Department of Pathology, University of British Columbia, Vancouver, British Columbia, Canada V6T 2B5
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ABSTRACT |
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Respirable ambient particles [particulate
matter <10 µm (PM10)]
are associated with both acute and chronic adverse health effects
including chronic airflow obstruction.
PM10 can induce expression of
inflammatory and fibrogenic mediators, but there is controversy about
the types and/or sizes of particles involved and, in particular,
whether ultrafine particles are the major toxic agents. To examine
whether particle size affects mediator generation, we exposed rat
tracheal explants, an inflammatory cell-free model of the airway wall,
to various concentrations up to 500 µg/cm2 of fine (0.12 µm) or
ultrafine (0.021 µm) titanium dioxide (anatase), maintained the
explants in an organ culture in air for 1-7 days, and used RT-PCR
to examine the expression of fibrogenic mediators and procollagen. No
increase in gene expression was seen at 1 or 3 days, but at 5 days,
ultrafine dust induced a small increase in procollagen. At 7 days, fine
titanium dioxide produced significantly greater increases for
platelet-derived growth factor (PDGF)-B, transforming growth
factor-, and transforming growth factor-
compared
with those by ultrafine dust; both dusts produced similar increases for
PDGF-A; and ultrafine dust produced increases in procollagen
expression, whereas fine dust had no effect. Expression levels were
dose related. Both dusts produced a similar decrease in expression of
PDGF receptor-
and a similar increase in PDGF receptor-
. These
observations suggest that ultrafine particles are intrinsically able to
induce procollagen expression even in the absence of inflammatory
cells; that chronic exposure to
PM10 may result in chronic airflow
obstruction, in part because of ultrafine particle-mediated increases
in airway wall fibrosis; and that chemically identical dusts of
differing size can produce quite different patterns of gene expression
in the airway wall. Differential upregulation of PDGF receptors does
not appear to explain dust-induced fibrosis in this model.
particulate matter less than 10 micrometers; air pollution; transforming growth factor-; transforming growth factor-
; platelet-derived growth factor; platelet-derived growth factor receptor
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INTRODUCTION |
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THERE IS NOW EXTENSIVE epidemiologic evidence that increases in the level of the respirable fraction of ambient atmospheric particulate pollutants [particulate matter <10 µm (PM10)] are associated with increased respiratory and cardiovascular morbidity and mortality (reviewed in Refs. 3, 12). In addition to acute effects, chronic exposure to high levels of PM10 appears to be associated with an increased risk of chronic airflow obstruction (1, 22). The mechanisms behind all of these effects are the subject of intensive investigation. How PM10 might produce chronic airflow obstruction, in particular, is at this point unclear, but one possibility is that such particles cause the release of mediators that lead to fibrosis of the airway wall. Airway wall fibrosis causes both airway distortion and a decrease in airway lumen size, and these features have been shown to be important mediators of the airflow obstruction seen in cigarette smokers and asthmatic patients (39).
One of the major questions regarding the effects of PM10 is the exact type and size of the particle involved. A variety of studies, initially from Oberdorster and colleagues (31, 32) and since confirmed by others (13, 16), have indicated that ultrafine particles (those <0.1 µm in diameter) are typically more pathogenic than larger particles of the same mineral type, even when the mineral involved, for example, titanium dioxide or carbon black, is an insoluble dust of low toxicity. For a given mineral species, ultrafine particles appear to be able to induce particularly intense inflammatory reactions, and somewhat indirect evidence suggests that they are also more fibrogenic than so-called "fine" particles, i.e., those between 0.1 and 2.5 µm in diameter (13, 16, 17, 26, 31, 32; also see DISCUSSION). There are also increasing data that suggest that ultrafine particles are particularly powerful generators of oxidants (13, 19, 26). It has been proposed that ultrafine particles are, in fact, the specific agents of PM10 toxicity (33, 36), although epidemiologic studies have not reached a consensus on this point (3, 12).
