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CARDIOVASCULAR
Departments of Biochemistry and Molecular Biology and Medicine, Division of Experimental Medicine, George Washington University Medical Center, Washington DC
Received October 26, 2005; accepted February 1, 2006.
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Abstract |
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-receptor blockers on iron overload and oxidative stress in endothelial cells (ECs) was assessed. Confluent bovine ECs were loaded with iron dextran (15 µM) for 24 h and then exposed to dihydroxyfumarate (DHF), a source of reactive oxygen species, for up to 2 h. Intracellular oxidant formation, monitored by fluorescence of 2',7'-dichlorofluorescin (DCF; 30 µM), increased and peaked at 30 min; total glutathione decreased by 52 ± 5% (p < 0.01) at 60 min. When the ECs were pretreated 30 min before iron loading with 1.25 to 10 µM D-propranolol, glutathione losses were attenuated 15 to 80%, with EC50 = 3.1 µM. D-Propranolol partially inhibited the DCF intensity increase, but atenolol up to 10 µM was ineffective. At 2 h, caspase 3 activity was elevated 3.2 ± 0.3-fold (p < 0.01) in the iron-loaded and DHF-treated ECs, and cell survival, determined 24 h later, decreased 47 ± 6% (p < 0.01). Ten micromoles of D-propranolol suppressed the caspase 3 activation by 63% (p < 0.05) and preserved cell survival back to 88% of control (p < 0.01). In separate experiments, 24-h iron loading resulted in a 3.6 ± 0.8-fold increase in total EC iron determined by atomic absorption spectroscopy; D-propranolol at 5 µM reduced this increase to 1.5 ± 0.4-fold (p < 0.01) of controls. Microscopic observation by Perls' staining revealed that the excessive iron accumulated in vesicular endosomal/lysosomal structures, which were substantially diminished by D-propranolol. We previously showed that propranolol could readily concentrate into the lysosomes and raise the intralysosomal pH; it is suggested that the lysosomotropic properties of D-propranolol retarded the EC iron accumulation and thereby conferred the protective effects against iron load-mediated cytotoxicity.
-adrenergic receptor blockers (-blockers) are known to possess varying degrees of membrane-partitioning properties that may bear potential biological significance (Pruett et al., 1980
; Herbette et al., 1983). We reported that the more lipophilic -blockers, such as propranolol, pindolol, and metoprolol, exhibit varying degrees of membrane antioxidant activity, which seems to correlate with their membrane partitioning property (Mak and Weglicki, 1988; Weglicki et al., 1990). Propranolol is a basic amphiphilic amine and is known to accumulate in various tissues (Bianchetti et al., 1980; Rodgers et al., 2005). At the cell level, propranolol is able to accumulate in various cell types up to a 1000-fold greater than the surrounding medium concentration (Cramb, 1986). In addition, this uptake process is nonstereoselective, and much of the intracellular accumulation is compartmentalized to the lysosome (Cramb, 1986). Red cells and platelets both lack lysosomes and did not take up significant levels of propranolol. Cardiac myocytes only take up propranolol slowly, perhaps because of the lower density of lysosomes (Cramb, 1986), but endothelial cells are more enriched in lysosomes. Recently, we observed that fluorescent-labeled propranolol could accumulate readily into the lysosomes of cultured endothelial cells (Dickens et al., 2002), but the potential biological consequence of this lysosomotropic action of propranolol remained unclear.
Because the lysosome is an important storage site of excess intracellular iron (Brun and Brunk, 1970), we wondered whether this might promote iron-mediated oxidative cytotoxicity. Iron is initially stored intracellularly in ferritin, a protein whose synthesis is induced by the influx of iron. Thereafter, iron is sequestered in acidic compartments (lysosomes) as iron-loaded ferritin, which is further degraded into hemosiderin (Brun and Brunk, 1970). Experimental evidence shows that iron loading in hepatocytes results in increased iron in the lysosomal compartment (LeSage et al., 1986). Recently, it has been suggested that the major pool of low-molecular weight iron, which is redox-active, resides within the lysosome (Yu et al., 2003). Presumably, during iron-overload disorder, the iron-binding sites of the lysosomal ferritin/hemosiderin are more saturated, thus leading to an increase in lysosomal content of redox-active iron available to amplify oxidant injury to the cell. To address this specific inquiry, the present study was designed to assess: 1) whether prior treatment of cultured endothelial cells with D-propranolol would attenuate iron overload-mediated cell injury in the presence of oxyradicals, and 2) whether such effects might be attributable to its influence on iron accumulation in the lysosome.
