© 1995 American Heart Association, Inc.
Articles |
Oxidized Low-Density Lipoproteins Facilitate Leukocyte Adhesion to Aortic Intima Without Affecting Endothelium-Dependent Relaxation
Role of P-Selectin
From the Departments of Medicine (B.Y., J.L.M.) and Pathology (S.K., J.B.H., C.S.), University of Florida College of Medicine, and the Veterans Affairs Medical Center, Gainesville, Fla.
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Abstract |
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Abstract Inflammatory cell deposition in atherosclerotic blood vessels has been thought to relate to loss of endothelium-derived nitric oxide (NO). To examine whether cell deposition correlates temporally with the loss of NO activity, rat aortic rings were incubated with buffer, native LDL (n-LDL), oxidized LDL (ox-LDL), or the endothelium-derived relaxing factor synthase inhibitor N
-nitro-L-arginine methyl ester
(L-NAME) for 2 hours, and vascular contractile response to
norepinephrine and relaxant response to acetylcholine,
thrombin, and calcium ionophore A23,187 were examined. Thereafter, the
rings were exposed to biotin-fluorescein
isothiocyanate-labeled fluorescent or unlabeled leukocytes
for 30 minutes. Cell adhesion was quantitated by fluorescent
microscopy as well as by scanning electron microscopy. Incubation with
n-LDL or ox-LDL did not affect either the contractile or the relaxant
response of rings. However, leukocyte adhesion increased markedly in
all ox-LDL–treated rings but not in those treated with n-LDL. Thus,
leukocyte adhesion occurred independent of NO activity. In keeping with
this concept, pretreatment of rings with the NO precursor
L-arginine failed to influence leukocyte adhesion to rings
incubated with ox-LDL. Treatment of rings with L-NAME also
resulted in adhesion of a large number of leukocytes. Furthermore, all
rings treated with ox-LDL or L-NAME demonstrated marked
expression of P-selectin leukocyte adhesion molecules, determined by
immunohistochemistry. Pretreatment of rings with the P-selectin
blocking antibody PB1.3 markedly decreased deposition of leukocytes in
rings exposed to ox-LDL. These data show that cell adhesion to vascular
intima exposed to ox-LDL shows no temporal relation with attenuation of
NO activity, although inhibition of NO synthesis leads to leukocyte
deposition. P-selectin expression on vascular rings exposed to ox-LDL
appears to be the basis of leukocyte deposition.
Key Words: leukocytes • low-density lipoproteins • nitric oxide
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Introduction |
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Elevated serum cholesterol level, especially a high level of LDL, is an important factor in atherogenesis.1 2 3 Studies by Steinberg and coworkers4 5 indicate that LDL undergoes oxidative modification when incubated in vitro with endothelial cells, smooth muscle cells, or macrophages or when exposed to solutions containing Cu2+. They demonstrated that ox-LDL is rapidly taken up by monocytes/macrophages, transforming them into "foam" cells, which are essential components of arterial atherosclerotic lesions. Nilsson et al6 also reported that ox-LDL is a key factor in the pathogenesis of atherosclerosis.
Accumulation of monocytes/macrophages in the subendothelial space has been clearly demonstrated to be an early event in atherosclerosis.4 5 Lehr et al7 8 9 reported that intravenous administration of ox-LDL results in an increase in leukocyte adherence to the vascular endothelium. Jeng et al10 showed that incubation of endothelial cells with ox-LDL enhances monocyte binding to the endothelial cells. The enhanced leukocyte adhesion to the vascular endothelium has been variably reported to be due to release of platelet-activating factor,7 leukotrienes,9 and chemotactic factors11 and expression of platelet-activating factor receptors7 and leukocyte adhesion molecules.8 10
There are also reports on the critical role of NO in the regulation of leukocyte adhesion to endothelium.12 13 Provost et al12 reported that endothelium-derived NO attenuates leukocyte adhesion to endothelium under arterial flow conditions. Kurose et al13 demonstrated that ischemia-reperfusion results in leukocyte adhesion to endothelial cells via a decrease in NO synthesis/activity in rat mesentery, which can be prevented by superfusion with NO donors. While hyperlipidemia14 and ox-LDL15 have been reported to impair endothelium-dependent vasorelaxation, data on the correlation between ox-LDL, NO activity, and leukocyte deposition are not available. This study was designed to address this issue.
