes pCMV-galactosidase (0·1 µg) and (ELAM)-NF-κB-luciferase cDNA (0·5 µg) using FuGene 6 transfection reagent (Boehringer Mannheim, Indianapolis, IN, USA) according to the manufacturer's instructions, as described previously [21]. After overnight transfection, cells were then stimulated for 6 h with 50 ng/ml LPS suspended in phosphate-buffered saline (PBS). Luciferase assay was performed to determine NF-κB and IFN-β promoter activation.

Supernatant cytokine and chemokine determination

IL-6 and IL-8 levels were determined in HMEC culture supernatants by enzyme-linked immunosorbent assay (ELISA) assay according to the manufacturer's instructions (BD Bioscience, Redford, MA, USA). Supernatant RANTES levels were measured by ELISA assay by using Quantikine RANTES immunoassay kit (R&D systems, Minneapolis, MN, USA) according to the manufacturer's instructions. All supernatants were kept frozen at  80°C until analysis; ELISA assays were performed in batches. All experiments were set up in triplicate.

Cell proliferation assay

We assessed HMEC proliferation by using Promega Cell Proliferation Assay (catalogue no. G3580, Madison, WI, USA), which employs a colorimetric method for determining the number of viable cells. Briefly, we plated HMEC in 96-well plates, 5000 cell/well in 100 µl media; treated them with 1α,25-(OH)2 D3 (10 nM in 5% serum) for different durations of time; added 20 µl/well aqueous one solution that contains a tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium, inner salt; MTS(a)] and an electron coupling reagent (phenazine ethosulphate; PES); incubated for 3 h at 37°C and read the value at 490 nm using an ELISA reader. The quantity of formazan product measured by the amount of 490 nm absorbance is directly proportional to the number of living cells in culture. The data are presented as the mean of three different measurements ± s.d.

Real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis

Total RNA was isolated with the RNeasy Protect Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. For quantitative real-time PCR 2 µg of RNA was reverse transcribed using Superscript II (Invitrogen) according to the manufacturer's instructions with random hexamer as primer. mRNA levels were quantified using the iCycler thermocycler (Bio-Rad, Hercules, CA, USA). Each PCR reaction was conducted in a final volume of 50 µl containing: 125 µM nucleotide triphosphates (NTPs), Hot-start Taq polymerase, reaction buffer and SYBR Green I dye (Molecular Probes). The reactions were supplemented with 3 mM Mg2+ and each gene-specific primer (sense and antisense). Primer-pairs for the human β-actin housekeeping gene were purchased from Clontech (Palo Alto, CA, USA) and gene-specific primers for TLR4 (GeneBank no. NM_003266) (TLR4; forward: 5′-AAG CCG AAA GGT GAT TGT TG-3′; reverse: 5′-CTG AGC AGG GTC TTC TCC AC-3′). Forty-five cycles of PCR reaction (5 s denaturation at 95°C; 10 s annealing at 60°C; and extension at 72°C) was run. To ensure that equal amounts of total RNA were loaded in each cDNA synthesis reaction, the TLR-primer-generated fluorescence data were normalized to the fluorescence values generated by human β-actin primers from the same cDNA sample.

TLR4 immunohistochemistry

For TLR4 immunocytochemistry, HMEC were grown on coverslips coated with 0·1% gelatine. Cells were fixed in 4% paraformaldehyde and blocked in PBS/1% bovine serum albumin (BSA), followed by incubation with 2 µg/ml of a monoclonal antibody against TLR4 (BD Pharmingen, cat. no. 551964) or irrelevant isotype-matched antibody followed by incubation with anti-mouse HRP antibody (the primary antibody is a mouse-anti-human; the secondary antibody should be an anti-mouse antibody). TLR staining was visualized using DAB substrate (Dako) and counterstained with haematoxylin.

