RAPID COMMUNICATION
TNF-alpha increases tracheal epithelial asbestos and fiberglass binding via a NF-kappa B-dependent mechanism

C. Xie, A. Reusse, J. Dai, K. Zay, J. Harnett, and A. Churg

Department of Pathology, University of British Columbia, Vancouver, British Columbia, Canada V6T 2B5


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Tumor necrosis factor (TNF)-alpha is released from alveolar macrophages after phagocytosis of mineral fibers. To determine whether TNF-alpha affects the binding of fibers to epithelial cells, we exposed rat tracheal explants to TNF-alpha or to culture medium alone, followed by a suspension of amosite asbestos or fiberglass (MMVF10). Loosely adherent fibers were removed from the surface with a standardized washing technique, and the number of bound fibers was determined by scanning electron microscopy. Increasing doses of TNF-alpha produced increases in fiber binding. This effect was abolished by an anti-TNF-alpha antibody, the proteasome inhibitor MG-132, and the nuclear factor (NF)-kappa B inhibitor pyrrolidine dithiocarbamate. Gel shift and Western blot analyses confirmed that TNF-alpha activated NF-kappa B and depleted Ikappa B in this system and that these effects were prevented by MG-132 and pyrrolidine dithiocarbamate. These observations indicate that TNF-alpha increases epithelial fiber binding by a NF-kappa B-dependent mechanism. They also suggest that mineral particles may cause pathological lesions via an autocrine-like process in which the response evoked by particles, for example, macrophage TNF-alpha production, acts to enhance subsequent interactions of particles with tissue.

tumor necrosis factor-alpha ; particle adhesion; nuclear factor-kappa B; MG-132; pyrrolidine dithiocarbamate


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ADHESION (binding) of mineral particles to the epithelial cell surface is the first step in a variety of mineral dust-induced pathological reactions. Some of these reactions are probably mediated directly on the cell surface. For example, Zanella et al. (27) reported that surface interactions of crocidolite asbestos with the epidermal growth factor receptor on rat pleural mesothelial cells induced autophosphorylation of the receptor, with subsequent upregulation of c-fos expression and apoptosis. Tsuda et al. (25) found that the adhesion of glass or crocidolite fibers to the surface of A549 cells (a model of type II cells) and the subsequent stretching of the cells caused them to elaborate enhanced amounts of the neutrophil chemoattractant interleukin (IL)-8.

Binding of fibers to the cell surface is also believed to be a prerequisite for fiber uptake, and fiber uptake leads to a wide variety of pathological processes including intracellular oxidative damage to lipids, proteins, and DNA; nuclear factor (NF)-kappa B activation, with subsequent induction of proinflammatory cytokines; and, depending on the nature of the particle, induction of fibrogenic mediators either within the epithelial cell or after passage of the particles through the cells to the underlying interstitial tissues (4, 7, 9, 10, 15).

Although the adhesion and uptake of mineral particles by pulmonary epithelial cells are processes seen with any particle that contacts the cell surface (7), the factors that govern adhesion are poorly defined. For positively charged particles such as chrysotile asbestos, carbonyl iron spheres, or aluminum spheres, binding to negatively charged sialic acid residues plays a role because binding can be abolished by pretreatment with neuraminidase (3, 13). There is some evidence to suggest that coating of the negatively charged asbestos fibers amosite and crocidolite with fibronectin or vitronectin and subsequent adhesion to cell surface integrins is important in increasing binding, although blocking the integrins does not completely abolish binding (2, 5, 6, 25). Heparin and polyinosinic acid but not polyanionic chondroitin sulfate have been found to decrease the binding of a variety of compact particles to A549 cells (23), suggesting that a macrophage scavenger-type receptor might be involved, and Palecanda et al. (21) have recently reported that TiO2, Fe2O3, and latex beads bind to hamster alveolar macrophages via a receptor that is analogous to the human macrophage scavenger receptor MARCO. Whether this receptor system functions on lung epithelial cells has not yet been determined. Churg et al. (8) observed that cigarette smoke, a source of reactive oxygen species (ROS), increased amosite asbestos binding to tracheal epithelial cells; this effect could be completely abolished by treatment of the fibers with deferoxamine and decreased with mannitol, suggesting that surface iron on the fibers played an important role, probably by generating the hydroxyl radical from ROS in the smoke.

