Department of General and Experimental Pathology, University of Vienna, A-1090 Vienna, Austria
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ABSTRACT |
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We investigated the effects of
1,25-dihydroxyvitamin D3
[1,25(OH)2D3]
on paracellular intestinal Ca2+
absorption by determination of transepithelial electric resistance (TEER), as a measure of tight-junction ion permeability and
bidirectional transepithelial
45Ca2+
fluxes in confluent Caco-2 cell cultures. The rise of TEER to steady-state levels of ~2,000
· cm2 was
significantly attenuated by
1,25(OH)2D3
(by up to 50%) in a dose-dependent fashion between
10
11 and
10
8 M. Synthetic analogs of
1,25(OH)2D3,
namely, 1
,25-dihydroxy-16-ene,23-yne-vitamin D3 and
1
,25-dihydroxy-26,27-hexafluoro-16-ene,23-yne-vitamin D3, exhibited similar biopotency,
whereas their genomically inactive 1-deoxy congeners were only
marginally effective. Enhancement of transepithelial conductance of
Caco-2 cell monolayers by vitamin D was accompanied by a significant
increase in bidirectional transepithelial 45Ca2+
fluxes. Although
1,25(OH)2D3
also induced cellular
45Ca2+
uptake from the apical aspect of Caco-2 cell layers and upregulated the
expression of calbindin-9kDa mRNA, no significant contribution of the
Ca2+-adenosinetriphosphatase-mediated
transcellular pathway to transepithelial Ca2+ transport could be detected.
Therefore stimulation of Ca2+
fluxes across confluent Caco-2 cells very likely results from a genomic
effect of vitamin D sterols on assembly and permeability of
tight-junctional complexes.
intestinal calcium absorption; 1,25-dihydroxyvitamin
D3; synthetic vitamin D compounds; vitamin D receptor; genomic action; ionic conductance; cellular calcium
uptake; calbindin-9kDa; calcium-adenosinetriphosphatase
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INTRODUCTION |
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IT IS STILL a matter of debate as to whether Caco-2
cells display vitamin D-dependent
Ca2+ transport. Giuliano and Wood
(21) have reported a stimulatory effect of the steroid hormone on
Ca2+ transport in the
mucosal-to-serosal direction but did not address the question of
whether the vitamin D-dependent
Ca2+ binding protein, or
calbindin, which is the sole mediator of vitamin D-dependent
transcellular Ca2+ transport (for
review, see Ref. 8) is involved in this process. Furthermore, Bindels
et al. (4) presented evidence that vitamin D also increases
Ca2+ transport to the same extent
in the serosal-to-mucosal direction, so that its effect on net
transepithelial Ca2+ transport was
zero. These conflicting views could be reconciled if one assumes that
vitamin D-dependent Ca2+ transport
across Caco-2 cell monolayers as measured by both groups proceeds
mainly on a paracellular route. In this respect, we and others (12, 16)
have previously shown that 1,25-dihydroxyvitamin D3
[1,25(OH)2D3]
in fact is able to increase paracellular ion permeability of the
intestinal epithelium.
It therefore appeared worthwile to reexamine the effect of vitamin D on intercellular ion permeability in Caco-2 cells to dissect the transcellular from the paracellular route of transepithelial transport.
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EXPERIMENTAL METHODS |
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Caco-2 cells. The Caco-2 cell clone AQ, which was used in the present study, was originated in our laboratory by subcloning of an established clone, Caco-2/15 (cf. Ref. 3) after passage 100 by dilution plating. The population doubling time of the Caco-2/AQ clone during the logarithmic growth phase was estimated as 24 h (vs. 36 h of Caco-2/15 clone). The activity of the differentiation marker, alkaline phosphatase, increased during 20 days of confluent growth from an average of 20 to 60 mU/mg cellular protein in Caco-2/AQ cells, whereas the respective values for the parent clone Caco-2/15 were 25 and 190 mU/mg protein.
