The nature of leukocyte shape changes in the pulmonary
capillaries
Darlene M.
Redenbach,
Dean
English, and
James C.
Hogg
University of British Columbia Pulmonary Research Laboratory, St.
Paul's Hospital, Vancouver, British Columbia, Canada V6T 2B5
 |
ABSTRACT |
The size discrepancy between leukocytes
[white blood cells (WBCs)] and pulmonary capillaries
requires WBCs to deform. We investigated the persistence of this
deformation on cells leaving the capillary bed and the role played by
the cytoskeleton. Isolated rabbit lungs were perfused in situ via the
pulmonary artery with effluent fractions collected from the left
ventricle. Washout curves from cell counts in each fraction confirmed
that WBCs are preferentially retained over erythrocytes. WBC
deformation present on exit from the circulation was compared with that
present after recovery in paired fractions, fixed either immediately or
60 min later. These cells were compared with cells recovered from the
capillary in perfused fixative or fixed in peripheral blood. Our
results show that leukocyte deformation persisted after the cells
exited the pulmonary circulation. This deformation was associated with
minimal submembranous F-actin staining, and microtubule distribution
and cell polarization were unchanged. We conclude that cytoskeletal
changes that occur during WBC deformation in the pulmonary capillaries
are minimal and differ from those known to occur in actively migrating
cells during chemotaxis.
neutrophil; pulmonary capillary; deformation; cell polarity; cytoskeleton; cell shape ; polymorphonuclear leukocytes
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INTRODUCTION |
THE PULMONARY CAPILLARY BED is made up of an
interconnecting network of ~1011 short segments ranging
in width from 1 to 15 µm with a mean diameter of 7.5 ± 2.3 (SD)
µm and length of 14.4 ± 5.8 (SD) µm (9, 24). The number of
segments in a single pathway, from an arteriole to venule, has been
variously estimated from 50 to 100 (14, 15), and 38% (human) to 67%
(rabbit) of them are too narrow to accommodate the spherical diameter
of a polymorphonuclear leukocyte (PMN; see Ref. 9). Assuming a normal
distribution of capillary segment diameters in the pathways from the
arteriole to venule, all of the pathways must contain one or two
segments that would restrict circulating cells. This assumption is
consistent with the recent report of Wiggs and co-workers (25) who
found that PMN with a mean diameter of ~7 µm are delayed to the
same degree as nondeformable beads 4 µm in diameter during a single
pass through the lung. This shows that the PMN have the capability of
rapidly reducing their minimum diameter by ~3 µm to negotiate the
restriction imposed by the capillary bed. Because the capillary bed is
made up of such a large number of parallel pathways, the pressure drop across individual segments is very small. This means that the cells may
negotiate the narrow capillary segments by active shape changes that
could involve active locomotion of the circulating WBCs through the
restrictions. A delay in restoring their spherical shape after
deformation is of interest because maintenance of the shape change
required to negotiate the first restriction would facilitate passage
through subsequent restrictions in a microvascular bed. The present
study was undertaken to examine the deformation and recovery of the PMN
in the pulmonary capillary bed and to determine the nature of the
cytoskeletal changes associated with these deformations.
 |
MATERIALS AND METHODS |
Materials
Buffers. Washout buffer was composed
of (in mM) 136 NaCl2, 2.7 KCl, 8.2 Na2PO4,
1.5 KH2PO4,
0.7 CaCl2, 0.5 MgCl2 · 6H2O,
and 5.5 glucose, pH 7.4. Washout buffered fixative was composed of 2.5% glutaraldehyde or 1.6% paraformaldehyde (EM grade; Electron Microscopy Sciences, Fort Washington, PA) made in washout buffer. All
buffers were used at 37°C.
