© 2000 by European Society of Cardiology
Copyright © 2000, European Society of Cardiology
β2-Adrenergic receptor overexpression driven by 
-MHC promoter is downregulated in hypertrophied and failing myocardium
Baker Medical Research Institute, PO Box 6492, St. Kilda Road Central, Melbourne, Victoria 8008, Australia
* Corresponding author. Tel.: +613-95-224-399; fax: +613-95-211-362 xiaojun.du{at}baker.edu.au
Received 15 December 1999; accepted 24 February 2000
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Abstract |
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 Top Abstract 1 Introduction2 Methods3 Results4 DiscussionReferences |
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Objective: The
-myosin heavy chain (-MHC) promoter is frequently used to direct cardiac specific transgene expression. We studied whether transgene expression controlled by this promoter was altered under conditions of cardiac hypertrophy and failure. Methods: Transgenic (TG) mice overexpressing human β2-adrenergic receptors (β2AR) and wild type (WT) controls were subjected to thoracic aortic constriction (TAC) or sham operation and studied at 1, 3 and 8 weeks after surgery. Results: Sham operated TG mice had higher heart rates and left ventricular (LV) contractility than WT (all P<0.01), demonstrating enhanced βAR activation. TAC at 1, 3 and 8 weeks produced progressive LV hypertrophy which was similar between WT and TG mice. Evidence of heart failure was more marked in TG mice with a greater increase in weights of the right ventricle and lungs and a higher prevalence of atrial thrombus (P<0.05 in each case). In hypertrophied TG hearts, endogenous -MHC mRNA transcripts in LV were maintained at 1 and 3 weeks, but were reduced by approximately 40% relative to the sham-operated group at 8 weeks after TAC. Transgene expression, measured as human β2AR mRNA, was reduced by 45% at 1 and 3 weeks and by 70% at 8 weeks after TAC. β2AR binding sites were reduced by 35, 47 and 65%, respectively, at 1, 3 and 8 weeks. Conclusion: Cardiac hypertrophy and failure cause downregulation of the endogenous -MHC as well as cardiac specific overexpression of the transgene directed by an -MHC promoter.
KEYWORDS Gene expression; Heart failure; Hypertrophy; Receptors
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1 Introduction |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
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The use of genetic manipulation to produce knockout and transgenic (TG) animal models has provided a powerful tool for the study of disease processes such as cardiac hypertrophy and failure. To date, the
-MHC promoter [1] has been commonly used to direct cardiac-specific overexpression of a wide range of transgenes, including tumor necrosis factor [2,3], nerve growth factor [4], tropomodulin [5], sarcoplasmic reticulum Ca2+ ATPase [6], angiotensin II receptors [7], 1B-adrenergic receptors (AR) [8,9], β1 or β2-ARs [10,11], βAR kinase (βARK1) [12], and GTP binding proteins [13–16]. The expression of some of these proteins has been shown to lead directly to cardiac hypertrophy and failure [2,3,9,11,13–17], while others modify the development of these conditions [18–20].
In the rodent, the -MHC gene is expressed continuously in the atria through the development whereas the expression of β-MHC is restricted to the ventricles during this period [1,21]. While expression of β-MHC is turned off shortly after the birth, expression of -MHC gene in the ventricular myocardium is upregulated and remains to be the predominant isoform in adults [1,21]. Under conditions of aging, cardiac hypertrophy and failure, -MHC expression is downregulated [22–26]. Thus, expression of the -MHC gene is altered in a variety of physiological and disease states.
Cardiac hypertrophy and failure are frequently the focus of research involving transgenic models that utilize the -MHC promoter to direct cardiac specific expression. An essential aspect of TG models is the need to ensure a predictable extent of tissue specific expression of a transgene. Therefore, an important question arises as to whether transgene overexpression remains constant in such disease states and to what extent regulation occurs. We have investigated this by studying TG mice overexpressing human β2AR directed by an -MHC promoter [10]. Levels of -MHC and human β2AR mRNA as well as β2AR density were measured at various time points after pressure overload. For comparison, cardiac hypertrophy and function were similarly studied in WT animals.
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2 Methods |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
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2.1 Mice and transgene screening
Parent TG mice (TG4) were generated at the Howard Hughes Medical Institute, Duke University Medical Center [10]. Male TG mice were crossed with female F1 mice from C57BLxSJL strains. Genomic DNA was extracted from mouse tail biopsy and expression of the transgene in offspring was detected by Southern blot hybridization, using a 32P-labeled HincII fragment of the transgene construct [10]. Male and female animals of TG and WT littermates, 3–5 months of age, were used in this study. All experimental procedures were approved by the local Animal Experimentation Ethics Committee.