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Materials and Methods |
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; Mak and Weglicki, 2004). Confluent cells (>90%) were loaded with 15 µM iron dextran in 2% calf serum for 24 h before experimentation. When the effects of D-propranolol or atenolol were studied, the cells were pre-incubated with the selected drugs at different concentrations (1.25–10 µM) for 30 min before the iron dextran loading. Control samples included incubation of dextran alone without iron.
Measurement of Intracellular Oxidant Generation
Intracellular generation of reactive oxygen species was determined by 2',7'-dichlorofluorescin (DCF) as described previously (Dickens et al., 1992; Wiles et al., 1997). The iron-loaded endothelial cells with or without drug pretreatment in 24-well plates were labeled for 30 min with DCF-DA probe (30 µM) in a balanced salt buffer containing 10 mM glucose, pH 7.2. At the end of the labeling, the cells were washed 2x with the balanced salt buffer to remove residual DCF-DA. All samples were then replaced with fresh buffer (as above, containing neither iron nor any drug) and subjected to free radical exposure generated from 0.83 mM dihydroxyfumarate (DHF) for up to 120 min. The primary oxygen free radical generated from DHF is superoxide anion (Goscin and Fridovich, 1972; Mak and Weglicki, 1994). Under these conditions, the initial rate of superoxide anions generated (determined by superoxide dismutase-inhibitable cytochrome c reduction) was 1.8 ± 0.30 nmol/min/ml. After 1 h of incubation, the rate decreased to 1.1 ± 0.26 nmol/min/ml (means of 4 ± S.D.). These rates of superoxide generation were not affected by the drug and/or iron treatments of the cells. After the addition of DHF, the time-dependent increases in the cellular DCF fluorescence intensity were measured by using a CytoFluor 2350 fluorescent plate reader (excitation 485/emission 530; Millipore Corp., Bedford, MA) with the intensity setting at level 2.
Measurements for Endothelial Glutathione, Caspase 3, and Cell Survival
Glutathione. For changes in the endothelial glutathione content, the cells were plated in six-well plates; total cellular glutathione as well as reduced and oxidized glutathione (GSH and GSSG, respectively) were determined by the established enzymatic "cyclic method" using glutathione reductase (Mak and Weglicki, 1994, 2004).
Caspase 3 Activity. After 2 h of free radical exposure, the cells (cultured in T-75 flasks) with different treatments were washed twice with PBS and lysed on ice with 25 mM HEPES buffer (pH 7.4) containing 2 mM EDTA, 0.1% CHAPS, and 5 mM dithiothreitol. The caspase 3 activity in the 16,000g cytosolic supernatant, the volume of which was adjusted to 200 µl per 3 to 5 x 106 cells, was determined by the Sigma's Caspase 3 Colorimetric Assay Kit using N-acetyl-Asp-Glu-Val-Asp-p-nitroanilide as the substrate. Ten microliters of the lysate was used for each assay sample, which was incubated for 2 h at 37°; p-nitroanilide released was measured at 405 nm. Protein determination was performed by using the Bradford reagent.
Cell Survival. Cell survival was determined by the colorimetric MTT assay 24 h later as described previously (Mak and Weglicki, 1994, 2004). Briefly, at the end of oxidant stress, all samples were replaced with fresh normal growth medium and returned to the incubation chamber. Twenty-four hours later, all samples were quantified for viable cells by the tetrazolium substrate MTT using the test wavelength of 570 nm and a reference one of 700 nm.