Immunohistochemical stains and
antibodies. Blood cells were stained with Geimsa stain
(BDH, Toronto, ON, Canada). Microtubules were identified with 5A6
anti-
-tubulin monoclonal antibody (final dilution 1:4,000 of a 10 mg/ml stock), a generous gift from Dr. David Brown, University of
Ottawa, ON, Canada (1). Filamentous actin (F-actin) was localized with
tetramethylrhodamine B isothiocyanate (TRITC)- or fluorescein
isothiocyanate (FITC)-conjugated phalloidin (reconstituted in 1 ml
methanol stock, evaporated and diluted 1:200; Molecular Probes, Eugene,
OR). Nuclei were identified with either Giemsa stain or
Hoechst 33342 fluorescent nuclear probe (diluted 1:200;
Sigma, St. Louis, MO). Secondary antibodies were either FITC- or
TRITC-conjugated mouse immunoglobulin G (IgG; Sigma).
Surgical Procedure
New Zealand White rabbits (2.62 ± 0.5 kg) were anesthetized with
40-60 ml of 0.25 g
-chloralose in 50 ml normal saline plus 20 ml of 32% urethan to deep anesthesia, which was maintained with 50%
urethan, intravenous fluid replacement, and 1,000 units heparin. The
rabbit was placed on a Harvard respirator via tracheostomy at an
initial respiratory rate of 25-30 cycles/min with a tidal volume
of 7 ml/kg and then was adjusted to establish physiological blood gas
parameters that were monitored from a cannulated carotid artery. The
heart and lungs were exposed via midsternal thoracotomy. A single tie
was placed around the pulmonary trunk and ascending aorta to isolate
the pulmonary circulation after which the pulmonary trunk and left
ventricle were cannulated.
Washout Procedure
The lungs were perfused with buffer at a rate of 20 ml/min using an
infusion pump (Cole-Palmer). In 11 experiments, effluent fractions were
collected from the left ventricle at 1-s intervals for 25 s, followed
by 5-s intervals to a total of 225 s. The pulmonary circulation was
then perfused with buffered fixative consisting of either 1.6%
paraformaldehyde for immunocytochemical studies or 2.5% glutaraldehyde
for morphological studies, with equal results. Preliminary experiments
were performed to verify that the pulmonary vascular and airway
pressures remained within the physiological range during the
experimental procedure. In a series of seven experiments, the effluent
was collected at 3-s intervals via a two-channeled cannula directly
into paired preweighed vials to compare cells fixed immediately with
those allowed to recover their shape. One of these vials contained
concentrated fixative at a volume estimated to give a final
concentration of 1.6% paraformaldehyde, and the other had fixative
added 60 min after collection to achieve the same concentration of
fixative. Immediately after fixation, tubes from both groups were
weighed, centrifuged, and resuspended in 1 ml of fresh 1.6%
paraformaldehyde.
Cell Counts
In experiments performed without fixation of the effluent, the number
of white blood cells (WBCs) and red blood cells (RBCs) in each buffer
fraction were counted on a Coulter counter (model S880). To determine
WBC and RBC counts in experiments in which cells were collected in
paired fractions, total cell counts were calculated using Coulter
counter values obtained from the samples before the addition of
fixative at 60 min. The cells in the effluent from lungs perfused with
fixative were counted using a hemocytometer.
Morphology
After the rabbit lungs were fixed by perfusion, they were immersed in
glutaraldehyde of the same concentration as that in the perfusate for a
minimum of 24 h. These lungs were then processed using a standard
procedure that has been previously described in this laboratory (9).
Cell shape analysis. Cell shape
analysis was performed on the first 20 intact WBCs encountered on
slides made from samples obtained at the peak, shoulder, and plateau of
the washout curve and from those washed from the lung by fixative at
the end of the experiment. Cells were stained in suspension with Giemsa
and were examined using a custom-designed morphometry program (BioView Colour Image Processing System; Infrascan, Vancouver, BC, Canada) to
locate the longest diameter and the centroid of the cell. The length
determination program was then used to measure the longest diameter and
the shortest diameter, defined as that passing through the centroid at
right angles to the longest diameter. The "shape ratio" was
calculated as the longest diameter divided by the shortest diameter.