2.2 Microsurgery
Mice were anesthetised by intraperitoneal injection with a mixture of ketamine (8 mg/100 g), xylazine (2 mg/100 g), atropine (0.06 mg/100 g) and a pain reliever zenecarp (0.1 mg/100 g). Animals were intubated and ventilated. With the aid of a surgical microscope, a midline incision was made at the sternum and the aorta was dissected between the right innominate and the left carotid arteries. The thoracic aorta was then constricted (TAC) by 60–70% to a lumen size of 0.4 mm by tying the vessel with a probe, as described previously [20,27]. The probe was removed promptly after the tie was secured. Sham operated mice were subjected to similar surgery except for the constriction of the aorta.
2.3 Functional measurements
Cardiac function was assessed by placing a catheter into the right carotid artery (proximal to the constriction site) and then the left ventricle (LV), as described previously [20]. Mice were anesthetized by intraperitoneal injection with a mixture of pentobarbitone (8 mg/100 g) and atropine (0.12 mg/100 g). When an animal was found to be sick (body weight loss, labored breath, motionless, etc.), the dose of pentobarbitone was reduced to 3–4 mg/100 g. Mice were placed in supine position on a heating pad. A cervical incision was made and the right main carotid artery dissected. A micro-tipped transducer catheter (1.4 F, Millar Instrument Co.), with the frequency response flat to 10 kHz, was inserted into the artery and then advanced into the LV [20]. The aortic blood pressure, LV pressure and the maximal rate of increase or decay of LV pressure, dP/dtmax or dP/dtmin, were recorded. Heart rate was derived from pulse signals using a Labview data acquisition system.
2.4 Organ weights
After the completion of the functional measurements, mice were killed by pentobarbitone overdose. The chest was opened to determine whether pleural effusion was present before isolation of the heart. The heart was immersed in saline on ice. The LV, right ventricle and atria were separated and weighed. The LV was frozen in liquid nitrogen for extraction of total RNA. Chronic thrombus in the left atrium was determined, under surgical microscope, by its yellowish color and tight adhesion to the atrial wall. When a thrombus was present, the weight of the thrombus (10–70 mg) was subtracted from atrial weight. The lungs and liver were weighed and the tibial length was measured.
2.5 Solution hybridization/RNase protection analysis of -MHC and β2AR mRNA
RT-PCR amplification was used to generate cDNA fragments of human β2AR (nucleotides 2116–2532; GenBank accession number M15169
[GenBank]
) and murine -MHC (nucleotides 3227–3450; GenBank accession number M76601
[GenBank]
). β2AR and -MHC cDNA fragments were gel purified, ligated into pGEM-T (Promega) and sequenced to confirm the identity of the cloned fragments. These plasmids were then used to generate 32P-labeled cRNA probes for use in solution hybridization/RNase protection analysis as previously described [28]. A mouse GAPDH cDNA clone was similarly used as a control probe. Protected RNA hybrids were then separated on non-denaturing polyacrylamide gels and analyzed on a Fuji BAS-1000 phosphorimaging system.
2.6 β2AR binding assay
Mouse left ventricles were homogenized using a Potter Elvejheim homogenizer in a binding buffer (50 mmol/l Tris–HCl, 10 mmol/l MgSO4, pH 7.4) containing 0.25 mol/l sucrose. The homogenate was centrifuged at 1000xg for 10 min at 4°C and the supernatant was re-centrifuged at 30 000xg for 30 min at 4°C. The resulting pellet was washed once in cold binding buffer and finally resuspended in the binding buffer at a final protein concentration of 3 µg/ml. Protein concentration was determined using the Bradford method. The binding assays included incubating membrane proteins (0.3 µg) with 40 pmol/l [125I](–)iodocyanopindalol (NEN, 2200 Ci/mmol) for 1 h at varying concentrations of a selective antagonist ICI 118,551 (10–11 to 10–5 mol/l, Sigma) in a total volume of 200 µl. The assay was done in duplicates at 22°C. The samples were then filtered through Whatman GF/C filters, followed by washes with 16 ml of the binding buffer. The dried filters were counted in a 
-counter.
2.7 Statistics
Results have been expressed as mean±S.E.M. For parametric data, between group comparison was made by two way analysis of variance followed by Fisher's PLSD or by unpaired Student's t-test. Receptor radioligand binding data were analyzed using ALLFIT program providing ‘best fit’ values for receptor concentration and affinity [29]. Incidences of events were compared using Fisher's exact test.