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Methods |
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Preparation of Aortic Rings
Male Sprague-Dawley rats (average weight, 250 g) were anesthetized with sodium pentobarbital (50 mg/kg IP). Blood was drawn from the carotid artery into 3.8% sodium citrate for separation of leukocytes. The thoracic aortas were quickly removed, placed in oxygen-saturated (95% O2+5% CO2) Krebs-Ringer buffer (composition in mmol/L: NaCl 118, KCl 4.7, CaCl2 2.5, KH2PO4 1.2, MgCl2 1.2, NaHCO3 12.5, Na-EDTA 0.01, and glucose 11.1, pH 7.4), cleaned of fat and loose connective tissue, and cut into 4- to 5-mm rings. Care was taken to avoid any unnecessary manipulation of vessels. The rings were then mounted onto wire stirrups connected to force transducers (Kistler Morse) and suspended in custom-designed tissue-organ baths filled with oxygen-saturated (95% O2+5% CO2) Krebs-Ringer buffer. The aortic rings were then stretched to and maintained at a preload of 5 g for approximately 30 minutes.16
Preparation and Characterization of Lipoproteins
We isolated n-LDL (d=1.025 to 1.063 g/mL) from human
plasma by discontinuous density gradient
ultracentrifugation as described
earlier.17 Briefly, the density of plasma was adjusted to
1.006 g/mL with NaCl medium, and the plasma was centrifuged at
150 000g for 24 hours. The VLDL- and chylomicron-rich
layer was discarded. The remaining fraction, after density was adjusted
at 1.063 g/mL with KBr medium, was centrifuged at
150 000g for 24 hours to isolate LDL fraction. The purified
LDL was dialyzed for 96 hours against PBS degassed with N2
and containing 0.3 mmol/L EDTA at 4°C. LDL was stored under
N2 at 4°C and used within 10 days, and suitable aliquots
were then oxidized in the presence of 5 µmol/L CuSO4 for
18 to 20 hours at 37°C.18 Oxidation was terminated by
refrigeration. Oxidation of LDL was confirmed by the presence of TBARS
with malondialdehyde bis(dimethyl acetal) as the standard. The TBARS
content of ox-LDL was 0.64±0.06 versus 0.22±0.02 nmol/100 µg
protein in the n-LDL preparation. Ox-LDL was used within 2 days of
preparation. Protein content was determined according to the method of
Bradford,19 with the use of bovine serum albumin
as the standard. In some cases LDL was purchased from Sigma Chemical
Co. Endotoxin contamination was checked with the E-Toxate kit (Sigma
Chemical Co) and was consistently 0.005 EU/mL or lower (lowest
detection limit).
Preparation of Leukocytes
Anticoagulated autologous rat blood was centrifuged
twice at 150g for 10 minutes. The platelet-rich
plasma was collected and centrifuged at 1000g for 15
minutes to obtain platelet-free plasma. The
platelet-free plasma was then added back to the remaining blood
to obtain platelet-poor blood. The platelet-poor blood
was then carefully layered on Histopaque-1077 medium (Sigma Chemical
Co) and centrifuged at 300g for 30 minutes. The
leukocyte layer, consisting mostly of neutrophils but also of some
monocytes and lymphocytes, was collected and washed with Hanks' buffer
without Mg2+ and Ca2+ (Flow
Laboratories, Inc).
Some leukocytes were labeled with biotin-FITC. The technique of labeling with fluorescent dye involved suspension of leukocytes in Ca2+- and Mg2+-free Hanks' solution (final concentration, 107 cells per milliliter) and incubation with ImmunoPure NHS-LC-Biotin (final concentration, 2 mmol/L) (Pierce) at room temperature for 30 minutes.20 The cells were then washed twice with Ca2+- and Mg2+-free Hanks' solution, incubated with streptavidin-FITC (final concentration, 10 µg/mL) (Sigma Chemical Co) in ice for 30 minutes, centrifuged, then washed once with Ca2+- and Mg2+-free Hanks' solution, and resuspended in Hanks' buffer (final concentration, 2x106 cells per milliliter). These cells were verified to be 90% alive by trypan blue exclusion, and 95% of cells picked up the label (by fluorescent microscopy). These cells were used to quantify cellular adhesion to rat aortic rings by fluorescent microscopy.