Immunohistochemistry
The cells perfused from the lung with buffer or buffered fixative were
processed for localization of tubulin (5A6 anti-
-tubulin, 1 mg/ml
stock diluted 1:400), F-actin (FITC- or TRITC-conjugated phalloidin,
1:50-1:200), and nuclei (Hoechst 33342, 1:200). Fixed cells were pelleted, the supernatant was removed, and cells were resuspended gently for washing and staining steps. All steps were carried out at room temperature unless otherwise noted. For each sample, 500-µl samples of fixed cells were rinsed three times in 1 ml
of perfusion buffer, incubated in 1 ml 0.2% Triton X-100 in
phosphate-buffered saline (PBS) for 15 min, rinsed two times in 1 ml of
0.1% bovine serum albumin (BSA)-PBS for 5 min each, and blocked for
nonspecific staining in 1.0 ml of 5.0% normal goat serum (NGS) in 1 ml
of 0.1% BSA-PBS for 30 min. Samples were then incubated in 200 µl of
5A6 anti-
-tubulin diluted in 0.1% BSA-PBS with 1% NGS for 60 min
at 37°C. After two 10-min rinses, cells were incubated in either
TRITC- for FITC-conjugated goat anti-mouse IgG (final dilution 1:200)
for 20 min. For dual-stained cells, the phalloidin conjugated with a
fluorochrome contrasting with goat anti-mouse secondary marker was
added during the last 20 min of the secondary antibody incubation (or
before the last rinse step with equal results). Finally, cells were
rinsed three times in 500 µl of 0.1% BSA-PBS and were
stored in either buffer or Dabco medium (to preserve fluorescence). For
observation, cells were allowed to settle onto glass slides after which
Dabco medium and then coverslips were added. Cells were visualized on a
Zeiss photomicroscope equipped for epifluorescence with filters for FITC (excitation: 450-490 nm; barrier: 510 nm; emission:
515-556 nm), TRITC (excitation: 546 nm; barrier: 580 nm; emission:
590 nm), and bis-benzamide (Hoechst
33342; Sigma; excitation 365: nm; barrier: 395 nm; emission: 420 nm).
Confocal Microscopy
The cytoskeleton of cells fixed in the capillary were observed on a
Biorad MRC 600 confocal laser scanner (Bio-Rad, Burlingame, CA)
attached to an upright Nikon Optiphot 2 microscope imaged with a
×60 oil immersion objective (Nikon, Mississauga, ON, Canada). With the use of 0.1-µm steps, three-dimensional fluorescent images were compiled from 120 consecutive optical sections to localize the
microtubule organizing center (MTOC) and the cellular distribution of
F-actin in fixed cells.
Data Analysis
For the shape ratio, data means were compared using a one-tailed
Student's t-test (significance,
P
0.05). For comparison between
groups on the washout curve, two-way analysis of variance was
determined (significance, P
0.5),
and specific comparisons were made using the sequential rejective
Bonferroni comparison. All statistical analyses were
performed using Systat software with errors expressed as ± SD.
 |
RESULTS |
Figure 1 shows data from a single
experiment in which cell counts are expressed as a percentage of the
total cells washed from the lung. The WBC-to-RBC ratio at the peak of
the curve (leading edge of perfusate from the left ventricle) at 1.77 ± 0.77 was not significantly different from that of peripheral
arterial blood (common carotid artery) at 1.68 ± 0.23. Figure
2 shows WBC and RBC curves constructed from
combined data from 11 experiments in which the values are expressed as
a percentage of their peak values. Comparison of the WBC and RBC curves
fitted with 95% confidence intervals showed that washout of WBCs was
delayed compared with RBCs. Increasing the flow after the WBC curve
reached a plateau resulted in a small increase in the number of cells
in the effluent with an immediate return to the plateau level
(n = 2, data not shown).

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Fig. 1.
Typical washout curve of leukocytes [white blood cells
(WBCs)] and red blood cells (RBCs) from the pulmonary
circulation. Data illustrate the washout curve from a representative
experiment. No. of cells is expressed as a percentage of the peak cell
number, and the volume perfused is indicated from the beginning of the
washout to a total of 50 ml perfused.
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Fig. 2.
Pulmonary circulation washout cumulative curve with 95% confidence
intervals. Solid line represents the best fit for all data points for
the WBC or RBC components, and broken lines indicate the 95%
confidence intervals for these data. WBCs were significantly delayed
with respect to RBCs in their transit through the pulmonary circulation
(n = 11).