Inhaled mineral particles also evoke a complex set of reactions in the alveolar spaces and airway lumens. All particles induce an alveolar macrophage influx, and phagocytosis of inhaled particles by macrophages is often postulated as an initial and central event in particle-associated pathology (19). One of the results of phagocytosis of most types of mineral particles by macrophages is the generation of tumor necrosis factor (TNF)-alpha , a proinflammatory cytokine that turns on cell signaling pathways and induces synthesis of other cytokines as well as of surface adhesion molecules (11, 19, 26). Production of TNF-alpha is also seen as a later event in epithelial cells in whole animal models of asbestos inhalation (18). In this study, we used amosite asbestos and a fibrous glass, MMVF10, as model particles to ask whether the TNF-alpha response can potentiate the reactions of fibers with epithelial cells by increasing surface binding.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Explant Preparation

Tracheal explants were prepared from 250-g Sprague-Dawley rats as previously described (8) and divided into treatment groups of 4 explants each. The following treatment groups were created.

Dust only. The explants were submerged in Dulbecco's minimal Eagle's medium (DMEM) without serum for 3 h (to keep total submersion time for all groups constant) at 37°C, placed on agarose-DMEM plates in an air-CO2 incubator at 37°C for 2 h (8), and then placed in a suspension of 500 µg/cm2 of International Union Against Cancer (UICC) amosite asbestos (kindly supplied by Dr. J. C. Wagner, Medical Research Council Pneumoconiosis Unit, Cardiff, UK) or a glass fiber, MMVF10 (Thermal Insulation Manufacturer's Association, Stanford, CT), for 1 h. The explants were subsequently washed to remove loosely adherent fibers and prepared for scanning electron microscopy (SEM) as described in Determination of Fiber Binding.

TNF-alpha plus dust and antibodies to TNF-alpha plus dust. The explants were submerged in DMEM alone for 1 h and then in DMEM containing variable amounts of recombinant human TNF-alpha (specific activity >2 × 107 U/mg; Life Technologies, Rockville, MD) for 2 h, followed by air-CO2 culture for 2 h, dust exposure as in Dust only for 1 h, and washing and preparation for SEM. To show specificity, additional experiments were performed in which TNF-alpha at 20 ng/ml was first mixed with excess goat polyclonal antibody to human TNF-alpha (Santa Cruz Biotechnology) for 1 h, and the explants were then carried forward as above. As a control, nonimmune serum was used instead of anti-TNF-alpha .

Proteasome inhibitor MG-132 plus TNF-alpha plus dust. The explants were submerged in 0.5 µM MG-132 (Peptide Institute, Osaka, Japan) in 0.1% DMSO-DMEM for 1 h, followed by TNF-alpha (20 ng/ml) for 2 h, air-CO2 culture for 2 h, and finally dust exposure for 1 h. To test the effects of MG-132 on baseline fiber adhesion, additional explants were exposed to MG-132 and dust but not to TNF-alpha .

NF-kappa B inhibitor pyrrolidine dithiocarbamate. The explants were submerged in 200 µM pyrrolidine dithiocarbamate (PDTC; Sigma) in culture medium for 2 h, and then the same protocol as in Proteasome inhibitor MG-132 plus TNF-alpha plus dust was followed but with the addition of PDTC to the medium.

Non-dust-exposed explants for Ikappa B Western blot and NF-kappa B gel shift assay. Three additional treatment groups of explants were created; initial studies showed that six explants per group were required to obtain reliable signals. One group was exposed to DMEM alone, another to DMEM followed by 20 ng/ml of TNF-alpha , and a third to 0.5 µM MG-132 for 1 h and then to 20 ng/ml of TNF-alpha . The explants were snap-frozen and assayed as described in Western Blots for Ikappa B. The same protocol was followed, with additional explants exposed to 200 µM PDTC.