Experimental models have demonstrated that many types of air pollutant
particles can induce cytokine mediators both in vitro and in vivo.
Experimental ultrafine and fine particles, whole PM10, and specific particulates
collected from ambient air, for example, diesel exhaust or residual oil
fly ash (ROFA), have been shown to lead to release of cytokine
mediators including tumor necrosis factor-, interleukin (IL)-1,
IL-6, IL-8, macrophage inflammatory protein-2, and monocyte
chemoattractant protein-1 as well as activation of nuclear factor-
B,
c-Jun kinase, and the activator protein-1 transcription factor in
tissue cultures of animal and human cells and in whole animal models
(4-6, 14, 15, 18, 23, 34, 35, 37, 38, 41). Both ROFA and Mexico
City PM10 are also able to
upregulate platelet-derived growth factor (PDGF) receptor (PDGFR)-
in cultured fibroblasts (6, 27, 29). However, there is very little
information available on the question of whether these effects are
generic ones related to the particle species in question or whether,
for a given particle type, fine and ultrafine particle types differ in
their ability to induce mediator expression. Finkelstein et al. (18)
briefly noted that exposure of cultured type II cells to fine and
ultrafine titanium dioxide at the same mass concentration resulted in
marked increases in tumor necrosis factor-
expression with ultrafine
dust and no effect with fine dust. Timblin et al. (38) observed that
ultrafine compared with fine titanium dioxide caused a greater increase
in phosphorylated c-Jun protein but no difference in bromodeoxyuridine
labeling in rat lung epithelial cells.
If rat tracheal explants are exposed to mineral dusts and maintained in organ culture, they will slowly take up dust from the cell surface and transport it through the cells to the interstitium. Such explants thus provide a model of the particle-exposed airway wall, and because the explants are free of exogenous inflammatory cells, they allow examination of the direct effects of dust on the airway wall. Dai et al. (11) have recently shown that, using RT-PCR, this explant model can be used to evaluate dust-induced cytokine and matrix protein gene expression. In this paper, we used tracheal explants to examine the effects of a fine versus an ultrafine particle of the same mineral type on the expression of growth factors, fibrogenic mediators, and procollagen. Because it has been proposed that induction of PDGFRs is an important mechanism of asbestos-related fibrosis (25) and that, as noted above, ROFA and PM10 can upregulate PDGFRs in monolayer culture (6, 27, 29), we also examined the effects of particle size on PDGFR gene expression.
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MATERIALS AND METHODS |
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Dusts. Fine titanium dioxide [anatase; geometric mean diameter 0.12 µm, geometric standard deviation (GSD) 1.4] was obtained from Aldrich (Milwaukee, WI). Ultrafine titanium dioxide (anatase; geometric mean diameter 0.021 µm, GSD 1.7) was a kind gift from Dr. G. Oberdorster (University of Rochester, Rochester, NY). Published data (38) and a personal communication from Dr. Oberdorster indicate that the ultrafine particles do not contain detectable levels of metals. The fine preparation was analyzed by inductively coupled argon plasma spectroscopy. This revealed concentrations of 4 parts/million (ppm) iron, 4 ppm vanadium, and 1 ppm zinc. Copper, lead, and cobalt were not detected. All dust suspensions were prepared in saline and extensively autoclaved before use. The dust suspensions were sonicated for 30 min and diluted into culture medium for use.
Explant preparation and culture. Two by two-millimeter tracheal explants were prepared from 250-g male Sprague-Dawley rats with a modification of the method of Mossman et al. (30) as previously described (11). Freshly prepared explants from several different animals were used for each experiment and mixed to ensure that all explants for a given data point did not come from the same animal.
Immediately after removal from the rat, the explants were submerged, epithelial side up, in a suspension of fine or ultrafine titanium dioxide at various concentrations in Dulbecco's modified Eagle's medium (DMEM) without serum for 1 h. Control explants were exposed only to the culture medium. At the end of this time, the explants were transferred to petri dishes containing DMEM in agarose supplemented with 1% glutamine, 1% penicillin-streptomycin-Fungizone, 1 µg/ml of insulin, 0.1 µg/ml of hydrocortisone, 1.5× amino acids, and 10% chicken serum.