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The switch from buffer to buffered fixative perfusion did not change
the number of leukocytes recovered in the effluent. Similar results
were obtained with either fixative. Microscopic examination of lung
tissue that had been fixed by perfusion after the washout procedure
showed that the majority of blood cells remaining in the perfused lung
were elongated leukocytes located in capillary segments (Fig.
3). Examination of the WBCs perfused from
the lung by buffered fixative, after the initial buffer washout, showed that those cells possess the same deformed shape as WBCs remaining in
the capillary (Fig. 3, inset).

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Fig. 3.
Blood cells left in the lung after washout of pulmonary circulation.
Blood cells left in the lung are highly enriched for WBCs after washout
(arrows). Blood cells recovered from the capillary after perfusion with
buffered fixative are shown in inset.
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Figure 4 shows cumulative washout data of
seven experiments in which the cells were perfused from the lung with
buffer and were collected in paired samples that were either fixed
immediately or were deferred for 60 min. Comparisons of shape analysis
(mean shape ratio) of cells that were fixed immediately with cells that were fixed 60 min later are shown in Fig.
5. Cells collected at the peak of the
washout curve showed a similar spherical shape with either immediate or
deferred (late) fixation. However, those cells collected at the
shoulder and plateau of the washout curve (see Fig. 4) and fixed
immediately were elongated compared with those that were allowed to
recover for 60 min before fixation (late fix). Grouping cells by size
reveals that cell elongation increased in frequency and degree over the
washout period (Fig. 6) but did not reach
values of cells perfused out of the lung by fixative.

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Fig. 4.
Pulmonary circulation washout cumulative curve
(n = 7) with 95% confidence
intervals. Washout cell numbers were determined from the control
fractions of divided washout experiment series for shape analysis. WBCs
are delayed in their transit. For shape analysis, cells were sampled
from the paired fractions immediate fix (IF) or late fix (LF) at points
indicated (peak, shoulder, and plateau). Fractions from perfused fix
effluent were considered "fixed in capillary" (FIC) and were
compared with cells from peripheral circulation.
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Fig. 5.
Mean shape ratio for cells from divided washout. Shape ratio was
determined as longest diameter/shortest diameter for each category.
Differences between mean shape ratio (±SD) in IF and LF groups were
significant (*) in shoulder, plateau, and FIC groups (P 0.0005) but
not significant in peak fractions (P 0.05).
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Fig. 6.
Grouped cell shape data from peak
(A), shoulder
(B), plateau
(C), and FIC
(D) samples for 7 experiments. Cells
in the IF groups were elongated but did not exceed a shape ratio of
2.0. Maximum shape ratio in LF and FIC cells reached shape ratio values
of 5.0.
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Although cells recovered by perfusing them out of the lung with
fixative were elongated, they showed no evidence of lamellipodia or
uropod formation, which is typical of a leading or trailing edge in
migrating cells. Furthermore, there was no consistent change in the
position of the nucleus, which was typical of a polarized pattern seen
in migrating WBCs (Fig. 7). In the deformed WBC, microtubules radiated from a perinuclear center and extended throughout the cell (Figs. 7 and 8). The
MTOC, which is considered to occur at the point of divergence of the
microtubule arrays, was localized in cells stained with anti-tubulin
antibodies (Fig. 8). The MTOC was most frequently located in a lateral
position adjacent to the nucleus and did not show any preference for a polarized position at either end of the nucleus of these elongated cells. Unlike the intense F-actin staining seen in activated
neutrophils, the F-actin staining in these cells was pale, appearing to
accumulate in the submembranous area occupied by the ectoplasm, an area
generally devoid of membranous organelles. The actin distribution at
the cell surface was explored further using anti-tubulin and F-actin probes (Fig. 9). The three-dimensional
images revealed the radiation of microtubules from randomly positioned
focal centers, with no evidence of preference for a leading or trailing
edge. These confocal images also verified that cortical F-actin was
distributed evenly and was not consistent with the intense F-actin
staining reported in activated or migrating cells.

