Hematology/Oncology Division, Internal Medicine Department, Wayne State University Medical School, Detroit, Michigan 48201
Submitted 13 December 2002 ; accepted in final form 31 May 2003
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ABSTRACT |
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Th1 cytokines; monokines
Zinc also plays important roles in immune functions. Zinc regulates several functions of lymphocytes, such as mitogenesis, antibody synthesis, the activation of T cells and natural killer cells, and more specifically cellular immunity (1, 10, 15, 19, 27). Zinc deficiency has been reported to impair cellular immune functions. Dysfunctions of cellular immunity resulting from zinc deficiency in humans induce frequent severe fungal, viral, and bacterial infections, thymic atrophy, anergy, reduced serum lymphocyte proliferative response to mitogens, a selective decrease in T helper cells, and reduced thymic hormone (thymulin) activity. Zinc supplementation to these individuals reverses all of these manifestations (9, 33).
The effect of a zinc-deficient state on cytokines has been examined in both animals and humans. Zinc deficiency decreased IL-2 production by the peripheral blood mononuclear cells (PBMC) in humans and animals (20, 30, 31). Decreased IL-2 production has been observed in zinc-deficient subjects with malignant diseases, patients on chronic hemodialysis, head and neck cancer patients, elderly human subjects, and healthy human subjects with zinc-restricted diets (2, 25, 26). Zinc supplementation administered to the zinc-deficient healthy human volunteers increased the production of IL-2 (26).
The number of studies of zinc deficiency on the production of proinflammatory cytokines such as TNF-, IL-1
, and IL-8 by mononuclear cells is limited. Henning and associates (11) examined a change in TNF-
production by using 1% FBS to induce zinc-deficient conditions. The results showed that zinc deficiency increased TNF-
production in endothelial cells. In certain diseases, such as Crohn's disease, rheumatoid arthritis, and alcoholism, patients with low blood zinc levels showed increased TNF-
production (34). However, other studies showed that zinc supplementation increased TNF-
production (16). Therefore, the results of these studies provide conflicting data.
We have previously induced a specific mild deficiency of zinc in human volunteers by dietary means, and in this model we have reported a decrease in the production of IL-2 and INF- by PBMC after p-phytohemagglutinin (PHA) stimulation (2). The production of IL-4, IL-6, and IL-10 was not affected by the zinc status (2). Zinc restriction, however, led to an increase in the production of IL-1
by PBMC (2). These changes were reversible by zinc supplementation (2). To define further the mechanism of zinc action on production of these cytokines, we have developed zinc-deficient and zinc-sufficient cell culture models.
We hypothesize that zinc upregulates the gene expression of cytokines such as IL-2 and INF- in Th1 cells and downregulates the gene expression of proinflammatory cytokines such as TNF-
, IL-1
, and IL-8 in monocyte-macrophage cells. HUT-78 cells have been previously used by us to study the effects of zinc defi-ciency on the cell cycle, deoxythymidine kinase activity, and its gene expression (25). In the present study, we used a zinc-deficient medium to examine the effects of zinc on the gene expression of IL-2 and IFN-
in HUT-78 cells (Th0 human malignant lymphoblastoid cell line) and in D1.1 cells (Th1 human malignant lymphoblastoid cell line) and IL-1
, TNF-
, and IL-8 in HL-60 cells (human malignant monocyte-macrophage cell line). We found that zinc deficiency decreased IL-2 and INF-
mRNAs and cytokine levels in Th0 and Th1 cells. In contrast, zinc deficiency increased TNF-
, IL-1
, and IL-8 mRNA and cytokine levels in the monocyte-macrophage cell line. Our study thus shows that the effect of zinc on the gene expression and production of cytokines is cell lineage specific. These observations also suggest that zinc may have a therapeutic role in the management of cell-mediated immune dysfunctions and disorders resulting from oxidative stress in humans. A Th2 cell line was not available for study; however, inasmuch as Th0 cells also produce IL-4 and IL-10, we used these cells for Th2 cytokine production in zinc-deficient cells. These cytokines were not affected by zinc deficiency.