Another set of unlabeled leukocytes was suspended in Hanks' solution (final concentration, 2x106 cells per milliliter) and used to quantify cell adhesion by scanning electron microscopy.
Protocol
Aortic rings hung in organ baths were incubated with buffer
alone or buffer containing n-LDL (100 µg protein per milliliter),
ox-LDL (100 µg protein per milliliter), NO synthase
inhibitor (L-NAME,
10-4 mol/L), or the NO precursor
L-arginine (10-4 mol/L) plus
ox-LDL (100 µg/mL) for approximately 100 to 120 minutes. During
incubation, the buffer containing lipids or other agents was
continuously aerated with 95% O2+5% CO2 and
replaced every 30 minutes.
Contractile reactivity of the aortic rings in response to norepinephrine (10-9 to 10-7 mol/L) and relaxant reactivity in response to acetylcholine (10-9 to 10-6 mol/L), calcium ionophore A23,187 (10-9 to 10-6 mol/L), and thrombin (0.1 U/mL) were examined.
Some aortic rings were then opened longitudinally, placed face-up
in gelatin-coated 35-mm dishes, and incubated with
biotin-FITC-labeled leukocytes (
2x106 cells per
milliliter) in Hanks' solution at room temperature for 30 minutes with
the dish rotated 120°/min. The medium was then aspirated and twice
replaced by fresh Hanks' solution without cells to remove nonadherent
cells. The aortic segments were then removed and placed on glass slides
with the endothelial side up. Adherent cells were
quantitated under fluorescent microscope from at least 10 sites
on each segment. Cell adhesion was scored as grade 0 (no cells), grade
1 (
5 cells per field), grade 2 (6 to 10 cells per field), grade 3 (11
to 30 cells per field), and grade 4 (>30 cells per field or clusters
of cells).
Some aortic rings were incubated with unlabeled leukocytes (as described above for fluorescent microscopy) and fixed in 4% glutaraldehyde and postfixed in 1% osmium tetroxide in 0.1% cacodylate buffer (pH 7.2). After several washes in cacodylate buffer, the tissues were dehydrated in graded alcohols. Specimens were then critical-point-dried in a critical-point dryer (model DCT-1, Denton Vacuum). Under a dissecting microscope, tissues were then cut longitudinally with a razor blade for full exposure of the luminal surface. Tissues were then coated with gold-palladium in a Hummer II Coating System (Technics). All specimens were examined by Dr S. Khan with a scanning electron microscope (model JSM 35C, JEOL) without knowledge of the treatment of vascular segments.16
Some aortic rings (treated with buffer, n-LDL, ox-LDL, or L-NAME) were subjected to immunohistochemistry.
Some aortic rings were treated with the murine P-selectin blocking antibody (PB1.3, Cytel Corp)21 along with buffer or ox-LDL. The concentration of PB1.3 was 20 to 100 µg/mL. After incubation for 2 hours, the rings were exposed to biotin-FITC-labeled leukocytes. The adhesion of cells was quantitated by fluorescence microscopy (see protocol 3).
Expression of P-Selectin in Rat Aortic Rings
Tissue samples were embedded in optimum cold temperature
compound (Miles Laboratory) and were quickly frozen in isopentane,
precooled in dry ice acetone. Blocks were stored at -80°C
before they were cryosectioned. Five-micrometer serial
sections were air dried and fixed in cold acetone. After rehydration
with PBS, sections were incubated with PB1.3 for 4 hours at room
temperature. A biotin-streptavidin detection system was used with
diaminobenzidine as the chromogen, as described
elsewhere.22 Briefly, slides were washed twice with PBS
and incubated with the linking reagent (biotinylated
anti-immunoglobulins) for 20 minutes at room temperature. After
they were rinsed in PBS, the slides were incubated with
peroxidase-conjugated streptavidin label for 20 minutes at room
temperature. The sections were again rinsed with PBS and incubated with
diaminobenzidine for 10 minutes in the dark. After chromogen
development, slides were washed in two changes of water for 8 minutes
each, dehydrated, cleared in xylene, and mounted with Permount. Mouse
serum applied instead of primary antibody was used as negative control
staining.
Data Analysis
Multiple studies in several rings from one rat are considered a
single experiment. Contraction of aortic rings is expressed as grams of
tone. Relaxation of aortic rings is expressed as percent decrease from
preexisting tone. All values are presented as mean±SEM.