Cell culture. Caco-2/AQ cells (between passages 20 and 50) were grown either in 24-well Falcon culture plates (Becton-Dickinson, Bedford, MA) or on filters with 0.4-µm pore size (Falcon cell culture inserts), as appropriate, in Dulbecco's modified Eagle's medium [supplemented with 10% fetal calf serum, 4.0 mM glutamine, 20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 50 U/ml penicillin, and 50 µg/ml streptomycin] at 37°C in a 5% CO2-95% air atmosphere.
Vitamin D compounds were dissolved in ethanol and added to cultures so that the final solvent concentration in the medium did not exceed 0.01%.Measurement of transepithelial electrical resistance.
Transepithelial electrical resistance (TEER) of the Caco-2 monolayers
was measured by a high-precision technique as described previously in
detail (30). Current pulses of 55 µA, 0.5-s duration, were passed
across the monolayers with Ag-AgCl electrodes from an ESCOM 486 SX
computer equipped with a high-performance Labcard (PCL-818). Resulting
voltages were recorded with the aid of a differential amplifier with a
high input resistance. Data were corrected for well area (given in
· cm2).
Transport studies. If not indicated otherwise, the medium used for determination of transepithelial transport of 45Ca2+, 86Rb+, [14C]mannitol, or [14C]inulin, respectively, contained (in mM) 134 NaCl, 4.1 KCl, 2.1 CaCl2, 1.0 KH2PO4, 1.0 MgSO4, and 12.3 HEPES. pH was adjusted to 7.4 with 1.0 N NaOH. Specific activity of radiotracers was 0.5 µCi/ml.
Transepithelial transport across confluent Caco-2 cells was evaluated as described by Giuliano and Wood (21). Briefly, at time 0, transport buffer containing the radiolabeled solute at an appropriate concentration, i.e., 2.1 mM 45Ca2+, 0.1 mM 86Rb+, 1.0 mM [14C]mannitol, or 36 µM [14C]inulin, respectively, was filled into the filter well (1.0 ml) or the outside compartment (3.0 ml) of the filter unit as appropriate. In each case, the concentration of the solute under investigation in the contralateral compartment was zero. The filter plates were shaken horizontally at a frequency of 50 oscillations/min at room temperature for 60 min. Linearity of transport rates was monitored by determination of radioactivity in 20-µl aliquots drawn from the contralateral solutions at 15-min intervals.Uptake studies. Caco-2 cells grown in 24-well plates were allowed to equilibrate with room temperature for 30 min before experimentation. After aspiration of the culture medium, 0.5 ml of a "low-sodium" mannitol buffer containing 45Ca2+ (0.5 µCi/ml) was added into each well. The buffer composition was (in mM) 198 mannitol, 25 KCl, 1.2 NaH2PO4, 25 NaHCO3, 1.2 MgSO4, 0.25 CaCl2, and 20 glucose (24). The uptake experiment was carried out for 10 min under horizontal shaking (50 oscillations/min) at room temperature. For termination of uptake, the transport medium was sucked off, and the cells were washed three times with 1.0 ml of ice-cold phosphate-buffered saline (pH 7.4). Cells were then suspended in 1.0 ml of 1.0 N NaOH and allowed to solubilize by overnight standing at 4°C.
Calbindin-9kDa mRNA isolation and Northern blotting.
Total RNA was prepared from cells grown on filters in six-well plates
until day 15 according to Ref. 11; 20 µg were used for Northern blotting (as described in Ref. 2). There
was no obvious need for the use of semiquantitative reverse
transcription-polymerase chain reaction (RT-PCR) as performed by Fleet
and Wood (19), since specific message was sufficient for
conventional Northern analysis of calbindin-9kDa (CaBP-9kDa) mRNA
expression. Probes for human (h) CaBP-9kDa were generated by RT-PCR.