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METHODS |
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Cell culture. The HUT-78 human malignant T lymphoblast cell line (NIAIDS), D1.1 human malignant T lymphoblast cell line (ATCC), and HL-60 human malignant monocyte-macrophage cell line (ATCC) were maintained in RPMI 1640 culture medium containing L-glutamine and supplemented with 10% regular FBS (without chelation), 100 U/ml penicillin, 100 µg/ml streptomycin, and 1.5 g/l sodium bicarbonate at 37°C in a humidified atmosphere of 5% CO2 in air. Cells (5 x 106/ml) were seeded in the culture medium described above. To initiate an experiment, cells were separated into two groups. One set of cells was further incubated in zinc-deficient medium, and the other set of cells was incubated in zinc-sufficient medium for 4 days.
Cellular zinc assay. Cellular zinc level was assessed according to our previously established technique using Varian Spectr AA-40 flameless atomic absorption spectrophotometry with a Zeeman background corrector (Varian Instruments, Palo Alto, CA). All samples were analyzed against bovine liver standard containing known amounts of zinc in a matrix (National Bureau of Standards, Washington, DC).
Measurements of cytokines. The zinc-deficient and zinc-sufficient cells (2 x 106/ml) were stimulated with 5 ng/ml PMA and 10 µg/ml PHA for 6 h. The supernatants from the cultures were saved for cytokine assays.
The concentrations of IL-1, IL-2, IL-4, IL-6, IL-8, IL-10, TNF-
, and IFN-
in the supernatant were measured by ELISA, using a Quantikine human cytokine kit (R & D Systems, Minneapolis, MN).
Northern blot analysis. Northern blot analysis was used to determine the relative abundance of IL-1, IL-2, IL-8, TNF-
, and IFN-
mRNAs in cells exposed to different concentrations of zinc.
Total RNA was isolated from fresh cell samples (40 x 106 cells for each treatment) by using RNA STAT-60 (Tel-Test "B," Friendswood, TX), a phenol-guanidinium thiocyanate method (5). The suspended cells were homogenized in 1 ml of STAT-60 solution and stored for 5 min at room temperature. The homogenate was mixed rigorously with 0.2 ml of chloroform and stored for 23 min at room temperature. After centrifugation in a Beckman GS-6KR centrifuge (Beckman Instruments, Palo Alto, CA) at 18,000 g for 30 min, the aqueous solution was transferred to one diethyl pyrocarbonate (DEPC)-treated Eppendorf tube, mixed with 0.5 ml of isopropanol, and kept at room temperature for 10 min. After centrifugation in a clinical microcentrifuge for 15 min, the RNA pellets were washed with 1 ml of 75% ethanol, air-dried, and dissolved in 2040 µl of DEPC-treated deionized water. The total RNA was quantitated by an absorbance at 260 nm using a spectrophotometer.
RNA sample (20 µg) was mixed in 25 µl of sample buffer containing 20 mM MOPS, 5 mM sodium acetate, 1 mM EDTA (pH 5.57.0), 6.54% formaldehyde, and 50% deionized formamide. Next, RNA sample along with 5 µl of loading buffer (0.2% bromphenol blue, 50% glycerol, and 0.5 µg of ethidium bromide) was heated to 65°C for 10 min. The RNA was separated electrophoretically on a 1.1% RNA agarose gel for 2 h at 100 V using 1x MOPS buffer (containing 20 mM MOPS, 5 mM sodium acetate, and 1 mM EDTA, pH 7.8) as running buffer.
Intact RNA on the 1.1% RNA agarose gel was transferred to a Gene Screen Plus Hybridization Transfer Membrane (DuPont NEN Research Products, Boston, MA) in 10x SSC solution (1x SSC contains 16 mM sodium chloride and 15 mM sodium citrate) by capillary blot (28) overnight and fixed to the membrane at 120 µJ with a GS Gene Linker UV Chamber (Bio-Rad, Hercules, CA) for Northern hybridization.