Determination of Fiber Binding

Fiber binding was determined as previously described (8). Exposure of the explants to the dusts resulted in initial coating of the epithelial surface with a layer of fibers, most of which were very loosely adherent. To remove fibers not bound to the surface, each explant was very slowly dipped once in four different containers of fresh culture medium. Churg et al. (8) have previously shown that this approach removes the vast majority of fibers and leads to quite reproducible levels of residual fibers bound to the epithelial surface; we also found that four washes were adequate, with little change in the number of adherent fibers if more than four washes were used. The explants were then dried under vacuum and examined by SEM. SEM photographs of randomly selected fields were taken at ×1,000 and printed. The proportion of the surface occupied by fibers (areal fraction of fibers) was determined with a 42-point transparent overlay by counting the points that fell on fibers versus the points that fell on tissue (8). Differences in the number of adherent fibers among treatment groups were determined by analysis of variance.

Western Blots for Ikappa B

Frozen tracheal explants were homogenized in a solution of 0.1% Triton X-100, 150 mM NaCl, 10 mM HEPES, pH 7.5, 1 mM EDTA, 0.5 mM 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), 1 µg/ml of leupeptin, 1 µg/ml of aprotinin, 10 µg/ml of soybean trypsin inhibitor, and 1 µg/ml of pepstatin A. The homogenate was incubated on ice for 5 min and then centrifuged at 5,000 rpm for 5 min. The nuclear pellet was collected for gel shift assay (see NF-kappa B Gel Shift Assay). A protein assay was carried out on the supernatant, and equal amounts of protein were used for each sample. The samples were separated on a 12% polyacrylamide gel and transferred to nitrocellulose membranes. The membranes were incubated in Tris-buffered saline (TBS) containing 0.05% Tween 20 and 5% skim milk powder for 18 h at 4°C. The membranes were then incubated in a 1:400 dilution of rabbit polyclonal anti-Ikappa B-alpha or Ikappa B-beta (Santa Cruz Biotechnology) in TBS with 0.05% Tween 20 and 5% skim milk powder. The membranes were washed three times in TBS with 0.05% Tween 20 and incubated in a 1:1,000 dilution of horseradish peroxidase-conjugated goat anti-rabbit IgG (ICN Biochemicals). Detection was by chemiluminescence, and densitometry was performed on the films.

NF-kappa B Gel Shift Assay

The pelleted nuclei were resuspended in 100-500 µl of 25% glycerol, 20 mM HEPES, 420 mM NaCl, 1.2 mM MgCl2, 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.5 mM AEBSF, 1 µg/ml of leupeptin, 1 µg/ml of aprotinin, 10 µg/ml of soybean trypsin inhibitor, and 1 µg/ml of pepstatin A and left on ice for a 30-min high-salt extraction of the nuclear proteins. The lysed nuclei were centrifuged at 2,000 rpm for 15 s, and a protein assay was carried out on the supernatant (1). A single-stranded NF-kappa B consensus oligonucleotide (5'-AGT TGA GGG GAC TTT CCC AGG C-3') was random labeled with [32P]CTP. Binding reactions containing equal amounts of protein (7 µg) and 6.7 pmol of oligonucleotide were performed for 20 min in binding buffer [10 mM Tris · HCl, 50 mM NaCl, 1 mM EDTA, 4% glycerol, and 67 µg/ml of poly(dI-dC)]. Reaction products were separated on a 5% polyacrylamide gel in 0.25× Tris-borate-EDTA buffer and analyzed by autoradiography and densitometry.