Previous experiments by Dai et al. (11) have shown that increased expression of fibrogenic mediators and matrix components is usually not detectable until 7 days, so the explants were maintained in an air plus 5% CO2 organ culture with basal feeding in an incubator at 37°C for this period and then harvested for RNA extraction. After examining the 7-day data, we went back and performed the same experiments with all dust doses (see Expression of growth factors, fibrogenic mediators, and matrix components by RT-PCR) for a 5-day incubation. Because this showed results only at the highest dust dose, only the highest dose was subsequently used for 1- and 3-day incubations.
Expression of growth factors, fibrogenic mediators, and matrix components by RT-PCR. Dai et al. (11) have previously found that to obtain reliable signals for gene expression in these very small tissues, it is necessary to use RT-PCR and to combine three explants to produce each data point. Each test group shown in the present experiments consisted of three such data points. In the previous study, Dai et al. also observed that dust concentrations of 5 mg/ml (500 µg/cm2) produced reliable levels of dust uptake and mediator expression at 7 days of culture, and we therefore used progressive halving doses of 5, 2.5, and 1.25 mg/ml for both fine and ultrafine dust in the current experiments.
Total RNA was extracted from cultured tracheal explants with the method of Chomczynski and Sacchi (8), and first-strand cDNA was synthesized with superscript RNase H reverse transcriptase (GIBCO BRL, Life Technologies, Grand Island, NY) according to the manufacturer's instruction. Briefly, 5 µg of RNA were added to a reaction mixture of 1× first-strand buffer; 200 ng of oligo(dT)12-18 primer; 0.5 mM each dATP, dTTP, dGTP, and dCTP; and 0.1 M dithiothreitol plus water to 49 µl. Two hundred units of superscript RT were added, and the reaction was incubated at 42°C for 1 h.
PCRs contained 1 µM primers, 1.5 mM Mg2+, 200 µM deoxynucleotide triphosphates, reaction buffer, 2.5 U of Taq DNA polymerase (Perkin-Elmer Cetus Instruments, Norwalk, CT), and 1 or 5 µl of cDNA in a final volume of 20 µl. The PCR temperature profile consisted of 25 or 28 cycles of denaturation at 94°C for 45 s, primer annealing at 60°C for 45 s, and extension at 72°C for 75 s followed by an additional 5-min final extension at 72°C. The PCR products were size fractionated on a 1.5% agarose gel and stained with ethidium bromide, and densitometry was performed on the ethidium bromide-stained gel with the Bio-Rad Gel Documentation System (Bio-Rad Laboratories, Hercules CA).
Primer design was based on sequences from the GenBank database (Table
1). We optimized the reaction conditions
(e.g., Mg2+ concentration,
thermocycler temperature) to produce the greatest amount of a single
PCR product. Test samples were run to ensure that the amount of cDNA
used and the number of cycles of amplification were within the linear
range. Specificity of the various amplification products was confirmed
by restriction digests. Expression of malate dehydrogenase (21) was
used as a control (housekeeping) gene. Experiments were repeated
several times, and representative data are shown.
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Statistics. Differences between fine and ultrafine particle-induced gene expression were determined by analysis of variance. P < 0.05 was considered significant.
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RESULTS |
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No increases were seen in the expression of any of the genes studied at
1 or 3 days (data not shown). At 5 days, there was an ~20% increase
in procollagen expression at the highest dust dose of ultrafine dust
but no effect with fine dust (Fig. 1); no
other gene showed increased expression levels. At 7 days, increases in
expression were seen with all of the genes studied. PDGF-A chain
expression (Fig. 2) was increased to about
the same degree (roughly 140% of control value) at the 5-mg dose of
both fine and ultrafine titanium dioxide and, to a small degree or not
at all, with lesser doses. For PDGF-B chain (Fig.