Human IL-1, IL-2, IL-8, TNF-
, and IFN-
cDNAs (ATCC, Manassas, VA) were labeled with [
-32P]dATP (3,000 Ci/mmol; DuPont NEN Research Products) by the random oligo-primer method, using the Prime-Gene labeling system kit (Promega Technical, Madison, WI).
The labeling reaction was incubated at room temperature for 60 min. After incubation, the labeled cDNAs were purified for Northern hybridization by using Chroma Spin-10 columns (Clontech Lab, Palo Alto, CA).
The Northern hybridization was performed according to Church and Gilbert (6). The fixed RNA membranes were prehybridized in 10 ml of prehybridization buffer (containing 0.25 M Na2HPO4, pH 7.2, and 5% SDS) in an Autoblot Micro Hybridization Oven (Bellco Glass, Vineland, NJ) at 55°C for 60 min, followed by hybridization in 10 ml of prehybridization buffer and the labeled cDNA probe overnight. The hybridized membrane was washed with 20 mM Na2HPO4, pH 7.2, and 3% SDS at 55°C for 45 min two times, followed by two other washings with 20 mM Na2HPO4, pH 7.2, and 1% SDS at 55°C for 30 min. The blots were exposed to X-ray film at 70°C for 2472 h. The intensity of the gene expression on the autoradiographed films was measured in HUT-78 cells on a densitometer with a laser scanner (Molecular Dynamics, Sunnyvale, CA). The relative abundance of IL-1, IL-2, IL-8, TNF-
, and IFN-
mRNAs was calculated against the intensity of 18S ribosome cDNA, a gift from Dr. O. C. Ikonomov (Dept. of Neuroscience and Behavior, Medical School, Wayne State University, Detroit, MI; see Ref. 13).
mRNA and protein synthesis inhibitors study. To investigate whether zinc affects the stability of IL-1, IL-2, IL-4, IL-6, IL-8, IL-10, TNF-
, and IFN-
mRNAs, actinomycin D (18), an mRNA synthesis inhibitor, was used to block mRNA synthesis under different levels of zinc concentration. Cycloheximide (CHX; see Ref. 18), a protein synthesis inhibitor, was used to assess whether new protein synthesis is necessary for the expression of IL-2, IL-2 receptor, and
and
genes in zinc-deficient and zinc-sufficient cells.
Zinc-treated cells were preincubated either for 20 min with 10 µg/ml actinomycin D (Sigma Chemical) or for 1 h with 40 µg/ml CHX (Sigma Chemical) before PMA/PHA stimulation. The concentrations of IL-2 and soluble IL-2 receptor peptides were determined by ELISA. Northern blot analysis of IL-1, IL-2, IL-4, IL-6, IL-8, IL-10, TNF-
, and IFN-
mRNAs from each treatment sample were performed as described above.
Statistical analysis. Data were expressed as means ± SD by three separate experiments. The differences between zinc-deficient and zinc-sufficient groups were determined using the Student's t-test. P values <0.05 were considered to be statistically significant.
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RESULTS |
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Without PMA/PHA stimulation, zinc-deficient and zinc-sufficient HUT-78 cells produced very small amounts of IL-2, 8 ± 2 and 21 ± 4 pg/ml, respectively (Table 1). However, after 6 h of PMA/PHA stimulation, zinc-deficient and zinc-sufficient HUT-78 cells produced 1,752 ± 498 and 2,854 ± 456 pg/ml of IL-2 (P < 0.05), respectively. In the presence of actinomycin D, IL-2 production in the zinc-deficient and zinc-sufficient stimulated cells was reduced to 118 ± 58 and 132 ± 43 pg/ml (P > 0.05), respectively (Table 1). In the presence of CHX, the stimulated cells produced 87 ± 52 and 112 ± 73 pg/ml IL-2 in zinc-deficient and zinc-sufficient conditions (P > 0.05), respectively (Table 1).
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Data in Fig. 2A indicate that zinc deficiency decreased 60% the relative abundance of IL-2 mRNA after 6 h of PMA/PHA stimulation compared with zinc sufficiency. In the presence of actinomycin D, the level of IL-2 mRNA was not detectable in either zinc-defi-cient or zinc-sufficient stimulated cells by Northern blot analysis. The presence of CHX significantly reduced the level of IL-2 mRNA in both zinc-deficient and zinc-sufficient stimulated HUT-78 cells (Fig. 2A). There was no significant difference between the two groups of cells (P > 0.05).