Cytotoxicity Assay

Groups of five explants were treated with culture medium only (control), amosite asbestos, TNF-alpha followed by asbestos, or MG-132 or PDTC followed by TNF-alpha followed by asbestos as described in Explant Preparation, and the supernatant from the last submersion was collected, concentrated, and assayed for lactate dehydrogenase with a commercial kit (Sigma). This assay produced values of 42, 33, 38, 33, and 35 B-B units/ml. These findings indicate that the procedures do not produce significant cytotoxicity.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Figure 1 shows the effects of increasing concentrations of TNF-alpha on amosite asbestos binding. Fiber adhesion significantly and progressively increased above the control levels with doses of 5 and 10 ng/ml of TNF-alpha ; at doses of 20 ng/ml, no further increase in binding was seen, perhaps suggesting that the possible expression or exposure of the actual binding agent (see DISCUSSION) can only be increased to a limited amount. Figure 2 shows the effects of pretreating TNF-alpha with polyclonal anti-TNF-alpha antibodies. Antibodies to TNF-alpha abolished the increase in fiber concentration. Nonimmune serum did not prevent the TNF-alpha effect (data not shown). These observations support the idea that increased adhesion is specifically driven by TNF-alpha . Figure 3 shows that MG-132 abolishes the TNF-alpha -mediated increases in amosite binding, implying that NF-kappa B activation is playing a role. Figure 4 shows the dose data and similar effects of MG-132 on MMVF10 binding. TNF-alpha increased the binding of this synthetic mineral fiber to the explants, although the dose response was somewhat different from that for amosite because greater TNF-alpha doses were needed to show an effect. MG-132 again prevented the TNF-alpha effect. Figure 5 shows that the NF-kappa B inhibitor PDTC also prevented increases in fiber binding after TNF-alpha exposure, again suggesting that this process is driven through NF-kappa B activation. To prove that TNF-alpha does activate NF-kappa B in this explant system and that the inhibitors prevented activation, gel shift assays for NF-kappa B and Western blots for Ikappa B levels were run after exposure of the explants to TNF-alpha or TNF-alpha plus MG-132 (Figs. 6 and 7) or TNF-alpha or TNF-alpha plus PDTC (Figs. 8 and 9). In both instances, the inhibitors prevented the increase in TNF-alpha -induced NF-kappa B nuclear translocation, as shown in the gel shifts, and ameliorated Ikappa B degradation, confirming that inhibition of fiber binding correlated with inhibition of NF-kappa B activation.


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Fig. 1.   Tumor necrosis factor (TNF)-alpha increases amosite asbestos adhesion. Ctrl, amosite without TNF-alpha (control). Values are means ± SD. Note the sensitivity to very low doses of TNF-alpha and the rapid achievement of a plateau above TNF-alpha concentrations of 10 ng/ml. * Significantly greater than Ctrl. Ten-nanogram value was also significantly greater than 5-ng value, but higher concentrations were not significantly greater than 10-ng value.



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Fig. 2.   Mixing TNF-alpha (20 ng/ml) with an excess of anti-TNF-alpha antibody and then exposing explants to this mixture totally prevented the TNF-alpha -driven increase in fiber adhesion. Values are means ± SD. * Significantly greater than Ctrl.



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Fig. 3.   The proteasome inhibitor MG-132 prevented the TNF-alpha -driven increase in amosite fiber adhesion. Ctrl, amosite without TNF-alpha ; Ctrl+MG-132, amosite plus MG-132 in the absence of TNF-alpha . Values are means ± SD. MG-132 did not affect binding in the absence of TNF-alpha . * Significantly greater than Ctrl.



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Fig. 4.   Effects of TNF-alpha and MG-132 on adhesion of MMVF10 fibers to epithelial cells. Ctrl, MMVF10 without TNF-alpha ; Ctrl+MG-132, MMVF10 plus MG132 in the absence of TNF-alpha . Values are means ± SD. Unlike amosite asbestos, MMVF10 binding was not increased at very low (5 ng/ml) doses of TNF-alpha . MG-132 was again protective (tested against 20 ng/ml of TNF-alpha ). * Significantly greater than Ctrl.



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Fig. 5.   The nuclear factor (NF)-kappa B inhibitor pyrrolidine dithiocarbamate (PDTC) prevented the TNF-alpha (20 ng/ml)-driven increase in amosite fiber adhesion. Ctrl, amosite without TNF-alpha . Values are means ± SD. * Significantly greater than Ctrl.