Measurement of Iron Content and Localization
Intracellular iron localization in isolated endothelial cells from different treatments was performed by Perls' staining according to Luna (1968). Briefly, endothelial cells were washed, resuspended in PBS, and spread onto microscopic slides to dry at room temperature. Cells were fixed in formalin vapors for 30 min in the closed chamber and air-dried. The staining for iron followed the Perls' protocol using 10% potassium ferrocyanide solution alone for 5 min and then mixed for 30 min (1:1) with 10% hydrochloric acid (Luna, 1968). Counterstained in nuclear fast red solution for 5 min, mounted preparations were viewed at 40 and 100x using a bright field microscope (Olympus; Optical Elements Corp., Dulles, VA), and multiple microphotographs were taken with a digital camera (Evolution Color MP; Media Cybernetics, Silver Spring, MD).
Total endothelial cell iron content was determined by atomic absorption (AA) flame emission spectroscopy using a Shimadzu 6200 AA spectrometer according to the procedure of Kreeftenberg et al. (1984). Briefly, cells grown in T-75 flasks ± drug treatment ± 24-h iron loading were isolated and washed 2x with PBS and suspended in 200-µl aliquots, which were then digested with 0.5 ml of destruction solution (perchloric acid/nitric acid, in a ratio of 4:1) overnight at 60°C (Kreeftenberg et al., 1984). The final sample aliquots were diluted 5x with distilled water, and the iron content was quantitated by AA analysis, with known iron reference standards in identical solution as for the experimental samples.
Statistical Analysis
All experiments were performed at least four times, and data were presented as means ± S.D. Statistical comparisons were performed by Student's t test when only two treatment groups were compared. Selected data were analyzed by one-way analysis of variance followed by a multiple comparison with a Tukey's test (SigmaStat for Windows, version 2.03, 1997; SPSS Inc., Chicago, IL) as described previously (Mak and Weglicki, 2004).
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Results |
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; Wiles et al., 1997). The probe DCF-DA would be taken up by the cells and deacetylated intracellularly to the nonfluorescent DCF, which reacts semiquantitatively with either oxidative species or peroxides to generate fluorescent 2,7-dichlorofluorescein within the cells. In the present study, iron loading for 24 h resulted in relatively low levels of DCF fluorescence, which is slightly higher than the control cells without iron loading [155 ± 35 versus 121 ± 18 RFU (relative fluorescence units)]. However, when the iron-loaded cells were exposed to DHF, the DCF intensity increased immediately and then appeared to peak at 30 min to 365 ± 39 RFU; afterward, the intensity leveled off and declined somewhat after 60 min (Fig. 1A). Without iron loading, exposure of the cells to DHF only resulted in a moderate increase in the DCF intensity (179 ± 22 RFU at 30 min; Fig. 1A). In separate experiments, adding superoxide dismutase (50–100 units/ml) to the medium had no significant effect on the DCF development; however, when catalase (50 units/ml) was added, the induced DCF intensity was inhibited >85%, suggesting that it is the hydrogen peroxide (H2O2) derived from DHF is the critical species promoting the oxidative stress. Both superoxide anions and H2O2 are considered to be only mildly oxidative; however, H2O2 is more permeable to the cell membrane. Once in the cell, H2O2 may react with redoxactive iron, resulting in the formation of more active oxyradicals (e.g., hydroxyl radicals and iron-oxygen complexes) that can generate additional oxidized species (such as alkoxyl and peroxyl radicals). DCF is relatively nonspecific and can react with H2O2, hydroxyl, alkoxyl, and peroxyl radicals. The elevation in DCF intensity in the iron-loaded cells might be interpreted as an increase in the sum total of all these oxidative species within the cells. Without iron loading, DHF only induced modest levels of DCF intensity in the EC, suggesting that the availability of intracellular redox iron was limited.
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The effects of D-propranolol and atenolol were assessed at the 30-min point of peak DCF fluorescence. Data from Fig. 1B indicate that D-propranolol at 2.5 to 10 µM partially attenuated the DCF intensity; significant effects were achieved by 5 and 10 µM; at 10 µM the DCF relative intensity was lowered to 192 ± 33 (p < 0.01). For comparison, atenolol at the same concentration range did not provided significant inhibition (Fig. 1B).
