Anti-TNF (Remicade) therapy protects dystrophic

skeletal muscle from necrosis

MIRANDA D. GROUNDS1 AND JO TORRISI
School of Anatomy and Human Biology, The University of Western Australia,
Western Australia, Australia 6009
Necrosis of skeletal muscle fibers in the
lethal childhood myopathy Duchenne muscular dystrophy (DMD) results from defects in the cell membraneassociated protein, dystrophin. This study tests the
novel hypothesis that the initial sarcolemmal breakdown resulting from dystrophin deficiency is exacerbated by inflammatory cells and that cytokines, specifically tumor necrosis factor- (TNF), contribute to
muscle necrosis. To block in vivo TNF bioactivity,
young dystrophic mdx mice (a model for DMD) were
injected weekly from 7 days of age with the anti-TNF
antibody Remicade before the onset of muscle necrosis and dystropathology that normally occurs at 21 days
postnatally. The extent of inflammation, muscle necrosis, and myotube formation was measured by histological analysis from 18 to 28 days and muscle damage was
also visualized by penetration of Evans blue dye into
myofibers. Data from Remicade-treated and control
mdx mice were compared with mdx/TNF(/) mice
that lack TNF. Pharmacological blockade of TNF
activity with Remicade clearly delayed and greatly
reduced the breakdown of dystrophic muscle, in
marked contrast to the situation in mdx and mdx/
TNF(/) mice. Remicade had no adverse effect
on new muscle formation. Remicade is a highly specific anti-inflammatory intervention, and clinical application to muscular dystrophies is suggested by this
marked protective effect against skeletal muscle breakdown.Grounds, M. D., Torrisi, J. Anti-TNF (Remicade) therapy protects dystrophic skeletal muscle
from necrosis. FASEB J. 18, 676 682 (2004)
ABSTRACT

Key Words: mdx mice dystropathology DMD

Duchenne muscular dystrophy (DMD) is an early
onset lethal X-linked disorder that occurs in 1 in 3500
boys, resulting in severe skeletal muscle degeneration
and death, usually by 20 years of age (1). In DMD and
the mdx mouse model for DMD, defects in the subsarcolemmal protein dystrophin initiate muscle pathology
(2, 3). There is no successful treatment for DMD. Much
research has focused on cell and gene therapies to try
and replace the defective dystrophin gene (4, 5). A
complementary approach is to try to render the dystrophic myofibers less susceptible to damage using booster
genes (6) to salvage the muscle integrity (3). For
example, reduction of dystropathology has been dem676

onstrated in mdx mice by overexpression of IGF-1 (7),

utrophin (8), cytotoxic T cell GalNAc transferase (9),
calpastatin (10), integrin- 7 (11), or ADAM-12 (12)
and in the absence of myostatin (13, 14). Many of these
(and other) observations are derived from experiments
using genetically engineered mdx mice; difficulties
arise in translating these promising observations into
the clinical situation. Beyond these strategies designed
to alter myofiber characteristics or extracellular interactions is the approach of targeting inflammatory cytokines; this is the focus of the present study.
Damage to the sarcolemma can result in small lesions
in the membrane that may normally be repaired rapidly
by patching to prevent myofiber breakdown, and it
has recently been demonstrated that dysferlin, the
defective gene in limb girdle type muscular dystrophy,
is a key protein involved in such membrane resealing in
skeletal muscle (15, 16). We hypothesize that in dystrophic muscle, membrane damage caused by the gene
defect can be exacerbated by proinflammatory cytokines, such as tumor necrosis factor- (TNF), that tip
the balance between patching of the minor lesion or
further breakdown leading to focal necrosis of the
myofiber. Strong evidence that inflammatory cells can
contribute to necrosis of healthy muscle cells comes
from studies investigating the role of neutrophils, macrophages, and oxidative damage in vitro (1720) and in
vivo (21, 22). It has been proposed that an inappropriate or excessive inflammatory response can directly
damage myofibers in myopathic conditions such as
dystrophies or myositis (2, 2325). TNF is an early and
potent proinflammatory cytokine that stimulates the
inflammatory response.
Even minor trauma to muscle will increase levels of
TNF by release from mast cells; TNF is also produced
by neutrophils, macrophages and lymphocytes that
accumulate rapidly at the site of injury. TNF increases
rapidly within damaged myofibers and is expressed by
myoblasts and myotubes (26 28). TNF is greatly elevated in damaged myofibers in injured normal (26, 27)
and myopathic skeletal muscle (29); it is chemotactic
1