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Fig. 7.
Cells perfusion fixed in pulmonary capillaries. Representative
leukocytes from FIC fraction. Lymphocytes
(A and
B) and neutrophils
(C and
D) are elongated, but the nucleus is
typically central. Tubulin (E and
G) and F-actin stain
(F and
H) are evenly
distributed.
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Fig. 8.
Microtubule organizing centers (MTOCs) in leukocytes from IF fractions.
MTOCs, the point of convergence of microtubules identified with
anti-tubulin staining, are randomly distributed in cells fixed after
being recovered from the pulmonary circulation.
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Fig. 9.
Confocal images of leukocytes from FIC fractions. WBCs dual stained
with tetramethylrhodamine isothiocyanate (TRITC)-phalloidin F-actin
(A,
C, and
E) and fluorescein isothiocyanate
(FITC)-5A6 antibody to tubulin (B and
D) have been imaged by confocal
microscopy and assembled as stereo-paired micrographs. Images
(A and
B) and
(C and
D) are from the same cells that were
perfused from the lung with fixative. Note the even distribution of
F-actin (A and
C) compared with the intense F-actin
accumulation that occurs in the presence of process formation
(E).
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|
 |
DISCUSSION |
These results confirm a previous report (9) showing that PMN are
deformed in the capillary bed and extend those observations by showing
that the deformation is maintained as PMN transit from the capillary
bed to the left ventricle. They also show that this cell deformation is
not accompanied by morphological evidence of actively migrating cells.
Previous studies have shown that PMN have a median transit time of 60 s
in the pulmonary capillary bed (14). Using direct observation by video
microscopy through lung windows, Gebb et al. (12) and Lien et al. (18,
19) have shown that the WBCs frequently stop during a single transit
through the vascular bed. This differs markedly from the RBCs that have
a median transit time of ~1 s (14) even though the maximum diameters
of RBCs and WBCs are similar (9). This difference in transit time is due to the RBC discoid shape, which allows it to fold rapidly and to
negotiate capillary restrictions ~300 times faster than leukocytes
(4, 5, 11), resulting in WBC margination in the lung (7, 8, 10).
Recent studies by Wiggs and co-workers (25) have shown that PMN have
the ability to undergo a rapid change in diameter from ~7 to 4 µm
during a single pass through the pulmonary circulation. Our purpose was
to determine the nature of this deformation, its persistence after the
cells leave the capillary bed, and the cytoskeletal changes that
accompany this change in shape.
Intracellular events associated with leukocyte migration have been
shown to be specific to the environment in which they migrate (21).
Nonpolarized leukocytes suspended in solution undergo a random
extension of actin-rich filopodia without becoming polarized (20, for
review, see Ref. 22). In contrast, cells exposed to chemoattractant
gradients reorganize their nuclei, Golgi apparatus, and microtubules.
If they are able to form contacts with a rigid substrate, they extend
actin-rich cell processes and migrate in the direction of the
attractant (17, 22, 23). Sullivan and Mandell (23) have shown that
cells in contact with a three-dimensional substrate behave quite
differently. PMN were placed on a firm substrate with a "gel"
overlay to provide three-dimensional contact and then were exposed to a
chemoattractant gradient. The cells moved slowly toward the
chemoattractant without accumulation of an F-actin-rich zone
in their leading edge. Cells not covered by the gel polarized, extended
F-actin-rich processes, and migrated. These data suggest that organelle
polarization is influenced by the presence of chemoattractants, whereas
the F-actin accumulation in cell processes depends on the nature of
surface contacts available.
Our data show that filopodia or lamellipodia formation, the hallmark of
active migration on a substrate, is lacking on WBCs recovered from the
pulmonary capillaries. However, the movement of WBCs surrounded by an
endothelial tube probably differs from cells migrating on rigid
surfaces, and this could explain the lack of F-actin-rich
filopodia or lamellipodia. Tensegrity, a structural system first
described by Buckminster Fuller (20a) and based on
tensional integrity provided by discontinuous compression elements, has
been proposed to explain the mechanical response of cells to
alterations in shape that are derived both externally and internally
(for review see Ref. 17). The tensegrity model of cell shape predicts
the formation of actin-dense stress fibers when cells exert a linear
pull on a rigid substrate but does not require F-actin redistribution
for changes in global shape when the inward pull is not focused on a
limited number of rigid cell-substrate contacts (16, 17). Our findings
are consistent with the tensegrity model of cell shape, which has the
single requirement that the cell surface be under continuous tension.