Statistics

In transfection and ELISA experiments, data shown are the mean ± s.d. of three or more experiments set up in triplicate. In transfection experiments, the data are reported as a percentage of LPS-induced NF-κB activation and cytokine release. Student's t-test was used to assess statistical significance between LPS treated cells pretreated with media or vitamin D.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Treatment of HMEC with 1α,25-(OH)2 D3 inhibits LPS-induced IL-6, IL-8 and RANTES release and interferon (IFN)-β promoter luciferase activation

1α-hydroxylase and vitamin D receptor are known to be expressed on venular and capillary endothelial cells of human skin biopsies [23,24]. Despite this, the data on the effect of 1α,25-(OH)2 D3 on endothelial cells is limited to 1α,25-(OH)2 D3 inhibition of endothelial cell proliferation [23–25]. Here we examined the effect of 1α,25-(OH)2 D3 on TLR-mediated innate immune activation of endothelial cells.

We pretreated HMEC with various concentrations of 1α,25-(OH)2 D3 for 48 h, stimulated them with LPS for 6 h and measured the supernatant IL-6 and IL-8 release by ELISA assay. We observed that 1α,25-(OH)2 D3 treatment alone did not induce cytokine release in HMEC (data not shown) and 1α,25-(OH)2 D3 pretreatment inhibited the LPS-induced IL-6 and IL-8 release in a dose-dependent manner (Fig. 1a, b). We then examined the effect of 1α,25-(OH)2 D3 pretreatment of HMEC on LPS-induced RANTES, CC-chemokine, release and observed that 1α,25-(OH)2 D3 blocks the LPS-induced RANTES release (Fig. 1c).

image

Figure 1. Vitamin D pretreatment inhibits lipopolysaccharide (LPS)-induced interleukin (IL)-6, IL-8 and regulated upon activation normal T cell exposed and secreted (RANTES) release in human microvessel endothelial cells (HMEC). We assessed the effect of vitamin D on LPS-induced cytokine release by measuring the supernatant IL-6, IL-8 and RANTES levels in cells treated with LPS (25 ng/ml) with or without vitamin D. We observed that pretreatment of HMEC with vitamin D inhibited the LPS-induced IL-6 (a), IL-8 (b) and RANTES (c) release in a dose-dependent manner. Treatment with vitamin D (200 nM) did not induce cytokine release in HMEC. In parallel, we plated HMEC in media with or without vitamin D; after 24 h, we transfected the cells with interferon-β promoter luciferase (0·2 µg) and β-galactosidase constructs overnight, as described under Methods. The cells were then treated with media or LPS for 5 h. Luciferase activity was measured to determine LPS-induced IFN-β promoter activation. The results were corrected for transfection efficiency, using galactosidase assay. The data was presented as fold increase above media-treated control (d). Each experiment was performed in triplicate. The data shown is the mean ± s.d. of three independent experiments (*P < 0·05 compared to LPS alone).

Download figure to PowerPoint

LPS stimulation of TLR4 can induce type 1 IFN-β release through a MyD88-independent signalling cascade [26,27]. Interferon regulatory factor (IRF)-3 mediates MyD88-independent TLR4 responses [28]. Next, we examined the effect of 1α,25-(OH)2 D3 pretreatment of HMEC on LPS-induced MyD88-independent signalling to activate IFN-β promoter, and observed that in HMEC transfected with β-galactosidase and IFN-β promoter luciferase cDNA pretreatment with 1α,25-(OH)2 D3 did not inhibit luciferase activity (Fig. 1d).

Because 1α,25-(OH)2 D3 binds to vitamin D binding protein in the plasma, and free 1α,25-(OH)2 D3 is the active component, we repeated the experiments by preincubating HMEC and 1α,25-(OH)2 D3 in media with different concentrations of FBS (5 and 10%). We observed a stronger inhibition of LPS-induced cytokine release in HMEC pretreated with 1α,25-(OH)2 D3 in media with 5% FBS compared to 10% FBS (data not shown). For the following experiments we used media with 5% FBS.