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Fig. 6.   Gel shift assay and corresponding densitometry showing activation of NF-kappa B by TNF-alpha and prevention of this effect by MG-132. Lane 1, Ctrl; lane 2, 20 ng/ml of TNF-alpha ; lane 3, TNF-alpha preceded by MG-132. Each data point is derived from 6 combined tracheal explants.



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Fig. 7.   Western blot and corresponding densitometry of Ikappa B-alpha (A) and Ikappa B-beta (B). Lanes 1, Ctrl; lanes 2, 20 ng/ml of TNF-alpha ; lanes 3, TNF-alpha preceded by MG-132. Nos. on left, molecular weight. Each data point is derived from 6 combined tracheal segments. TNF-alpha decreased both Ikappa B-alpha and Ikappa B-beta levels; this effect was ameliorated by MG-132.



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Fig. 8.   Gel shift assay and corresponding densitometry showing activation of NF-kappa B by TNF-alpha and prevention of this effect by PDTC. Lane 1, Ctrl; lane 2, 20 ng/ml of TNF-alpha ; lane 3, TNF-alpha preceded by PDTC. Each data point is derived from 6 combined tracheal explants.



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Fig. 9.   Western blot and corresponding densitometry of Ikappa B-alpha (A) and Ikappa B-beta (B). Lanes 1, Ctrl; lanes 2, 20 ng/ml of TNF-alpha ; lanes 3, TNF-alpha preceded by PDTC. Nos. on left, molecular weight. Each data point was derived from 6 combined tracheal segments. TNF-alpha decreased both Ikappa B-alpha and Ikappa B-beta levels; this effect was ameliorated by PDTC.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we looked at the effects of exogenous TNF-alpha on fiber binding to tracheal epithelial cells. A word about our technical approach is in order. We are defining a "bound" particle as one that is resistant to removal by a simple washing technique. Review of the literature shows that there is no generally utilized method for measuring fiber binding. A wide variety of approaches including examination of cultured cells by confocal scanning microscopy to attempt to separate surface from internalized particles (2); a fairly simple SEM technique similar to ours (3, 13); fractionation of cells with radiolabeled membranes and subsequent measurement of radioactivity adherent to centrifuged fibers (5, 6); and measurement of changes in light scattering by flow cytometry (23) have all been reported. Not only are these methods quite different and the results difficult to compare, but with any of these methods, harsh enough treatment will remove all fibers from the cells. Thus the definition of a bound fiber is in many respects arbitrary. Nonetheless, the method of Churg et al. (8) is simple and reproducible and shows consistent effects with various treatments, suggesting that it provides useful data on binding. An additional advantage of using tracheal explants for evaluating binding is that fiber uptake is very slow, with very few fibers seen in the epithelial cells even at 24 h (9); thus we do not need to be concerned that the surface assay has missed bound fibers that have already entered the cells.

Our present data indicate that TNF-alpha increases fiber binding to the surface of tracheal epithelial cells and that this process appears to be mediated by TNF-alpha -induced activation of NF-kappa B. NF-kappa B consists of a cytoplasmic complex of two proteins (typically p50 and p65) and an inhibitory protein, Ikappa B-alpha or Ikappa B-beta . TNF-alpha and other inflammatory or oxidant mediators can activate NF-kappa B by causing ubiquitination and subsequent degradation of Ikappa B, allowing the p65/p50 complex to translocate into the nucleus and bind to recognition sequences in the promoters of a wide variety of genes important in acute inflammatory responses (1, 16). MG-132 is a proteasome inhibitor that prevents degradation of Ikappa B and thus inhibits the TNF-alpha -driven pathway of NF-kappa B activation (16). Our gel shift and Western blot data confirm that TNF-alpha does cause Ikappa B degradation and NF-kappa B activation and that MG-132 prevents NF-kappa B activation and Ikappa B degradation in our explant system. The fact that a second NF-kappa B inhibitor, PDTC, also prevents the effects of TNF-alpha further adds to the idea that increased adhesion is driven through a NF-kappa B-dependent mechanism. It is, of course, possible that increases in adhesion and NF-kappa B activation are two TNF-alpha -driven but unrelated effects, but this seems to be a much less likely explanation for our observations.