This force is provided by the F-actin-based contractile system, which
provides centripetal force on the cell membrane. Microtubules, on the
other hand, provide an opposing centrifugal force that may be regulated
locally. The tensegrity model predicts actin-rich lamellipodia
formation only in cell deformation associated with adhesion to a rigid
substrate but not when adhesion does not occur as in leukocyte transit
under physiological conditions.
Signals provided by externally applied deformation of the cell membrane
have been proposed to be capable of initiating changes in local
actin-membrane interaction through a number of proposed second
messenger pathways (for review, see Ref. 6). The formation of
microfilament structures is regulated in at least two ways. Filament
length is regulated by actin monomer sequestering, filament capping
proteins, and actin filament severing proteins, whereas three-dimensional microfilament structures are modulated by changes in
microfilament cross-linking by actin cross-linking proteins. In
leukocytes, cytosolic actin binding proteins sequester monomeric actin,
effectively raising the cytosolic critical concentration for actin
polymerization to 800 µM from 8 µM of purified actin, conferring a
powerful buffering capacity on actin monomer to polymer exchange.
Deformation of the leukocyte membrane could initiate second
messenger-mediated modulation of actin cross-linking events through a
separate pathway from actin binding proteins, providing for local
buffering of "stiffening" without increased polymer formation
(see Ref. 6). This is consistent with our observation of transiently
retained shape change without formation of actin filament bundles and
with earlier observations by others that PMN retain deformation
relative to the degree and duration of deformation (2). Retention of
the shape beyond the capillary segment, which would facilitate passage
through the subsequent segment, may be mediated by the time course of
such actin-associated cross-linking events (3). These observations are
consistent with the findings of Wiggs and co-workers (25) in which PMN having a mean diameter of 7 µm succeeded in mimicking the passage of
rigid beads 4 µm in diameter, implying a limited deformablity through
rapid reduction in cross-sectional diameter. Furthermore, our results
are consistent with the findings of Gebb and co-workers (12) in which
leukocytes passing through canine pulmonary capillaries, visualized
directly under the visceral plural surface through a window in the
chest, were observed to elongate as they crossed capillary segments at
the pleural surface, a requirement predicted by earlier modeling of
leukocyte transit in pulmonary capillaries by these investigators (13).
In summary, our data show that leukocytes passing through the pulmonary
capillary segments undergo deformation, which they retain beyond the
capillary segment without evidence of cytoskeletal reorganization or
cell polarization. These results suggest that the rapid reduction in
PMN diameter required to negotiate restrictions in the capillary bed
are different from those observed in cells migrating toward a
chemoattractant. We propose that these events may be initiated by
membrane deformation signals that mediate internal buffering of
stiffness to facilitate leukocyte transit through narrow capillaries in
the circulation.
 |
ACKNOWLEDGEMENTS |
We are indebted to Dr. David Walker for helpful discussions, Dr.
Mick Okazawa for help with pressure measurements, Michael Weiss for
help in preparing the confocal images, Lorri Verburgt for statistical
analysis, Fanny Chu for technical assistance, and Stuart Greene for
assistance with photography. The 5A6 monocloncal antibody to tubulin
was the generous gift of Dr. David Brown, University of Ottawa.
 |
FOOTNOTES |
This work was supported by a Postdoctoral Fellowship from the Canadian
Lung Association (to D. M. Redenbach) and by Medical Research Council
Grant 4209 (to J. C. Hogg).
Address for reprint requests: D. M. Redenbach, T325-2211 Wesbrook
Mall, School of Rehabilitation Science, University of British Columbia,
Vancouver, BC, Canada V6T 2B5.
Received 6 August 1996; accepted in final form 11 June 1997.
 |
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