1α,25-(OH)2 D3 inhibition of cytokine release is not due to increased cell death or inhibition of cell proliferation

We assessed whether 1α,25-(OH)2 D3 inhibition of LPS-induced cytokine release was due to increased cell death by measuring lactate dehydrogenase release in the supernatant, and observed that 1α,25-(OH)2 D3 pretreatment did not increase cell death above control levels (data not shown). Exogenous 1α,25-(OH)2 D3 and its precursor, 25-hydroxyvitamin D3, are known to inhibit human umbilical vein endothelial cell proliferation [23]. However, we did not observe inhibition of endothelial cell proliferation in HMEC pretreated with 10 nM of 1α,25-(OH)2 D3 for various durations of time (Fig. 2).

image

Figure 2. Vitamin D did not inhibit human microvessel endothelial cells (HMEC) proliferation at the doses used. We hypothesized that vitamin D treatment HMEC proliferation may be inhibited in wells treated with vitamin D and this may lead to decreased lipopolysaccharide (LPS)-induced interleukin (IL)-6, IL-8 and regulated upon activation normal T cell exposed and secreted (RANTES) release. In order to test this hypothesis we assessed cell proliferation by using a Promega cell proliferation assay and observed that treatment with vitamin D for various durations did not block cell proliferation. Data are presented as the absorbance at 490 nM  ± s.d. of three independent experiments.

Download figure to PowerPoint

1α,25-(OH)2 D3 treatment of HMEC blocked the LPS-induced NF-κB activation

Vitamin D receptor ligands have been shown to down-regulate IL-12 release and NF-κB activation in macrophages and dendritic cells [29]. 1α,25-(OH)2 D3 treatment of peripheral human mononuclear cells blocks phytohaemaglutinin (PHA)-induced NF-κB activation [30]. Next, we hypothesized that 1α,25-(OH)2 D3 treatment of HMEC will block LPS-induced NF-κB activation.

In order to test this hypothesis, HMEC were pretreated with 1α,25-(OH)2 D3 for 24 h, transfected with ELAM-NF-κB luciferase and β-galactosidase constructs overnight, as described in Methods. Cells were stimulated with LPS for 5 h and luciferase activity was measured to assess NF-κB activation. Colorimetric β-galactosidase assay was performed to correct for transfection efficiency. We observed that pretreatment of cells with 1α,25-(OH)2 D3 inhibited the LPS-induced NF-κB activation in a dose-dependent manner (Fig. 3). These data suggest that 1α,25-(OH)2 D3 inhibition of LPS-induced IL-6, IL-8 and RANTES release is secondary to its inhibitory effect on NF-κB activation.

image

Figure 3. Vitamin D pretreatment blocks the lipopolysaccharide (LPS)-induced NF-κB activation. In order to assess the effect of vitamin D treatment on LPS-induced NF-κB activation we transfected human microvessel endothelial cells (HMEC) with NF-κB-luciferase construct and measured luciferase activity upon LPS-stimulation (50 ng/ml) by using a luminometer, as described in the Methods. Calorimetric galactosidase assay was performed to correct for transfection efficiency. LPS-induced NF-κB activation was considered as 100%. Each experiment was set up in triplicate, and data are presented as percentage of LPS-induced luciferase activity ± s.d. of three independent experiments (*P < 0·05 compared to LPS alone).

Download figure to PowerPoint

1α,25-(OH)2 D3 treatment does not modulate HMEC-TLR4 expression

1α,25-(OH)2 D3 treatment is known to modulate cell surface molecule expression in immune system cells. Both in vivo and in vitro studies have shown that 1α,25-(OH)2 D3 treatment of bone marrow-derived dendritic cells and monocyte-derived dendritic cells down-regulate the expression of co-stimulatory molecules such as CD40, CD80 and CD86 [10]. In human HL-60 promonocytic leukaemia, cell line treatment with 1α,25-(OH)2 D3 increased CD14 mRNA expression [31].