We have previously demonstrated that the loss in total glutathione is a sensitive indicator of acute endothelial oxidative stress (Mak et al., 1992; Mak and Weglicki, 1994). In the present study, iron loading for 24 h alone resulted in no change in the level of glutathione compared with the normal controls. However, when the iron-loaded cells were exposed to oxyradicals generated from DHF for 60 min, a 53% decrease in total glutathione occurred (Fig. 2). Because >95% of the total glutathione in all samples was in the reduced (GSH) form, the GSH decrease essentially reflected the depletion. Without iron loading, exposure of the cells to DHF alone only resulted in a 20 ± 4% decrease of the glutathione (data not shown). The ability of D-propranolol and atenolol to prevent the loss of cellular glutathione was compared. As represented by Fig. 2, D-propranolol provided dose-dependent attenuation of the GSH loss induced after the addition of DHF. Five micromoles of D-propranolol preserved GSH to approximately 80% of control. With additional concentrations, the EC50 for D-propranolol to attenuated GSH loss is 3.2 µM. Atenolol at the highest level (10 µM) only provided a modest and insignificant attenuation.
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It has been suggested that iron overload may promote apoptosis both in vivo (Oudit et al., 2004) and in vitro (Tampo et al., 2003). Because caspase 3 activation is considered the ultimate step leading to apoptotic cell death, the effect of iron loading with or without subsequent oxyradical stress on caspase 3 activity was examined. The initial time course study indicated that significant activation of caspase 3 activity occurred 2 h after the oxidant stress. As represented by Fig. 3A, iron loading alone only resulted in a modest but insignificant increase in the caspase 3 activity. However, after 2 h of exposure to the oxyradicals, a 3-fold elevation (p < 0.05) of caspase 3 activity was evident; D-propranolol, but not atenolol, produced significant attenuation of the activation (Fig. 3A). Cell survival was determined 24 h later by the MTT assay. Iron loading and superoxide exposure resulted in a 47% reduction of cell viability, and treatment with 
5 µM D-propranolol significantly preserved cell survival (Fig. 3B); atenolol was without significant effect.
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2.5 µM. In data not shown, we found that D,L-propranolol was equally effective in reducing the endothelial iron accumulated. To further approach the mechanism by which propranolol may block iron uptake, we have assessed the effects of two additional agents: Trolox (water-soluble vitamin E; Hoffman-La Roche, Nutley, NJ) and methylamine. Under our conditions, Trolox at 10 µM had minimal effect (<10% effect) in blocking the iron accumulation as assessed quantitatively by AA. Because Trolox is >10-fold more potent than propranolol as a membrane antioxidant, the data suggest that the antioxidant activity of propranolol plays a minor role in reducing the iron accumulation, although Trolox (10 µM) did provide partial attenuation of the losses of GSH and cell survival (approximately 30% effect) induced by DHF, possibly attributable to the antioxidant activity of vitamin E. When the cells were incubated with methylamine (1–3 mM), a well established lysosome-alkalinizing agent at high concentrations (Cramb, 1986), iron accumulation was blocked up to 90% (determined by AA). Nevertheless, the iron-related cytoprotective effect of methylamine could not be accurately ascertained at such high concentrations required. Methylamine alone was toxic to the cells (>35%); cell death occurred at 24 h. The combined data lend further support to the notion that the potent lysosomotropic and organelle alkalinization properties of D-propranolol conferred the attenuation of iron accumulation. As opposed to propranolol, atenolol at 5 µM had no effect, but at 10 µM, a modest inhibition (22%, p < 0.05) of iron accumulation was achieved (Fig. 4), perhaps because of some residual lysosomotropic activity.