3), a progressive increase was seen with
increasing doses of fine dust (~140% of the control value at 5 mg/ml
of dust), but there was essentially no increase (<110% of control
value at 5 mg/ml) with ultrafine dust. Figure
4 illustrates the dose response for
transforming growth factor (TGF)- expression. An increase was seen
with both dusts and was significantly greater for fine dust (~200%
of the control value) than for ultrafine dust (~150% of the control
value) at 5 mg/ml as well as at a dose of 2.5 mg. Figure
5 shows similar data for TGF-
expression. Again, the effects of fine dust are significantly greater
(190% of the control value at 5 mg/ml) compared with ultrafine dust
(140% of the control value at 5 mg/ml). Figure 6 demonstrates the effects of the dusts on
procollagen expression. No effect at all was seen at any concentration
of fine dust, but ultrafine dust induced a marked increase in
expression (roughly 190% of the control value at 5 mg/ml and a lesser
increase at 2.5 mg/ml).
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Figure 7 shows expression of procollagen,
PDGFR-, and PDGFR-
with a 5 mg/ml dose of dust in a separate
experiment. Again, only ultrafine dust caused an increase in
procollagen expression. Both dusts produced a similar decrease in
PDGFR-
expression (to roughly 80-85% of the control value),
and both dusts produced an increase in PDGFR-
expression, with a
slightly greater increase for fine compared with ultrafine dust (190 vs. 160%).
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DISCUSSION |
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In these studies, we used a tracheal explant model of the airway wall
to examine the effects of particle size on the expression of a variety
of growth factors, fibrogenic mediators, and matrix components.
Explants offer several major advantages for this type of study. First,
they provide a model in which both epithelial and interstitial
(subepithelial connective tissue including fibroblasts and interstitial
macrophages) tissues are present in their normal anatomic
arrangement. Over time, dust is taken up by the epithelial cells (9,
11) and then moves through the epithelial cells into the interstitium,
exactly mimicking the process in vivo (20). Second, because both
compartments are present, mediators produced in one compartment are
able to affect the other compartment, and this is particularly
important in examining the effects relating to fibrogenesis because
mediators such as TGF-, TGF-
, and PDGF are either constitutively
expressed in the epithelial cells or upregulated in those cells after
dust exposure (10, 11, 28, 29) but potentially affect matrix production
in the interstitium. Matrix production is likely also upregulated by
dusts reaching the interstitium and interacting directly with
fibroblasts and interstitial macrophages (2). Moreover, coculture
models of epithelial and mesenchymal cells show clearly that each
compartment modulates the response of the other to noxious stimuli,
whereas the same stimulus applied to pure epithelial or mesenchymal
cell cultures can produce quite different patterns of cytokine
expression (24; see below). Thus if one wishes to examine a realistic
model of potential chronic fibrogenic effects in the airway
wall, coculture or organ culture models are particularly
useful. Equally important, the explant model is free of
circulating inflammatory cells and the numerous air space inflammatory
cells evoked by dusts and thus can be used to examine the direct
effects of dust on the airway wall. This is very difficult to do
with whole animal models.
Tracheal explants also have a number of limitations for this type of study, and these limitations need to be kept in mind. Dust uptake is very slow in vitro as it is in vivo. Churg et al. (9) and Dai et al. (11) have previously observed that incubation periods of 7 days are generally required to see mediator expression, and this slow induction period appears to correspond to the time it takes dust particles to penetrate into the tissues in significant numbers. Because of the very small amount of tissue available, we needed to use RT-PCR to obtain a signal, and quantification of gene expression levels with RT-PCR must always be interpreted cautiously. Nonetheless, we do find that our results are linear and reproducible. The absolute magnitude of an increase in gene expression varies somewhat from run to run, but the pattern of increases for a given gene and dust dose is always similar, and there is a consistent increase in expression as dust dose is increased. These observations suggest that we were able to detect significant differences in gene expression.