Differences between specific means were tested by ANOVA with Student's
t and Newman-Keuls tests. A value of P<.05 was
accepted as statistically significant.
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Results |
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Lipoproteins and Aortic Ring Reactivity
Treatment of rings with n-LDL had no effect on either contractile or relaxant response to various stimuli. Similarly, incubation of aortic rings with ox-LDL for 100 to 120 minutes did not affect the aortic ring contraction in response to norepinephrine (Fig 1
). Aortic ring relaxation in response to
acetylcholine was also not influenced by incubation with ox-LDL.
Similarly, relaxant responses to calcium ionophore A23,187 or thrombin
remained unaffected (Fig 1).
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Treatment of rings with L-arginine before incubation with
ox-LDL did not affect the contractile and relaxant responses to
different stimuli (Table).
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Treatment of rat aortic rings with L-NAME caused a marked
increase in contractile response to norepinephrine and
diminution in relaxation in response to acetylcholine (Table
).
Lipoproteins and Leukocyte Adhesion to
Endothelium
Fluorescent Microscopy
As shown in Fig 2, leukocyte adhesion to vascular
intima as quantitated by fluorescent microscopy was not
affected by incubation of rat aortic rings with n-LDL. However,
incubation of aortic rings with ox-LDL caused a marked increase in
adhesion of leukocytes to vascular intima. The increased leukocyte
adhesion to the intima in rings incubated with ox-LDL, however, was not
affected by prior treatment of rings with L-arginine.
Interestingly, treatment of rings with L-NAME caused marked
deposition of cells.
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Scanning Electron Microscopy
Fig 3 shows representative examples
of leukocyte adhesion to vascular intima determined by scanning
electron microscopy. While treatment of aortic rings with n-LDL did not
affect leukocyte adhesion to endothelium, treatment of
aortic rings with ox-LDL markedly increased the adhesion
(P<.05). Treatment of aortic rings with L-NAME
also revealed a marked increase in cell adhesion to the
endothelial surface of rat aortic rings.
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Immunohistochemistry
When reacted with anti-P-selectin antibody, tissues showed various
staining patterns. There was only weak staining on the luminal surface
of endothelial cells of n-LDL–treated aortic rings. In
contrast, consistently intense staining on the luminal surface
of endothelial cells was observed in ox-LDL– and
L-NAME–treated rings. No immunostaining
was observed in the negative control rings. In some cases, a weak brown
immunoreaction was observed in the intima, which we considered
nonspecific (Fig 4).
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Effect of P-Selectin Blocking Antibody on Leukocyte
Deposition
Ox-LDL–treated rings coincubated with PB1.3 showed a marked
decrease in leukocyte deposition on the vascular intima. The reduction
in leukocyte deposition was dependent on the concentration of PB1.3
(Fig 5).
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Discussion |
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The present study demonstrates that incubation of rat aortic rings with n-LDL or ox-LDL for 100 to 120 minutes did not affect either the contractile response to norepinephrine or the relaxant response to three endothelium-dependent vasorelaxants: acetylcholine, calcium ionophore A23,187, and thrombin. Despite the lack of effect on vasoreactivity, incubation of aortic rings with ox-LDL, but not n-LDL, markedly enhanced leukocyte adhesion to the endothelium. Furthermore, stimulation of cell adhesion by ox-LDL was not affected by pretreatment of rings with the NO precursor L-arginine but was blocked by P-selectin blocking antibody PB1.3. Interestingly, treatment of aortic rings with the NO synthase inhibitor L-NAME stimulated leukocyte adhesion to the endothelium, comparable in magnitude to that with ox-LDL. This study, perhaps for the first time, indicates that ox-LDL stimulates leukocyte adhesion to endothelium without affecting NO bioactivity.