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After PMA/PHA stimulation, zinc-deficient cells and zinc-sufficient cells produced 22.5 ± 8.6 and 44.3 ± 11.2 pg/ml IFN- cytokine, respectively (P < 0.05; Table 1). Both actinomycin D and CHX inhibited the production of IFN-
in HUT-78 cells. There was no significant difference in IFN-
production between zinc-deficient cells and zinc-sufficient cells in the presence of either actinomycin D or CHX (P > 0.05; Table 1). Zinc deficiency decreased 40% the relative abundance of IFN-
mRNA after 6 h of PMA/PHA stimulation compared with zinc sufficiency (Fig. 2B).
HUT-78 cells also produced IL-4 and IL-10 cytokines after 6 h of PMA/PHA stimulation (Table 1). Without PMA/PHA stimulation, zinc-deficient cells and zinc-sufficient cells produced 56 ± 25 and 73 ± 33 pg/ml IL-4 cytokine, respectively (P > 0.05; Table 1). With 6 h of PMA/PHA stimulation, zinc-deficient cells and zinc-sufficient cells produced 1,350 ± 245 and 1,552 ± 283 pg/ml IL-4 cytokine, respectively (P > 0.05). The presence of either actinomycin D or CHX significantly reduced the production of IL-4 cytokine in both groups of the PMA- and/or PHA-stimulated HUT-78 cells. However, there was no significant difference between the two groups of cells (P > 0.05).
Without PMA/PHA stimulation, zinc-deficient cells and zinc-sufficient cells produced 411 ± 221 and 730 ± 323 pg/ml IL-10 cytokine, respectively (P > 0.05; Table 1). After 6 h of PMA/PHA stimulation, zinc-deficient cells and zinc-sufficient cells produced 5,252 ± 1,725 and 5,556 ± 1,157 pg/ml IL-10 cytokine, respectively (P > 0.05). The presence of either actinomycin D or CHX significantly reduced the production of IL-10 cytokine in both groups of the PMA- and/or PHA-stimulated HUT-78 cells. However, there was no significant difference between zinc-deficient and zinc-sufficient conditions in the presence of either actinomycin D or CHX (P > 0.05; Table 1).
PMA/ionomycin stimulation induced the production of IL-2 and IFN- in D1.1 cells (Table 1). After 6 h of PMA/ionomycin stimulation, D1.1 cells produced 127 ± 29 and 192 ± 36 pg/ml IL-2 cytokine (P < 0.05) in zinc-deficient vs. zinc-sufficient cells, respectively (Table 1). Figure 3A shows the effect of zinc on IL-2 mRNA in D1.1 cells. Without stimulation, the level of IL-2 mRNA was not detectable by Northern analysis. After 6 h of PMA/ionomycin stimulation, D1.1 cells expressed IL-2 mRNA in both zinc-deficient and zinc-sufficient conditions. However, zinc deficiency decreased 50% the relative abundance of IL-2 mRNA after 6h of PMA/ionomycin stimulation compared with zinc sufficiency.
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Without PMA/ionomycin stimulation, zinc-deficient and zinc-sufficient D1.1 cells produced 6.9 and 8.1 pg/ml IFN-, respectively (P > 0.05; Table 1). After 6 h of PMA/ionomycin stimulation, zinc-deficient and zinc-sufficient D1.1 cells produced 83.1 ± 11 and 105.5 ± 14 pg/ml (P < 0.05) of IFN-
cytokine, respectively. We examined the effect of zinc on IFN-
mRNA in D1.1 cells. Figure 3B shows that zinc deficiency decreased 40% the relative abundance of IFN-
mRNA after6hof PMA/ionomycin stimulation compared with zinc suffi-ciency.