The following primers were selected from hCaBP-9kDa cDNA: Cal9.1.1 (1. for coding),
; Cal9.2.1 (1. for coding),
; Cal9.3.0 (0. for reverse),
; Cal9.4.0 (0. for reverse),
. The primers' specificity was
determined by searching data bases with the respective sequences (FASTA), and primers with 100% homology to only hCaBP-9kDa were accepted. Products of ~380 and 290 bp with primers Cal9.1. and Cal9.4. ("outside") and Cal9.2. and Cal9.3. ("inside"),
respectively, were obtained after 30 cycles. The longer PCR product was
then reamplified with the inside primers. The expected length together with a characteristic restriction fragment pattern definitely identified the product as hCaBP-9kDa (RT-PCR from human brain cDNA
yielded no product and thus served as a negative control). The PCR
product was cloned into a pCRII vector (Invitrogen) and used as probe
for the expression of hCaBP-9kDa mRNA thereafter.
Data presentation and statistical analysis. In each experimental series, at least three separate experiments were performed. Data are presented as means ± SE. Differences were considered statistically significant at the 5% confidence level with P values calculated by Student's unpaired t-test.
Materials.
25-Hydroxyvitamin D3 [25(OH)D3] was
purchased from Sigma (Deisenhofen, Germany). 1,25-Dihydroxyvitamin
D3 was generously supplied by Hoffmann-LaRoche (Basel,
Switzerland). Synthetic vitamin D analogs,
25-hydroxy-16-ene,23-yne-vitamin D3
[25(OH)-16ene,23yne-D3], 25-hydroxy-26,27-hexafluoro-16-ene,23-yne-vitamin D3
[25(OH)-26,27-F6-16ene,23yne-D3], 1
,25-dihydroxy-16-ene,23-yne-vitamin D3
[1,25(OH)2-16ene,23yne-D3], and 1
,25-dihydroxy-26,27-hexafluoro-16-ene,23-yne-vitamin
D3 [1,25(OH)2-26,27-F6-16ene,23yne-D3],
were a generous gift from Dr. Milan R. Uskokovi
(Roche, Nutley,
NJ). D-[1-14C]mannitol
(sp act 2.1 GBq/mmol), 45CaCl2, and
86RbCl were purchased from New England Nuclear
(Vienna, Austria) and
[carboxyl-14C]inulin (sp act
2.05 mCi/g) from ARC (St. Louis, MO).
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RESULTS |
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Development of paracellullar permeability in confluent Caco-2 cells.
Mucosal-to-serosal transepithelial transport of extracellular markers,
namely, [14C]inulin
(~5,000 mol wt) and
[14C]mannitol (182.2 mol wt), was determined in parallel to
45Ca2+
transfer across confluent Caco-2 cell layers. As shown in Fig. 1, exposure of Caco-2 cells to
108 M
1,25(OH)2D3
for 2 wk past confluence had a distinct effect on the extent to which
extracellular markers of different molecular weight could penetrate the
Caco-2 cell layer. Although exposure to the steroid hormone had no
effect whatsoever on transfer of the high molecular weight compound
inulin, a small but significant vitamin D-related increment of
transport of the considerably smaller molecule mannitol could be
observed. Expectedly,
1,25(OH)2D3
elicited an approximately threefold rise in transepithelial transport
of 45Ca2+
(Fig. 1).
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Effect of
1,25(OH)2D3
analogs on TEER of confluent Caco-2 cells.
Caco-2 cells respond not only to
1,25(OH)2D3
but also to a number of its synthetic analogs by changes in growth and
morphological appearance, provided that those compounds bear a
1-hydroxy group (5), which is a prerequisite for binding to the
vitamin D receptor (VDR) and hence for genomic activity. We therefore
tested the effect of
1,25(OH)2D3
and of two potent antimitogenic compounds, 1,25(OH)2-16ene,23yne-D3
and
1,25(OH)2-26,27-F6-16ene,23yne-D3, as well as of the respective 1
-deoxy compounds,
[25(OH)D3,
25(OH)-16ene,23yne-D3], and
25(OH)-26,27-F6-16ene,23yne-D3,
on TEER and transepithelial Ca2+
transport across Caco-2 cell layers. Figure
5 depicts the effect of the vitamin D
compounds at 10
8 M on the
development of TEER of postconfluent Caco-2 cell layers. Although
25(OH)D3, with the exception of
day 5, had no significant effect,
25(OH)-16ene,23yne-D3 and
25(OH)-26,27-F6-16ene,23yne-D3 from day 8 or 5 on, respectively, significantly reduced TEER by an average of
10%.