The effect of ox-LDL on NO release/activity varies from cell to cell
and depends on the conditions used for study. Hayashi et
al14 23 reported that hyperlipidemia
impairs vascular endothelium-dependent relaxation
in pig coronary arteries. Simon et al15 reported
that exposure of pig right coronary arterial rings
to ox-LDL results in enhanced contraction and inhibition of
endothelium-dependent relaxation via an oxygen free
radical-related mechanism. Studies by Tanner et al24
indicated that ox-LDL activates the scavenger receptor on
endothelial cells and inhibits the
receptor-operated NO formation in epicardial but not in
intramyocardial coronary arteries. Chin et al25
reported that exposure of bovine aortic endothelial
cells to ox-LDL decreases NO activity measured by bioassay. Jacobs et
al26 showed that ox-LDL inhibits relaxation in response to
both endothelium-derived and exogenous NO in rabbit
aortic rings. Schmidt et al27 and Galle and
Bassenge28 suggest that inhibition of
endothelium-dependent vascular relaxation by ox-LDL
is due not to reduced formation of NO but to a diminished
responsiveness of soluble guanylate cyclase. Yang et
al29 recently reported that ox-LDL inhibits NO synthase
activity in macrophages. On the other hand, there are reports
on the upregulation of NO synthesis by cholesterol in
arterial smooth muscle cells30 and
neutrophils.31 In the present study incubation of rat
aortic rings with ox-LDL for 100 to 120 minutes did not show any
evidence of alteration of the vascular contractile response to
norepinephrine or the relaxant responses to
receptor-mediated endothelium-dependent
vasorelaxant acetylcholine and thrombin as well as
receptor-independent vasorelaxant calcium ionophore A23,187. These
observations indicate that treatment with ox-LDL for a short period of
time (2 hours) does not affect NO synthesis, release, and activity in
isolated rat aortic rings.
Alteration of NO activity has been postulated to be involved in leukocyte-endothelium interactions. Provost et al12 reported that endothelium-derived NO attenuates leukocyte adhesion to endothelium under arterial flow conditions. Kurose et al13 demonstrated that ischemia-reperfusion results in leukocyte-endothelium adhesion via a decrease in NO synthesis/activity in rat mesentery arteries, which could be prevented by superfusion with NO donors. Studies by Niu et al32 showed that incubation of HUVECs with the NO synthase inhibitor L-NAME causes an increase in neutrophil adhesion to HUVECs, which can be abolished by the NO precursor L-arginine, NO donors, or intracellular oxygen free radical scavengers. Our studies confirm the increase in leukocyte adhesion to rat aortic rings treated with the NO synthase inhibitor L-NAME. Tsao et al33 reported that hypercholesterolemia enhances the adhesion of monocytes to aortic endothelium in the rabbit, which was attenuated by L-arginine. Liao and Granger34 also reported that L-arginine significantly reduces leukocyte adhesion to the endothelium of venules in ox-LDL–treated rat mesentery preparations. All these findings indicate that decrease in NO synthase or activity participates, at least in part, in the process of leukocyte-endothelium interaction.
In the present study incubation of rat aortic rings with ox-LDL did not affect NO activity and yet markedly increased leukocyte adhesion to endothelium and caused a modest degree of endothelial disruption. Treatment of rings with L-NAME resulted in a degree of cell adhesion similar to that of incubation with ox-LDL, suggesting that a similar mechanism, ie, inhibition of NO synthesis, may underlie enhanced leukocyte adhesion to endothelium. However, the ox-LDL–induced increase in leukocyte adhesion to endothelium was not affected by L-arginine, suggesting that it is either not due to the decrease in NO release/synthesis or the cell adhesion is evident before a decrease in NO activity becomes apparent. Thus, there does not appear to be a significant temporal correlation between loss of NO activity and leukocyte adhesion to the endothelium, at least in rat aortic rings. The modest endothelial disruption in rings treated with ox-LDL or L-NAME may be one of the mechanisms of cell adherence, although the endothelial disruption was not severe enough to cause alterations in vasoreactivity. In addition, Ohara et al35 36 reported that hypercholesterolemia and lysophosphatidylcholine increase superoxide anion production in vascular endothelium. Incubation of aortic rings with ox-LDL may have led to similar oxidative injury to the endothelium, which may be one of the mechanisms for the ox-LDL–stimulated leukocyte adhesion to the endothelium in the present study.
LDL scavenger receptors on macrophages recognize and uptake n-LDL and ox-LDL and transform themselves into foam cells,4 5 which are present in human and rabbit atherosclerotic lesions.37 38 Previous studies have indicated that ox-LDL activates the scavenger receptor on endothelial cells and inhibits the receptor-operated NO formation in epicardial coronary arteries.24 These observations imply the contribution of LDL scavenger receptors to the progression of atherosclerosis. In preliminary studies we examined the role of the LDL scavenger receptor on endothelium as a basis for enhanced leukocyte adhesion. Nonetheless, we failed to observe any effect of the LDL scavenger receptor antagonist dextran sulfate on ox-LDL–stimulated cell adhesion (data not shown).