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). In contrast with the normal control samples (Fig. 5A), 24 h of iron loading resulted in a prominent staining of intracellular iron; the positive iron granules seemed to colocalize with endosomal/lysosomal vesicles (Fig. 5B). For comparison, such intensity of iron staining for the cells pretreated with D-propranolol (10 µM) before iron loading was substantially attenuated (Fig. 5C). In data not shown, no appreciable effect was achieved by atenolol (10 µM).
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Discussion |
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; Mak and Weglicki, 2004). In those studies, hydroxyl radicals were generated extracellularly by an iron-dependent, superoxide anion-driven (Fenton reaction-like) system. Under these conditions, the effective protective concentrations of propranolol were in high micromolar levels (25–200 µM), and the EC50 for the endothelial cells was 
50 µM (Mak and Weglicki, 2004). The presumed mechanism of protection was a direct membrane "chain-breaking" action of propranolol, mediated by its membrane partitioning and intrinsic antioxidant properties (Mak et al., 1990). In the present study, the endothelial cells were iron-loaded overnight in the absence or presence of D-propranolol. The cells were then exposed to a superoxide-generating system (from DHF) in the absence of extracellular iron. Severe oxidative stress indices were induced in the cells loaded with iron alone but were significantly attenuated by D-propranolol. Because superoxide alone or its dismutated molecule, H2O2, is considered to be only mildly oxidative, the presumed damaging oxidative species (such as hydroxyl radicals) must be formed intracellularly between H2O2 and redox-active iron. The apparent protective effect of D-propranolol is related to its ability to block iron accumulation rather than to a direct antioxidant action as described previously. Therefore, the mode of protective action provided by propranolol in the present study is clearly different; the cytoprotective efficacy is also more potent: the estimated EC50 is approximately 3 µM (Figs. 2 and 3), which is closely related to its efficacy to attenuate cellular iron accumulation (Fig. 4).
Primary (hereditary) and secondary transfusion iron overload due to treatment of anemia are now recognized as growing global epidemics (Weatherall and Clegg, 1996; Andrews, 1999; Siah et al., 2005). Parenteral iron therapy is also often used to decrease the need to use red cell transfusions in patients with iron-deficiency anemia (Silverstein and Rodgers, 2004). Iron dextran is thought to be a relatively safe parenteral iron product because of its stable chemistry (Silverstein and Rodgers, 2004). However, animal studies have shown that its prolonged use causes tissue iron accumulation and results in impairment of the cardiac function (Voogd et al., 1992; Voogd, 1993; Yang et al., 2003). Histological evidence following iron loading in the rat shows that the enrichment of iron in the heart occurred prominently in the endothelial cells and to some degree in vascular pericytes (Voogd et al., 1992; Voogd, 1993). Our present study demonstrated that exposure of cultured endothelial cells to a relatively low concentration of iron dextran, which is within the clinical range reported for patients receiving the iron supplementation (Silverstein and Rodgers, 2004), resulted in a 3- to 4-fold increase in total cellular iron. The morphological analysis by Perls' staining suggests that much of the excessive iron is stored in the lysosomal/endosomal vesicles. Because the lysosome may provide the major intracellular source of redoxactive iron (Yu et al., 2003), increased accumulation of iron in this organelle would presumably enhance this pool of iron available to synergize with oxidants to promote cell injury. Increased oxidants may be generated extracellularly from activated white cells or intracellularly from mitochondria or from drug metabolites. In our case, we used DHF as a convenient source of superoxide anions generated to mimic extracellular oxidant stress (Mak et al., 1992, 1995; Mak and Weglicki, 1994, 2004; Dickens et al., 2002). In the present study, iron loading alone did not seem to cause significant cytotoxic effect. However, when exposed to the oxyradicals, the iron-loaded endothelial cells revealed enhanced levels of oxidant formation (DCF fluorescence) followed by significant depletion of glutathione, increased apoptotic activity, and loss of cell viability. A previous study by others (Lin et al., 1983) using the Chinese hamster cell line V79 reported that increased cellular uptake of iron derived from iron dextran in the cultured medium enhanced the bleomycin toxicity; it was presumed that the oxidant source was generated intracellularly from bleomycin (Lin et al., 1983). Both cases support the notion that the cultured cells exposed to extracellular iron dextran resulted in elevated intracellular redox-active iron, which in turn synergized with oxidants to promote cell death.