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3 Results |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
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3.1 Outcome of surgery
A total of 114 mice were operated and ten were lost during the surgery. Of the remaining mice, 11 (three WT, eight TG) died at various times after surgery due to severe heart failure, indicated by postmortem findings (massive pleural effusion, severe lung congestion, chronic thrombus in the left atrium and cardiac dilatation). A total of 95 mice, 42 WT (18 males and 24 females) and 53 TG (23 males and 30 females), were studied at various time points (n=9-16 per group). Sham operated mice were similarly studied at various time points with results pooled to form a single group. All groups were matched for male/female ratio.
Our previous studies showed that in animals with TAC for 9 weeks pleural effusion and chronic atrial thrombus are closely associated with the development of heart failure [20]. In all groups with TAC, TG mice had higher incidences of pleural effusion (58.1%, 25/43 vs. 24.1%, 7/29) and atrial thrombus than WT mice (55.8%, 24/43 vs. 27.6%, 8/29, both P<0.02, Fig. 1). At the time of hemodynamic measurements, one WT (week 8) and 12 TG mice (one at week 1, three at week 3 and nine at week 8) died soon after intraperitoneal injection with a reduced dose of pentobarbitone (P=0.015 for the difference in the incidence between WT and TG mice, Fig. 1). Failure in catheterization occurred in eight animals. Therefore, functional data were obtained from 36 WT and 35 TG mice (n=10–11 for sham-operated and 6–10 for aortic constricted groups).
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P<0.05 vs. respective WT group.3.2 Organ weights
There was no difference in body weight between TG and WT groups at the time of surgery. At week 1 after TAC, WT and TG animals had reduced body weights, probably reflecting the insult of surgery. At weeks 3 and 8, only TG mice, but not WT mice, showed reduced body weight relative to sham-operated group (P<0.01, Fig. 2). Tibial length was similar in all groups (data not shown). For these reasons organ weights are expressed as net values.
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TAC resulted in significant and progressive increases in lung and heart weights in both WT and TG mice (Figs. 1 and 2
). LV weight increased progressively and in a similar manner in TG and WT animals following TAC, being already significantly greater at 1 week than in the corresponding sham-operated animals. Atrial and lung weights increased more rapidly and to a greater extent in TG animals than WT following TAC (Figs. 1 and 2). Thus RV, atrial and lung weights were already greater in TG than WT mice at week 1 following TAC (all P<0.05, Fig. 2). RV and atrial weights also increased to a greater degree at week 8 in TG animals (P<0.05). These changes in organ weights indicate the onset of LV failure resulting in congestion in the left atrium and lungs and, at a later stage, increased afterload to the RV. Liver weight was significantly lower in both groups at week 1 following surgery. This was reversed by week 3 and 8 in the WT group, but remained lower in the TG groups at week 3 and 8, probably reflecting the more severe hemodynamic disturbance in TG mice.
3.3 Hemodynamics
Hemodynamic changes following TAC are shown in Fig. 3. Systolic arterial pressure and pulse pressure proximal to the constriction site were significantly and similarly increased in all WT and TG groups relative to respective controls, indicating a similar degree of pressure overload. Heart rate and LV dP/dt were significantly greater in sham-operated TG animals than the corresponding WT group (P<0.001 in each case). The difference in heart rate was maintained following TAC (Fig. 3). dP/dt was reduced in TG mice at all time points following TAC compared with the sham-operated group (P<0.05). In contrast, in WT mice with TAC, LV dP/dt levels were increased at week 1 (P<0.01), maintained at week 3 but reduced at week 8 (P<0.05) compared with the sham-operated group (Fig. 3). Following TAC LV end-diastolic pressure (LVEDP) increased significantly in all TG groups but only in the week 8 WT group (Fig. 3). Since substantial number of TG mice died of severe heart failure after TAC, the levels of LV function measured from surviving TG mice actually overestimated the true inotropic state as a whole group.
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3.4 Expression of endogenous
-MHC mRNA and transgenic β2AR mRNAExpression of
-MHC mRNA was identical in WT and TG mice that were sham-operated (Fig. 4A). Following TAC, -MHC mRNA levels in LV myocardium from TG mice were not significantly altered on week 1 or week 3, but were reduced to 57% of those found in the sham-operated group by week 8 (P<0.01, Fig. 4A).
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A highly specific RNase protection assay was used to quantitate expression of transgenic human β2AR mRNA; endogenous murine β2AR mRNA transcripts were not detected under the assay conditions employed. Following TAC, transgenic human β2AR mRNA levels in LV myocardium were significantly reduced to approximately 60% of sham-operated TG hearts on weeks 1 and 3, and were further reduced to 29% of those found in the sham-operated group by week 8 (P<0.0001, Fig. 4B). Expression of GAPDH mRNA in the same LV RNA samples remained constant between WT and TG sham-operated groups and was not influenced by TAC (Fig. 4C).