We examined the effect of a single 100 µg/mL concentration of n-LDL and ox-LDL in these experiments. Whereas this concentration of n-LDL is in the physiological range, ox-LDL does not circulate in the plasma. It is, however, likely that the concentration of ox-LDL in the growing atherosclerotic lesion is quite high. We did not examine the effect of higher pharmacological concentrations of ox-LDL, which may have affected aortic ring reactivity and probably caused much greater endothelial disruption and leukocyte adhesion. The lack of any effect on vascular reactivity despite marked cell adhesion on rings treated with 100 µg/mL of ox-LDL clearly suggests that loss of NO activity is not the basis of enhanced adhesion to endothelium.
In the absence of a temporal correlation between leukocyte deposition and loss of NO activity, we wondered whether expression of leukocyte adhesion molecules relates to deposition of cells. Since expression of E-selectin, ICAM-1, and VCAM-1 occurs several hours after exposure of endothelial cells to injurious stimuli,39 40 41 we hypothesized that early expression of P-selectin on endothelial cells42 exposed to ox-LDL may be the basis of marked cell deposition. P-selectin is synthesized by vascular endothelial cells and localized in Weibel-Palade bodies.43 Previous studies have shown rapid expression of P-selectin in the venules of pulmonary vascular endothelium of rats subjected to infusion of cobra venom factor44 and myocardial venules of cats subjected to ischemia and reperfusion.45 In the present study the rat aortic rings expressed a large number of P-selectin adhesion molecules soon after exposure to ox-LDL. The importance of expression of P-selectin adhesion molecules became evident in blocking experiments in which PB1.3 inhibited leukocyte deposition in ox-LDL–treated rings.
The expression of P-selectin adhesion molecules in ox-LDL–treated rings was similar to that in the L-NAME–treated rings. Nonetheless, the mechanism of the two agents on P-selectin expression and leukocyte adhesion remains dissimilar. In our preliminary studies we observed that ox-LDL causes a similar expression of P-selectin adhesion molecules on human platelets (A.M. et al, unpublished data, 1995). Thus, increased adhesion of leukocytes to ox-LDL-treated vascular tissues involves early expression of P-selectin. It is noteworthy that some n-LDL–treated aortic rings also showed a weak P-selectin expression, which may be attributed to some oxidation of LDL during the period of incubation with aortic rings.
Lehr et al46 reported that ox-LDL elicits leukocyte rolling and adhesion on the endothelium of both arterioles and venules and promotes the formation of aggregates tumbling down the microvasculature and firmly adhering to the microvasculature in hamsters. The aggregates consisted of leukocytes and dendritic platelets. The adhesion of leukocytes to the endothelium as well as formation of clusters was significantly reduced by pretreatment of hamsters with anti–P-selectin antibody. In the present study leukocyte clusters were often observed, similar to the observation of Lehr et al.46 Since platelets may not have been completely separated from the leukocyte preparation and platelets do become activated upon contact with ox-LDL, activated platelets may also be involved in the deposition of a large number of leukocytes and the formation of leukocyte clusters.
In summary, incubation of rat aortic rings with ox-LDL stimulates leukocyte adhesion to the intima of rat aortic rings without affecting vascular contractile and relaxant responses. In accordance with this concept, ox-LDL–stimulated leukocyte adhesion to endothelium was not modulated by the NO precursor L-arginine. Under the conditions we used, leukocyte deposition within 2 hours of exposure of rat aortic rings to ox-LDL appears to be a result of expression of P-selectin leukocyte adhesion molecules. It should be mentioned that the rat aorta used in this study is not a model for ox-LDL–mediated atherosclerosis but may represent a simple model of ox-LDL–induced endothelial injury and leukocyte deposition.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by the American Heart Association, Florida Affiliate, St Petersburg, Fla. Asha Mehta conducted this study while a junior at Eastside High School, International Baccalaureate Program, Gainesville, Fla. The authors thank Britt Isaac for expert secretarial assistance in the preparation of this manuscript.
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Footnotes |
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Reprint requests to J.L. Mehta, MD, PhD, Box 100277, JHMHC, University of Florida, Gainesville, FL 32610.
Received April 4, 1995; accepted September 15, 1995.
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