After 6 h of PMA stimulation, zinc-deficient and zinc-sufficient HL-60 cells produced 218 ± 50 and 58 ± 21 pg/ml TNF- cytokine (P < 0.05), respectively (Table 1). Either actinomycin D or CHX inhibited the production of TNF-
cytokine in both zinc-deficient and zinc-sufficient HL-60 cells after 6 h PMA stimulation (Table 1). There was no significant difference between the two groups of HL-60 cells (P > 0.05). Figure 4A shows that zinc-deficient conditions caused a 60% increase in the relative abundance of TNF-
mRNA after 6 h of PMA stimulation compared with zinc sufficiency. In the presence of actinomycin D, the level of TNF-
mRNA was not detectable in any stimulated cells by Northern blotting analysis (Fig. 4A).
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After 6 h of PMA stimulation, zinc-deficient and zinc-sufficient HL-60 cells produced 2.3 ± 0.4 and 1.1 ± 0.2 pg/ml IL-1, respectively (P < 0.05; Table 1). Either actinomycin D or CHX inhibited the production of IL-1
cytokine in both zinc-deficient and zinc-suffi-cient PMA-stimulated cells (Table 1). There was no significant difference between zinc-deficient and zinc-sufficient conditions (P > 0.05). We also examined the effect of zinc on IL-1
mRNA in HL-60 cells by Northern blot analysis. Figure 4B shows that zinc deficiency increased 50% the relative abundance of IL-1
mRNA after 6 h of PMA stimulation compared with zinc sufficiency. In the presence of either actinomycin D or CHX, the level of IL-1
mRNA was not detectable in either group of PMA-stimulated HL-60 cells by Northern blot analysis (Fig. 4B).
After 6 h of PMA stimulation, zinc-deficient and zinc-sufficient HL-60 cells produced 812 ± 114 and 63 ± 23 pg/ml IL-8 (P < 0.05), respectively (Table 1). In the presence of actinomycin D, both zinc-deficient and zinc-sufficient stimulated cells produced only 121 ± 35 and 89 ± 16 pg/ml (P > 0.05), respectively (Table 1). In the presence of CHX, the level of IL-8 produced by zinc-deficient and zinc-sufficient HL-60 cells was not detectable. The effect of zinc on IL-8 mRNA in HL-60 cells was examined by Northern blot analysis (Fig. 4C). The data show that zinc deficiency increased 50% the relative abundance of IL-8 mRNA after 6 h of PMA stimulation compared with zinc sufficiency. In the presence of either actinomycin D or CHX, the level of IL-8 mRNA was not detectable in any stimulated cells after 6h of PMA stimulation by Northern blot analysis.
Table 1 also shows that zinc-deficient and zinc-suffi-cient HL-60 cells produced 149 ± 46 and 172 ± 53 pg/ml soluble TNF receptor 1 (sTNF-r1; P > 0.05), respectively, after 6 h of PMA stimulation. In the presence of actinomycin D, the PMA-stimulated HL-60 cells produced 102 ± 34 and 98 ± 28 pg/ml sTNF-R1 in zinc-deficient and zinc-sufficient conditions (P > 0.05), respectively. In the presence of CHX, the PMA-stimulated HL-60 cells produced 79 ± 21 and 68 ± 37 pg/ml sTNF-R1 in zinc-deficient and zinc-sufficient media, respectively. There was no significant difference between the two groups of cells (P > 0.05).
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DISCUSSION |
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Actinomycin D, an mRNA synthesis inhibitor, significantly inhibited the level of IL-2 mRNA equally in both zinc-deficient and zinc-sufficient cells. The presence of actinomycin D might reflect the status of the half-life or stability of mRNAs. However, actinomycin D significantly inhibits IL-2 mRNA and production in stimulated cells, and there was no significant difference in IL-2 mRNA between zinc-deficient and zinc-sufficient conditions in the presence of actinomycin D. These data suggest that the decreased level of IL-2 mRNA in zinc-deficient cells might not be the result of the decreased stability of IL-2 mRNA affected by zinc-deficient conditions, and it is probably the result of the decreased gene expression of IL-2 in zinc-deficient cells. We have observed that zinc deficiency decreased the activation of zinc-dependent transcription factors for IL-2 gene expression, such as NF-B, activator protein-1, and surfactant protein-1 (23), and decreased the level of newly synthesized IL-2 mRNA in the nucleus of HUT-78 cells by nuclear run-on assay (24). Therefore, the decreased level of IL-2 mRNA is responsible for the decreased gene expression of IL-2, and the stability of IL-2 mRNA is not affected by zinc.