We have shown previously that TLR4 mediates LPS signalling in HMEC [32]. We hypothesized that the effect of 1α,25-(OH)2 D3 in HMEC to inhibit LPS-induced NF-κB activation was secondary to its effect on TLR4 expression. In order to test this hypothesis we examined TLR4 expression in HMEC treated with media control versus 1α,25-(OH)2 D3 by using quantitative real-time PCR and immunohistochemistry methods and observed that 1α,25-(OH)2 D3 did not modulate TLR4 mRNA or protein expression, respectively in HMEC (data not shown).

In order to respond efficiently to LPS, TLR4 requires an accessory protein, MD-2, which is a 30-kDa glycoprotein that binds to the extracellular domain of TLR4 [33]. MD2–/– mice do not respond to LPS and do survive endotoxic shock [34]. An MD-2 mutation generated by replacing Cys95 with Tyr95 abolishes LPS responses [35], and a coding mutation that has been identified recently within the first exon of the human MD-2 gene, resulting in an amino-acid exchange from Thr35 to Ala results in decreased LPS signalling in peripheral blood mononuclear cells [36]. We examined whether 48 h 1α,25-(OH)2 D3 treatment of HMEC modulates the MD-2 mRNA expression, and observed that that there was no change in the expression of MD-2 short or long isoform mRNAs (data not shown). Although our data suggest that 1α,25-(OH)2 D3 does not modulate surface TLR4 expression, it may still modulate total cellular TLR4 and MD2 concentrations.

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Until the discovery of expression of vitamin D receptor (VDR) on immune system cells in the early 1970s, 1α,25-(OH)2 D3 was thought to play a role only in calcium, phosphorus and bone metabolism. Noting vitamin D3 receptor (VDR) in promyelocytes, Abe and Tanaka et al. first demonstrated that 1α,25-(OH)2 D3 can suppress proliferation of promyelocytes and cause their differentiation [37,38]. It is now well known that 1α,25-(OH)2 D3 has immunomodulatory effects on antigen-presenting cells and lymphocytes [1].

Synthesis of the active form of vitamin D, 1α,25-(OH)2 D3 from its precursor, 25-(OH) D3, is a pivotal feature of calcium homeostasis. This endocrine process is catalysed by the enzyme, 25(OH) D3−1 α-hydroxylase (1 α-OHase), the expression and regulation of which is best described in the kidney [39]. However, enzyme activity studies using a variety of tissues have suggested that synthesis of 1,25-(OH)2 D3 occurs at several key peripheral sites, including the immune system and skin [40,41]. Recently, 1α-OHase expression was detected in endothelial cells from human renal arteries and primary cultures of human umbilical vein endothelial cells (HUVEC) [23]; the enzyme activity in HUVEC increased after treatment with TNF-α and LPS [23]. In HMEC calcitriol treatment inhibited the TNF-α and IL-1β-induced IL-6 production [42]. Here we show for the first time that 1α,25-(OH)2 D3 modulates the innate immune responses of human microvessel endothelial cells and inhibits TLR4 agonist LPS-induced MyD88-dependent NF-κB activation and proinflammatory cytokine (IL-6 and IL-8) and CC-chemokine RANTES release.

Previously published data and our own data suggest an autocrine/paracrine immune modulatory role for 1α,25-(OH)2 D3 in endothelial cells, where LPS stimulation leads to the release of proinflammatory cytokines and induces 1α-OHase activity and consequently 1α,25-(OH)2 D3 expression, which then inhibits NF-κB activation and IL-6, IL-8 and RANTES expression in an autocrine/paracrine fashion to ‘turn off’ the immune activation. This may potentially suggest the presence of dysfunctional 1α,25-(OH)2 D3 signalling in a group of patients who exhibit uncontrolled immune activation to microbial antigens and develop post-infectious immune-mediated complications. Interestingly, vitamin D receptor polymorphism was found to be more common in Japanese patients with multiple sclerosis, which is a debilitating autoimmune disease of the central nervous system (CNS) that develops in genetically susceptible individuals who are exposed to undefined environmental risk factors [43], and 1α,25-(OH)2 D3 prevents the in vivo induction of murine experimental autoimmune encephalomyelitis [44].