In contrast,
1,25(OH)2D3
and both synthetic 1
-hydroxylated analogs reduced TEER to
50% of
control levels at any time point. It should be noted that the decrement
in TEER induced by the synthetic 1
-hydroxyvitamin D
compounds tended to be even higher than that induced by
1,25(OH)2D3.
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Time course of 1,25(OH)2D3 effects on TEER and transepithelial Ca2+ transport. The increase in TEER during growth of confluent Caco-2 cells in all likelihood reflects the development of tight junctions (cf. Ref. 28). It was therefore of interest to know whether vitamin D would affect not only the assembly of intercellular junctions during confluent cell growth but also influence their barrier function in a more advanced state of development. Thus, in another series of experiments, Caco-2 cells were allowed to grow for 12 days past confluence before treatment with 1,25(OH)2D3 was begun. Table 1 shows that, after 48-72 h, a highly significant reduction of TEER with a concomitant rise in transepithelial Ca2+ transport could be observed. It should be noted that Ca2+ transport in the mucosal-to-serosal as well as in the opposite direction was influenced by the hormone to the same extent (Table 1).
Effect of
1,25(OH)2D3 on
cellular
45Ca2+
uptake and CaBP-9kDa mRNA expression.
To evaluate a possible contribution of the transcellular route to
vitamin D-related transepithelial
Ca2+ transport as measured, we
determined the effect of
1,25(OH)2D3 and analogs on Ca2+ uptake by
Caco-2 cells at different growth stages (Fig.
6). Basal cellular
Ca2+ uptake in vitamin D-free
control cultures conspicuously increased during transition from the log
growth phase into the confluent state. During this time period, the
1-hydroxylated vitamin D compounds under investigation were most
effective in raising cellular 45Ca2+
accumulation, whereas the 1
-deoxy compound,
25(OH)-16ene,23yne-D3, had no
effect at all.
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Ca2+-adenosinetriphosphatase inhibition and transepithelial Ca2+ transport. Because the last step of mucosal-to-serosal transcellular Ca2+ transport involves extrusion of Ca2+ across the basolateral aspect of the cell by the Ca2+-adenosinetriphosphatase (ATPase), we sought to evaluate the contribution of active Ca2+ pumping to net transepithelial transport by blocking the activity with a potent inhibitor, calmidazolium. The data collated in Table 2 show that, apart from the fact that pretreatment with the inhibitor had no influence on TEER in either controls or 1,25(OH)2D3-treated Caco-2 cells, a block of the Ca2+ pump did not change the extent of basal mucosal-to-serosal transepithelial Ca2+ transfer as measured but, even more important, by no means reduced its 1,25(OH)2D3-related increment.
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1,25(OH)2D3 and transepithelial 86Rb+ transport. Further proof for the assumption that 1,25(OH)2D3 can modulate ion transport on the paracellular route was obtained when we measured transepithelial 86Rb+ transport across confluent Caco-2 cell layers (Table 3). 86Rb+ is widely used as a substitute for K+ for assessment of Na+-K+-ATPase activity in whole cell preparations. It should be noted that, under the experimental conditions employed, transepithelial 86Rb+ transport was completely insensitive to ouabain treatment (Table 3). Because this excludes any contribution from the Na+-K+-ATPase-mediated transcellular pathway, 86Rb+ transport as measured mainly reflects ion flux on a paracellular route. The data collated in Table 3 therefore strongly suggest that stimulation of 86Rb+ transport across confluent Caco-2 cell layers in the serosal-to-mucosal direction by the steroid hormone occurs in parallel with reduction of TEER during postconfluent cell growth.
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DISCUSSION |
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Caco-2 cells, though originally derived from a human colon adenocarcinoma, are still able to undergo spontaneous differentiation into enterocyte-like cells. Thereby Caco-2 cells form confluent monolayers consisting of well-polarized cells with tight junctions and a typical apical brush border (28).