What is not as yet clear is the mechanism by which TNF-alpha or NF-kappa B increases surface binding. TNF-alpha increases the production of a variety of substances that are expressed on the epithelial cell surface or exported into the lumen. In bronchial epithelial cells, TNF-alpha , via NF-kappa B, is known to induce the adhesion molecule intercellular adhesion molecule-1 in bronchial epithelial cells (22), and TNF-alpha also induces mucus secretion (12) in these cells. Other candidate binding substances include fibronectin, which is produced in increased amounts in A549 cells after coal dust plus TNF-alpha exposure (17), and MARCO-type scavenger receptors, which probably exist on pulmonary epithelial cells (21, 23), although this has yet to be confirmed. This list is not comprehensive and other possible candidate molecules, particularly cell surface integrins such as alpha vbeta 5 (2) or even the TNF-alpha -responsive beta 1- and beta 2-integrins that bind neutrophils and eosinophils to bronchial epithelial cells (14), certainly exist. This list is, of course, speculative, but it is of interest that amosite asbestos and MMVF10 show a different dose response to TNF-alpha , and it is possible that the binding agents differ for these two fibers.

Our observations also suggest the novel possibility that the interaction of dusts and macrophages or epithelial cells is not the one-way street that is typically assumed in most hypothetical schemes of events after dust inhalation; i.e., what is usually proposed is that dusts interact with both macrophages and tissues to elicit a variety of cytokine and oxidant mediators that are then assumed to be sufficient in and of themselves to establish subsequent biochemical and molecular responses and eventual pathological changes such as interstitial fibrosis, with the dust particle that evoked the response becoming (conceptually) almost irrelevant to the process. (19).

The notion that particle-evoked responses can influence subsequent interactions of particles and pulmonary epithelial cells has been little explored. What we observe here suggests that a dust-evoked cytokine, TNF-alpha , acts to increase dust binding to epithelial cells and thus presumably to increase both surface interactions, as described in the introduction, and particle uptake, with, eventually, increased activation of cell signaling pathways and increased mediator production. With asbestos and glass fibers, one of those increased intracellular mediators is TNF-alpha (18, 20, 26); thus not only does TNF-alpha affect downstream interactions of particles with the epithelia, but by doing so, it potentially upregulates epithelial TNF-alpha production in a type of autocrine-like feedback loop.

It is interesting in this context that Stringer and Kobzik (24) showed that TNF-alpha primes A549 cells for IL-8 release after subsequent particle contact. Stringer and Kobzik intended their experiments to mimic the effects of air pollutant particles in individuals with preexisting inflammatory disease. However, the Stringer and Kobzik data could be viewed in much the same way as ours, in that TNF-alpha increases release of IL-8 after particle adhesion; IL-8 attracts neutrophils that release ROS, and, as Churg (7) has discussed in detail elsewhere, ROS cause respiratory epithelia to increase the uptake of most mineral particles and thus again to increase mediator (including TNF-alpha ) production.

This study has only looked at the effects of TNF-alpha on fiber binding, but the same phenomena might occur with compact particles including air pollutant particles. We propose that mineral particles may cause pathological lesions not only by an initial reaction with macrophages and epithelial cells that results in mediator production but also via a feedback loop in which the dust-evoked mediator increases subsequent interactions of the dust with epithelial cells and thus magnifies the response.


    ACKNOWLEDGEMENTS

This study was supported by Grant MA8051 from the Medical Research Council of Canada.


    FOOTNOTES

Address for reprint requests and other correspondence: A. Churg, Dept. of Pathology, Univ. of British Columbia, 2211 Wesbrook Mall, Vancouver, BC, Canada V6T 2B5 (E-mail: achurg{at}interchange.ubc.ca).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Received 7 January 2000; accepted in final form 14 May 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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