3.5 β2AR binding
β2AR binding was assayed using [125I](–)iodocyanopindalol and receptor density was quantitated as binding sites with high affinity for the selective β2-antagonist ICI 118551. The concentration of endogenous β2ARs in WT hearts was 6±0.2 fmol/mg protein. β2AR density in sham-operated TG mice was 4460±810 fmol/mg protein. The concentration of LV β2ARs in TG mice was progressively reduced at weeks 1, 3 and 8 after TAC to 65% (P=0.16), 53% (P<0.05) and 35% (P<0.01), respectively, of the value of sham-operated group (Fig. 5). The degree of reduction in the receptor density correlated well with human β2 mRNA levels (Fig. 4) based on group means (r=0.979, P<0.01). The affinity of receptors for ICI 118,551 was 5.0±1.4 nM in sham-operated TG hearts and there was no significant change in the receptor affinity in hypertrophied hearts from TG mice (data not shown).
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4 Discussion |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
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Downregulation of
-MHC expression in favor of the β isoform has been reported in a number of models of cardiac hypertrophy and failure [23,25,26]. Since the -MHC has frequently been used to target cardiac specific expression of a variety of transgenes [2–16], we hypothesized that this downregulation would extend to transgenes driven by this promoter. The novel finding in experiments on TG mice with TAC is that expression of β2AR transgene under the -MHC promoter is progressively downregulated as early as week 1 after TAC, in parallel with the reduction in β2AR density. Falls in endogenous -MHC mRNA levels were also observed 8 weeks after TAC. As we are aware, this is the first study to describe the time-dependent downregulation in the expression of a transgene and endogenous -MHC, both are controlled by the same promoter, in hearts with induced hypertrophy and failure.
The use of TG models provides a means for studying the effects of overexpressing specific genes on physiological and disease processes. Correct interpretation of such experiments requires an understanding of potential interactions between the phenotype under investigation and degree of expression of the gene being manipulated. Because cardiac hypertrophy and failure are frequently the subject of studies involving transgenic models that utilize the -MHC promoter, our findings have important implications for a wide range of studies, specifically that transgene expression cannot be assumed to be constant in the presence of hypertrophy or heart failure. Full appreciation of the downregulated transgene overexpression is also necessary in determining the transgene dose–effect relationship.
Interestingly, a similar transgene downregulation has been observed in other two different TG models using the same promoter. In a recent study by us on TG mice expressing constitutively active mutant (CAM) 1BAR, pressure overload by TAC for 6 weeks resulted in a 
40% reduction in transgene mRNA levels (our unpublished data). Another group reported a downregulation of CAM Gq transgene expression with the development of cardiomyopathy [16]. These findings suggest that downregulation of transgene expression under the -MHC promoter is not strain-specific but may pertain to all TG lines using the same promoter. We observed an earlier reduction in mRNA levels of the transgene in comparison to that of endogenous -MHC. Such differences in the regulation of expression are likely to reflect differences in the stability and half-lives of the endogenous -MHC and β2AR transgene mRNA transcripts, however, the influence of additional regulatory elements not present in the transgene construct cannot be ruled out. In addition, it is possible that insertional effects may play a role to modulate the expression of the transgene, however, these effects are probably minimal given that similar data were recently obtained in mice expressing a CAM Gq [16] or CAM 1BAR genes (our unpublished data) under the control of the -MHC promoter.
Compared with sham-operated TG group, LV dP/dt levels in TG mice with TAC were significantly suppressed throughout the experiment period. This can be explained by a facilitated transition from compensatory hypertrophy to heart failure in TG mice and a partial loss of the β2AR density following the downregulation of the transgene may also play a role. The β2AR TG model is unique for its extremely active overexpression of the transgene with several hundred-fold increase in the receptor concentrations that fully activates cardiac function [10,19]. In the present study, a 65% reduction in β2AR density was observed 8 weeks after TAC. Despite this, TG mice maintained a functional phenotype (higher heart rates and LV dP/dt vs. respective WT groups). This contradiction may be explained by functional overestimation in TG group with TAC after substantial loss of mice with critical heart failure, and that β2AR concentrations in the remaining TG animals were still higher enough to maintain functional activation. A number of TG models that utilize the -MHC promoter result in more modest transgene expression at the protein level ranging from 1.75- to 40-fold [2–9,12–16]. Thus, partial loss of functional phenotypes is likely to occur with the development of cardiac hypertrophy and failure in TG models in which the -MHC promoter directs more modest transgene overexpression. A recent study provides a good example. Mice that express CAM Gq under the -MHC promoter develop cardiac dilatation, hypertrophy and fibrosis starting within a few weeks of age. Interestingly, there was a progressive decline in the mRNA transcripts of CAM Gq during 2–10 weeks of age as well as the expression of -MHC [16]. By week 10 the trangene products were undetectable. Thus, the maintained cardiac abnormalities in this model after the attenuation in CAM-Gq expression should be attributable to other factors rather than CAM-Gq [16].