It is generally accepted that transepithelial electric conductance across Caco-2 cell layers is mainly determined by the ionic permeability of the intercellular junctions which develop during postconfluent cell growth (28). The fact that the paracellular route is the predominant pathway of transepithelial ion flux can be inferred from the observations that ~80% of total electrical resistance of Caco-2 cells is located in the mucosal membrane and that, in addition, its Na+ conductance is very limited (22). Changes in TEER can also not be explained by activation of Na+-D-glucose cotransport, because the Caco-2 cell clone used in the present study is devoid of any Na+-dependent D-glucose transport activity (10, 22), and in addition, no D-glucose was present in the incubation medium. Thus the decline in TEER largely reflects an effect on tight junction-mediated paracellular ion permeability (27). The present study documents that 1,25(OH)2D3 as well as its synthetic D-ring and side-chain-modified analogs substantially reduce TEER of confluent Caco-2 cells. This must be considered as clear evidence for the ability of genomically active vitamin D compounds to increase bidirectional paracellular flux of all ion species including Ca2+. In this respect, it is interesting to note that Favus et al. (16) had observed that 1,25(OH)2D3 caused a significant increase of tissue conductance and, most notably, also of bidirectional mannitol fluxes in the duodenum and descending colon of rats, whereas Cross et al. (12) reported on stimulation of paracellular ion transport, i.e., Na+, K+, and Rb+, in organ-cultured embryonic chick small intestine.
Caco-2 cells express VDR mRNA and protein during the log growth phase
as well as after confluence (20, 23). This is apparently the basis for
the action on paracellular ion permeability of vitamin D compounds,
since all 1-deoxyvitamin D compounds under investigation were either
completely ineffective in reducing TEER, namely,
25(OH)-D3, or, like the two
synthetic compounds,
25(OH)-16ene,23yne-D3 and 25(OH)-26,27-F6-16ene,23yne-D3,
showed only marginal activity compared with their 1
-hydroxylated
congeners (cf. Fig. 5). Because the 1
-hydroxy group mediates
high-affinity binding to the VDR (cf. Ref. 6), it is reasonable to
assume that the observed effects on TEER of
1,25(OH)2D3
and its two synthetic side-chain- and D-ring-modified analogs result
from a genomic rather than from a nongenomic action. The latter
possibility seems unlikely also for a number of other reasons. First,
typical nongenomic effects of
1,25(OH)2D3
(for review see, e.g., Ref. 7) involve interactions with plasma
membrane activities and are observed within seconds or minutes, whereas
reduction of TEER requires at least 48-h exposure to the hormone (cf.
Table 1). Second, a rapid membrane action of
1,25(OH)2D3
cannot be easily reconciled with the observation that the sensitivity
of Caco-2 cells varies with ongoing differentiation between
days 4 and
12 past confluence (cf. Fig. 3). It
has been shown, however, that the expression of genomic effects of
1,25(OH)2D3,
particularly in enterocytes, can depend to a large extent on the degree
of their differentiation (13). Third,
25(OH)-16ene,23yne-D3 was shown to
be most potent in eliciting nongenomic effects such as activation of
voltage-gated Ca2+ channels in rat
osteosarcoma cells (15), whereas the same analog was only weakly
effective in attenuating TEER (cf. Fig. 5). Fourth, it is conceivable
that the observed small effects of synthetic 1-deoxyvitamin D compounds
on TEER of Caco-2 cells reflect their small genomic potency due to the
ability to bind weakly to the VDR (6, 15) or, fifth, result from
conversion into genomically active 1-hydroxy compounds. Although
substantial 25-hydroxyvitamin D3-1-hydroxylase activity has been
observed only in serum-free cultures of Caco-2 cells (14), it is
conceivable that, even under the culture conditions employed in the
present study, a small fraction of the 25-hydroxy compounds tested is
converted to respective 1
-hydroxy derivatives, which could then be
responsible for the observed effects on TEER (Fig. 5).