More problematic is the issue of dust dose. With explants, there is a very narrow dose range in which effects occur and the level of dust required is quite high, considerably higher than is required to see effects in monolayer cultures. It would be useful to be able to translate these doses into human terms, but the very different nature of the ex vivo explant compared with an in vivo inhalation system, the problems of deciding what generation of human airway (a parameter that significantly affects deposited particle number) should be used for comparison, and especially the problems of particle size and particle aggregation documented below make such a comparison unfeasible. Moreover, even at these high doses, the level of dust uptake in the explants is relatively low (see Ref. 11 for illustrations). The reason for this requirement for high dust loading is unclear. However, high dust levels are used in many experimental models to elicit potentially important and measurable effects, with the understanding that such effects in humans at realistic dose levels must proceed on a barely measurable and much slower course. Thus our intent in these experiments was to show ways in which fine compared with ultrafine particles could affect the airway wall. The fact that we observed both a dust dose and a time dependency for upregulation of expression suggests that the model produces potentially useful data.
The question of dose is quite complex in other ways. Ambient ultrafine particles occur as singlets near point sources of emission but rapidly aggregate to form "fine" particles, and it is believed that away from point sources relatively few singlet particles are present (40). As noted by Oberdorster (31), singlet particles appear to be more active than aggregates in their ability to evoke a response. However, the dusts used here occur as aggregates in aqueous suspension and are extremely difficult to disaggregate; we found that 30 min of sonication compared with no sonication in culture medium resulted in geometric mean aggregate diameters (GSD) of 3.5 µm (2.3) compared with 3.0 µm (2.1) for ultrafine particles and 0.59 µm (2.0) compared with 0.65 µm (2.0) for fine particles when the particles were simply collected on electron microscope grids. Very few singlet particles of either type were observed. We also examined procollagen expression using sonicated and nonsonicated fine and ultrafine dust after 7 days of culture. Fine dust again produced no increase in procollagen expression, and expression levels after exposure to sonicated and nonsonicated ultrafine dust were within 8%. Our model system thus may not be reflective of events that occur in the airway wall in regions where large numbers of ambient singlet particles are present.
A further complication is that we, like most workers who have examined fine versus ultrafine effects (13, 16, 17, 31), used equal mass doses of the two different-size particles. Ideally, one would like to use equal numbers of fine and ultrafine particles, which, for these dusts, would require a dose difference of ~200-fold. This assumes that the particles all disaggregate into singlets. In practice, the problem of aggregation makes it impossible to determine what dose would produce "equal" numbers of fine and ultrafine particles, and a previous electron-microscopic analysis of particles taken up by tracheal epithelial cells in this system by Churg et al. (9) showed that, in the epithelium and subepithelium, almost all the particles occurred as aggregates, with very few singlets visible.
Given these considerations, one has to ask whether our results reflect particle size or applied particle number. It is difficult to rule out particle number in a very broad sense because the current data on gene expression correlate with previous observations by Churg et al. (9) and Dai et al. (11) that the number of particles (or number of aggregates) of both dusts entering the tissues and reaching the subepithelial spaces increases slowly over time, with by far the greatest number of particles seen in the tissue at 7 days. But if gene expression were simply a matter of applied particle number or, as reviewed by Oberdorster (31), the closely related measure of total particle surface area, we should (assuming that the particles all dissociated) be observing greater increases in expression of every mediator at every dust dose of the ultrafine compared with the fine dust. In fact, only procollagen expression is greater with ultrafine dust, and even here, no effect is seen at the lowest dust dose. Again, the reality is not this simple because almost all the particles are aggregated and we cannot determine how many particles the tissue actually "sees"; moreover, as Churg et al. (9) previously showed in tissue, aggregate sizes change over time in different ways for the different dusts. However, what this really says is that the two different-size dusts, administered in equal mass doses, behave differently in terms of tissue entry and transepithelial transport and the resulting pattern of gene expression.