Hemodynamics and organ weights were compared in WT and TG mice following TAC. In WT mice, LV contractility (dP/dt) was increased at week 1, maintained at control levels at week 3 but reduced at week 8 following TAC. Significant falls from control levels in the LV contractility were observed in TG mice with TAC at all time points studied. In addition, TG mice exhibited significantly greater increase in atrial and lung wet weights, higher incidence of pleural effusion and atrial thrombus as well as greater loss of animals due to critical heart failure. Thus, further to our recent finding of a more severe heart failure in TG mice following TAC for 9 weeks [20], these results demonstrate that an enhanced β2AR activity accelerates the functional deterioration and development of heart failure following TAC. One of the possible reasons for this deleterious outcome is that the substantial increase in heart rate in the TG animals accentuated the hemodynamic effects of the elevated afterload, causing early LV failure and a fall in contractility. Indeed, it is well known that rapid heart pacing can result in heart failure in large laboratory species and high heart rate levels could limit the diastolic filling and increase the energy expenditure of the myocardium. It should be pointed out that this adverse outcome was observed in the TG strain with a very high level of β2AR overexpression. Recent studies have shown that cardiac overexpression of β2AR at a lower level (30-fold) in Gq TG mice by cross-breeding restored ventricular dysfunction and inhibited hypertrophy whereas excessive overexpression (1000-fold) was deleterious [19].
Activation of βAR stimulates expression of a number of immediate early genes [30,31] and myocardial hypertrophy [32]. Although TG mice overexpressing β1AR and Gs in the heart developed hypertrophy and cardiomyopathy [17,19], TG mice with overexpression of β2AR apparently do not have a hypertrophic phenotype at least within the age studied, although expression of atrial natriuretic peptide is elevated [20]. In the present study, TAC resulted in time-dependent development of LV hypertrophy which was similar in WT and TG mice. Thus, an enhanced β2AR activity does not affect the extent of hypertrophy in vivo following pressure overload.
In conclusion, our results demonstrate downregulation of -MHC and the β2AR transgene using an -MHC promoter in murine hearts with hypertrophy and failure following pressure overload. Such downregulation is a time-dependent process leading to progressive and substantial reduction in β2AR density. Further, this study provides evidence for a facilitated transition from hypertrophy to heart failure in β2AR overexpressing mice subjected to pressure overload.
Time for primary review 26 days.
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Acknowledgements |
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We are grateful to Dr R.J. Lefkowitz for providing the transgenic line. This work was supported by the National Health and Medical Research Council of Australia, and a grant from Merck-Sharp and Dohme Research Foundation (Australia). We thank the staff at Biological Research Unit for their help in the mouse breeding program and Brian Jones for help in imaging. Dr Sheridan thanks the Imperial College, London, and the Baker Institute, Melbourne, for the support during his sabbatical leave.
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Notes |
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1 Present address: Imperial College School of Medicine, St. Mary's Hospital, London W2 1NY, UK.
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H. Kiriazis, Y. Sato, V. J Kadambi, A. G Schmidt, M. J Gerst, B. D Hoit, and E. G Kranias Hypertrophy and functional alterations in hyperdynamic phospholamban-knockout mouse hearts under chronic aortic stenosis Cardiovasc Res, February 1, 2002; 53(2): 372 - 381. [Abstract] [Full Text] [PDF]
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X.-J. Du Sympathoadrenergic mechanisms in functional regulation and development of cardiac hypertrophy and failure: findings from genetically engineered mice Cardiovasc Res, June 1, 2001; 50(3): 443 - 453. [Full Text] [PDF]
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K.-D. Schluter, X.-J. Du, D. J. Autelitano, R. Dilley, B.-H. Wang, A. M. Dart, and E. A. Woodcock {beta}2-Adrenergic Receptor Overexpression Exacerbates Development of Heart Failure After Aortic Stenosis Response Circulation, January 16, 2001; 103 (2): e11 - e11. [Full Text] [PDF]
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