Both assembly and barrier properties of tight junctions depend on the
formation of a bipartite functional complex with adjacent adherens
junctions as well as on an appropriate organization of the latter with
the actin cytoskeleton (1, 26). Fialka et al. (17) showed that
estrogen-related upregulation of the c-Jun oncoprotein diminishes TEER
in mammary epithelial cells and, at the same time, disrupts the
polarized expression of the tight junction-associated protein zonin-1
as well as of the constituents of adherens junctions, E-cadherin and
-catenin. Because the c-jun protooncogene is also a well-known target for signaling from the VDR
(9, 25), we suggest that upregulation of c-Jun expression could also
explain the observed effects of genomically active vitamin D compounds
on tight-junctional permeability of Caco-2 cells. In fact, we have
obtained evidence from Western blot analysis that treatment with
10
8 M
1,25(OH)2D3
for 5 days leads to reduced expression of E-cadherin in Caco-2 cells
(unpublished results).
A strong argument for the notion that vitamin D stimulates transepithelial Ca2+ transport by an increase in junctional ion permeability rather than by stimulation of transcellular calbindin-mediated transport, as suggested by Fleet et al. (18, 19), can be derived from the following observations: 1) an identical relationship between TEER or conductance, respectively, and Ca2+ transport exists in untreated and vitamin D-treated Caco-2 cell cultures, and hence no conductance-independent vitamin D-related increment exists; 2) vitamin D has an identical effect on apical-to-basolateral as well as on basolateral-to-apical Ca2+ fluxes, which would not be the case if there were a major contribution from vectorial transcellular calbindin-mediated transport that proceeds exclusively in the apical-to-basolateral direction; and 3) the effect of vitamin D is not specific for Ca2+ transport but is visible also on bidirectional Rb+ fluxes, which are certainly not calbindin mediated.
As far as the existence of a major transcellular Ca2+ path in confluent Caco-2 cells is concerned, we were able to confirm the observation of Surendran et al. (29) that blocking Ca2+ extrusion across the basolateral membrane by Ca2+-ATPase inhibition does not alter the extent of transepithelial Ca2+ transport. Because this is valid also for 1,25(OH)2D3-treated Caco-2 cells (cf. Table 2), this observation must be considered as additional support for the assumption that vitamin D affects Ca2+ transport mainly through its effect on tight-junctional ion permeability.
In probing the vitamin D sensitivity of the consecutive steps of apical-to-basolateral transcellular Ca2+ transport, we could show that cellular Ca2+ uptake from the apical aspect of confluent Caco-2 cell layers involves a genomic action of vitamin D sterols. Furthermore, consistent with the results of Fleet et al. (18, 19), 1,25(OH)2D3 upregulates CaBP-9kDa mRNA levels (Fig. 7). However, it must be borne in mind that as long as direct measurement of human CaBP-9kDa protein in Caco-2 cells is not available, it remains questionable whether the vitamin D actions on mucosal Ca2+ influx and CaBP-9kDa mRNA are of a magnitude to efficiently raise the rate of transcellular transport of Ca2+.
Another explanation for the difference in the interpretation of our results and those of Fleet et al. (18, 19) lies in the fact that these authors did not observe any effect of vitamin D sterols on transepithelial transfer of phenol red, which they used as a marker for paracellular permeability. However, the relatively high molecular weight and negative charge of this compound may have compromised its use to detect changes in paracellular permeability of ions with a much smaller atomic radius, such as Ca2+. Because of the lack of any substantial contribution of the transcellular route to transepithelial Ca2+ transport, the Caco-2 system could serve as an excellent model for the study of vitamin D effects on intestinal Ca2+ absorption via the paracellular route (for review, see Ref. 31).
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ACKNOWLEDGEMENTS |
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The authors thank Teresa Manhardt for skillful technical assistance.
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FOOTNOTES |
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These investigations were supported by Grant P-10133-MED from the Austrian Science Foundation.
M. V. Chirayath was on leave of absence from Dept. of Physiology, PSG Medical College, Coimbatore, South India, and was the recipient of a fellowship of the International Academy of Pathology, Austrian Section.
Address for reprint requests: M. Peterlik, Dept. of General and Experimental Pathology, Waehringer Guertel 18-20, A-1090 Vienna, Austria.
Received 12 December 1996; accepted in final form 20 October 1997.
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