RANTES expression is increased following cellular activation of fibroblasts, T cells, monocytes and endothelial and epithelial cells [45]. RANTES attracts monocytes, eosinophils, basophils, natural killer cells and T cells, including memory T cells, during inflammation and immune response, arguing for a role of this chemokine in virus-related or unrelated diseases [46]. The promoter of RANTES contains transcription factor binding elements for both NF-κB and IRF3 [47], and LPS can activate RANTES promoter through both MyD88-dependent and -independent signalling pathways. Here we show that in addition to proinflammatory cytokines, IL-6 and IL-8, 1α,25-(OH)2 D3 inhibits LPS-induced RANTES release, and this is mediated through 1α,25-(OH)2 D3 inhibition of NF-κB activation. Psoriasis vulgaris is a chronic cutaneous inflammatory disease. The expression of RANTES was shown to be increased in psoriatic lesions and suggest the involvement of this chemokine in the outcome of cutaneous inflammatory diseases. Indeed, Tacalcitol [1α,24(R)-dihydroxyvitamin D3], an active vitamin D3 analogue, has been shown to inhibit RANTES and IL-8 production in cultured normal epidermal keratinocytes [48].

In HMEC, 1α,25-(OH)2 D3 inhibition of LPS-induced NF-κB activation and cytokine/chemokine release was not due to decreased TLR4 or MD2 expression. The vitamin D receptor (VDR) is a transcription factor that transmits incoming 1α,25-(OH)2 D3 signalling via combined contact with co-activator proteins and specific DNA binding sites, vitamin D response elements (VDREs) [49,50]. VDREs result ultimately in activation or repression of transcription (reviewed in [41]). There are five known mammalian Rel/NF-κB proteins, rel (c-Rel), p65 (RelA), RelB, p50 and p52, that function as dimers held inactive in the cytoplasm by inhibitor proteins IκB [51]. Recently VDREs were identified within promoter regions of human and mouse relB genes, and treatment of dendritic cells (DC) with 1α,25-(OH)2 D3 inhibited RelB expression [52]. In normal human lymphocytes 1α,25-(OH)2 D3 treatment inhibited PHA-induced p50 and p105 expression [53]. In endothelial cells, 1α,25-(OH)2 D3 inhibition of LPS-induced NF-κB activation may be secondary to 1α,25-(OH)2 D3 suppression of Rel/NF-κB expression.

The immune regulatory properties of 1α,25-(OH)2 D3 and synthetic VDR ligands hold great potential for the treatment of autoimmune diseases, cancers and transplant rejection. The results of our experiments suggest that 1α,25-(OH)2 D3 and its analogues may exert immune regulatory functions on endothelial cells as well. Because microbial antigen-induced endothelial cell activation plays a key role in sepsis [54,55] and atherosclerosis (reviewed in [56]), our findings suggest a potential use for 1α,25-(OH)2 D3 and its analogues for the treatment of sepsis and/or atherosclerosis.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

No commercial interest or other conflict of interest exists between the authors and the manufacturers of the products used in this study. The study on which this paper is based was conducted while Dr Alan Shapiro was a fellow in the Division of Pediatric Infectious Diseases at Mattel Children's Hospital at UCLA and before he joined the staff of the Food and Drug Administration. The views expressed are those of the authors. No official support or endorsement by the Food and Drug Administration is provided or should be inferred. This work was supported by UCLA NIH Child Health Research Center Award P30HD34610 and NIH KO8 Award AI51216 (to O.E.)

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References