We have shown that D-propranolol at low micromolar concentrations can provide effective cytoprotective effects against endothelial iron loading and related cytotoxicity. Although the pharmacological and physiochemical properties of propranolol have been extensively studied (Pruett et al., 1980; Herbette et al., 1983; Nies, 1990), to our knowledge this represents the first report that propranolol is able to block iron accumulation in endothelial cells. However, the molecular events that enable D-propranolol to block iron uptake/accumulation remain unclear. Because transferrin is absent in the extracellular medium, it is presumed that the iron uptake is mediated through a metal iron carrier system similar to the divalent metal transporter-1 described for uptake of nontransferrin-bound iron (Burdo et al., 2001). The possibility of transport by direct endocytosis of iron dextran has also been suggested (Jonas and Riley, 1991). The two processes may not be mutually exclusive because it has been observed that uptake of nontransferrin-bound iron by the divalent metal transporter-1 also involves endocytotic steps shared by the transferrin receptor pathway for the uptake of iron transferrin (Chua et al., 2004). In our previous work (Dickens et al., 2002), we found that propranolol can concentrate readily into the lysosomes of the cultured endothelial cells used in the present study. Because the cellular uptake of iron involves endocytosis, one may speculate that the presence of propranolol, which raises the pH in the lysosomes/endosomes, may interfere with this process. In addition, propranolol-loaded lysosomes may be unable to perform their normal function of intracellular internalization of iron-loaded ferritin. The net result would be a substantial reduction in the iron accumulated in the lysosomes. To support such a scenario, the morphological images for the iron-loaded samples reveal the prominent presence of cytosolic granular aggregates (by Perls' staining), which are almost absent in the D-propranolol (10 µM)-treated cells. However, because dual staining with a lysosomal marker is difficult and was not performed, one may only speculate that the granular aggregates colocalize with the lysosomes (Fig. 5B). In addition, others have indicated that the release of redox-active iron from lysosomes is acid pH-dependent (Newman et al., 1994); D-propranolol would also prevent this release by raising the lysosomal pH. Because D,L-propranolol was found equally effective, the effects of D-propranolol described here were probably mediated by its lysosomotropic property, which is nonstereospecific. For comparison, atenolol, which displayed minimal lysosomotropic activity (Cramb, 1986), was found largely ineffective within the same concentration range used for D-propranolol.
In summary, D-propranolol at low micromolar concentration was able to attenuate iron overload-mediated endothelial cell oxidative injury; its effects on DCF intensity, glutathione loss, increased apoptotic activity, and cell death seem to be secondary to its primary action of blocking iron accumulation. We tentatively conclude that the primary effect was conferred by its lysosomotropic properties. We also speculate that the low effective levels may bear potential pharmacological relevance because it was demonstrated that the plasma propranolol levels could reach 10–6 M range in patients receiving high doses of D,L-propranolol (Walle et al., 1980). Because D-propranolol lacks -receptor blockade activity, one may assume that a much higher dosage of the drug can be clinically tolerated. Thus, although the exact molecular interaction(s) remain to be elucidated, we submit that this dramatic ability of D-propranolol to prevent iron accumulation deserves further exploration for its use as a potential adjunctive therapeutic agent in vivo for iron overload disorders.
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Acknowledgements |
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Footnotes |
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ABBREVIATIONS: MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; DCF, 2',7'-dichlorofluorescin; DA, diacetate; DHF, dihydroxyfumarate; GSH, reduced glutathione; EC, endothelial cell; PBS, phosphate-buffered saline; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; AA, atomic absorption; RFU, relative fluorescence unit.
Address correspondence to: Dr. I. Tong Mak, Dept. of Biochemistry and Molecular Biology, Division of Experimental Medicine, George Washington University Medical Center, 2300 Eye Street, N.W. Ross Hall, Rm 443, Washington DC 20037. E-mail: itmak{at}gwu.edu
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