Zinc deficiency induced by an experimental diet in human subjects decreased the level of IFN-, and zinc supplementation to these zinc-deficient subjects reversed the level of IFN-
cytokine in PBMC after PHA stimulation (2). In this study, zinc-deficient HUT-78 (Th0) and D1.1 (Th1) cells exhibited decreased levels of IFN-
mRNA and cytokine compared with zinc-suffi-cient HUT-78 and D1.1 cells, which suggests that zinc may mediate the gene expression of IFN-
in Th0 and Th1 cells.
IL-4 and IL-10, mainly produced from Th2 cells, are involved in the hypersensitive delayed reaction. It has been reported that zinc may not be involved in regulation of function of Th2. The human Th2 cell line was not available. However, in this study, we found that HUT-78 cells produced IL-4 and IL-10 cytokines and that zinc did not affect the production of IL-4 and IL-10 in HUT-78 (Th0) cells.
Proinflammatory cytokines, IL-1, IL-8, and TNF-
, are primarily produced from the monocyte-macrophage lineage. An increased level of IL-1
cytokine has been observed in the human volunteers with zinc restriction and head and neck cancer patients with low zinc status (26, 2). Our present data demonstrate that zinc-defi-cient cells show increased levels of IL-1
, IL-8, and TNF-
cytokines and their mRNAs in HL-60 cells. Results from the actinomycin D study suggest that the increased levels of IL-1
, IL-8, and TNF-
mRNAs in zinc-deficient HL-60 cells are most likely the result of increased gene expression of IL-1
, IL-8, and TNF-
and not the result of altered stability of their mRNAs affected by zinc. One report demonstrated that zinc supplementation to zinc-deficient endothelial cells decreased NF-
B activity and also caused a marked attenuation in IL-8 expression in response to TNF-
stimulation (7), which suggests that the zinc-sufficient conditions decreased generation of IL-8 in endothelial cells.
The role of zinc in regulation of the gene expression of these proinflammatory cytokines has not yet been clearly defined. A novel zinc finger protein, A20, has been proposed to inhibit the activity of proinflammatory cytokines via TNF receptor-associated factors in cells (32, 12). A20 encoding 790 amino acids is originally identified as a TNF--responsive gene in endothelial cells. A20 is expressed in various types of cells in response to a number of stimuli, such as TNF-
, IL-1
, PMA, Epstein-Barr virus latent membrane protein, and other stimuli (8). A20 expression primarily protects cells from TNF-
-induced cytotoxicity in endothelial cells (17). The evidence shows that A20 protein inhibits the activation of NF-
B for IL-1
and TNF-
gene expression in endothelial cells (14). Thus A20 may play an important role in regulating the gene expression of IL-1
, IL-8, and TNF-
affected by zinc. Our unpublished data show that zinc increased the level of A20 protein and mRNA in PMA-stimulated HL-60 cells. Further studies regarding the mechanism of zinc action on these proinflammatory cytokines are being undertaken in our laboratory.
Evidence is accumulating that oxidative stress is an important contributing factor in several chronic human diseases, such as atherosclerosis and related vascular diseases, mutagenesis and cancer, neurodegenerative disorders, immunological diseases, and the aging process (4). In view of the fact that cytotoxic cytokines such as TNF-, IL-1
, and IL-8 generate increased amounts of free radicals (21, 3), our observation that zinc supplementation may reduce the gene expression of these cytotoxic cytokines suggests that zinc may have a therapeutic role in some of the diseases that are caused by oxidative stress. Our observation in this regard should stimulate further research.
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DISCLOSURES |
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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. Section 1734 solely to indicate this fact.
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REFERENCES |
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