Redox active metals are an important component of
PM10 and can affect gene
expression. However, the dusts used here had no or relatively low
levels of metals, levels to
of those seen
in actual PM10 samples from Mexico
City (6) or in ROFA (7). Differences in formation of active oxygen
species on the basis of metal contamination of the two different dust
samples appear unlikely to explain the differences we observed.
All of these issues mandate that conclusions be carefully drawn, but
the most likely conclusion from our study is that we are seeing effects
specifically related to particle size (arguably, particle size as it
affects aggregate size or some other property of the aggregates) and
that, for titanium dioxide, a relatively insoluble and nontoxic dust,
particle size does influence mediator and matrix gene expression.
However, our results are also surprising in that different genes show
quite different expression patterns with the two dusts. PDGF-B,
TGF-, and TGF-
expression are more marked with fine dust at all
doses; PDGF-A chain and both PDGFR-
and PDGFR-
expression are
increased or decreased to about the same degree with both dusts; and,
as noted, procollagen expression is markedly upregulated with ultrafine
dust and not affected at all by fine dust. These observations support
the idea that particle size plays a major role in the way the airway
epithelium and wall (and perhaps the alveolar epithelium and wall)
respond to dusts and are consistent with observations in other systems
that morphological changes and oxidant injury also vary with particle
size (13, 16, 31). These are, as noted, intrinsic reactions of the
airway wall to dusts; production of cytokine mediators by dust-evoked inflammatory cells probably acts to additionally upregulate or downregulate responses.
Ferin and colleagues (16, 17) reported a series of experiments in which they exposed rats to fine and ultrafine titanium dioxide and concluded by measuring the unlavagable dust fraction, that ultrafine particles entered the tissues more readily than fine particles. Ultrafine particles were also more fibrogenic than fine particles as assessed by histology. These experiments were elegant but somewhat hard to interpret because of the indirect method used for assessing particle uptake and also because, as is true of all whole animal models of dust exposure, the evoked inflammatory response masks the intrinsic reaction of the epithelium and interstitium to dust. Our present findings, although based on airway rather than parenchymal tissues, support the conclusions of Ferin and colleagues that ultrafine titanium dioxide is more fibrogenic than fine titanium dioxide and suggest again that this may be an inherent property of ultrafine dusts because it occurs in the absence of exogenous inflammatory cells.
The fact that ultrafine but not fine dust elicits an increase in
procollagen expression, whereas the reverse pattern applies to PDGF-B,
TGF-, and TGF-
, suggests that, although all of these mediators
are known to affect mitogenesis of epithelial and/or mesenchymal cells
or to affect matrix production, no one or no combination of these
mediators is driving fibrogenesis in this system. This conclusion is
supported by the observation that increases in procollagen expression
appear by day 5, a time when there are no increases in any of the other mediators examined. As noted above,
ROFA and Mexico City PM10 have
been reported to increase PDGFR-
in cultured fibroblasts (6, 27,
29), and Lasky et al. (25) have recently reported that, in
asbestos-exposed but not in carbonyl iron-exposed animals, there is
evidence of upregulation of PDGFR-
. Because asbestos but not iron
causes increased matrix production in their model, they suggested that increased expression of PDGFR-
may be playing a crucial role in
fibrogenesis. Our findings are quite different: here PDGFR-
is
downregulated and PDGFR-
is upregulated to about the same degree
with both dusts. The vastly different dusts, doses, methods of
administration, and, of course, whole animal or monolayer culture versus explant nature of the models make comparison difficult, but
PDGFR upregulation also does not appear to explain the increased procollagen expression seen in our studies with ultrafine titanium dioxide. It is possible that increased procollagen expression might be
an effect of ultrafine particles reaching the interstitium and
interacting directly with fibroblasts, without the requirement for an
intermediate agent.
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ACKNOWLEDGEMENTS |
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This work was supported by Grant MT8051 from the Medical Research Council of Canada.
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FOOTNOTES |
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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.
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).
Received 17 December 1998; accepted in final form 13 June 1999.
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