Correspondence: School of Anatomy and Human Biology, The University of Western Australia, 35 Stirling Hwy.,
Crawley, Western Australia, Australia 6009. E-mail:
mgrounds@anhb.uwa.edu
doi: 10.1096/fj.03-1024com
0892-6638/04/0018-0676 FASEB

for myoblasts (30) and is mitogenic for satellite cells in

vivo, suggesting a direct role in myogenesis of regenerating muscle (28).
The role of the cytokine TNF in muscular dystrophy
has been investigated using mdx/TNF(/) mice,
and histopathological analysis has found that the absence of TNF in vivo resulted in equivocal findings as
opposed to amelioration of muscle pathology as predicted (31), although long-term deletion of TNF
appeared beneficial in older (12 months) mdx/
TNF(/) mice (32). Cytokine networks are complex, and when TNF is removed from an in vivo
system, as in mdx/TNF(/) mice, other proinflammatory cytokines such as IL-12 and IFN (33) may be
up-regulated to overcome the TNF deficiency. The
confounding situation of altered cytokine profiles in
TNF knockout mice is further demonstrated by studies of regenerating whole muscle grafts in these mice,
where predictions of impaired skeletal muscle regeneration in the absence of TNF are not supported (26).
As an alternate approach, we have used the antibody
Remicade to neutralize activity of the TNF protein in
mdx mice to assess whether blockade of TNF in
patients with DMD has therapeutic value. It was expected that this strategy to block TNF activity in vivo
would avoid the problems of compensatory up-regulation of other proinflammatory cytokines and silence
the inflammatory cell response. Strong support for the
in vivo efficacy of Remicade comes from histological
analysis of whole muscle autografts in C57Bl/10SnSc
mice (the parental strain for mdx) pretreated with
Remicade, where grafts examined at 5 days after
transplantation contain very few inflammatory cells and
consist almost entirely of persisting necrotic muscle
tissue with no myotubes, in striking contrast to the
advanced regeneration seen in control grafts where
necrotic tissue has been removed by inflammatory cells
and largely replaced by myotubes at 5 days (M. Grounds
et al., unpublished results). However, whereas inflammatory cell infiltration and associated regeneration are
delayed, they are not prevented since new muscle is
formed within 7 days in these Remicade-treated mice.
Clinically, the TNF neutralizing antibody Remicade
is highly effective at reducing symptoms of inflammatory diseases such as rheumatoid arthritis (34) and
Crohn's disease (35), and is being extended to myositis.
The clinical success of Remicade makes it attractive as
a potential drug to treat muscular dystrophies, and so it
was tested in the present study on dystrophic mdx mice
in vivo. It was hypothesized that TNF plays a role in
the breakdown of dystrophic myofibers and that neutralizing TNF activity in vivo will result in delayed
onset and/or decreased intensity of dystropathology in
mdx mice.
In mdx mice, the absence of dystrophin results in an
abrupt onset of skeletal muscle necrosis at 21 days of
age (36, 37), and our studies focus on this early acute
transitory phase of dystropathology. This critical time
provides a sensitive assay for factors that shift the
threshold of breakdown of dystrophic muscle. Muscle
ANTI-TNF (REMICADE) THERAPY

breakdown peaks at 28 days, then decreases markedly

to stabilize around 12 wk of age to a relatively low level
of damage (37), with symptoms of dystropathology
being cumulative and more pronounced in older (15
months) mdx mice (38, 39). Any delay in the acute
onset of damage at 19 21 days, indicating a shift in the
threshold of muscle breakdown, is readily detected.
The onset of damage is assessed histologically (evidence of necrosis and myotubes formed 3 days after
muscle breakdown) and by injection of Evans blue dye
(EBD) as a sensitive and early marker of muscle damage/leakiness (40, 41). Although it is often stated that
the marked difference in severity of pathology between
mdx mice and DMD boys is due to a greater capacity for
regeneration in the mice, it would seem that the
reduced muscle breakdown in mature mdx mice (after
the transitory acute phase has ceased) may be the key
issue to consider. For if muscle necrosis does not occur,
regeneration is not required.

MATERIALS AND METHODS

Overview
Mdx mice were injected intraperitoneally once weekly from
day 7 after birth with 10 g (1 L) Remicade (ScheringPlough Pty. Ltd., Sydney, Australia) per gram body weight or
equivalent volume of control mouse serum albumin (MSA);
all mice were weighed before injection. Remicade-treated
and control (MSA) mdx mice were also compared with
untreated mdx/TNF(/) mice. At 24 h before sampling,
mice were intraperitoneally injected with 1% Evans blue dye
(EBD) at 1% volume relative to body mass to identify damaged/leaky sarcolemmal membranes (41). Mice were killed
(by halothane anesthesia) and TA muscles sampled from 18
to 28 days of age and at 12 wk. The TA muscles were either
frozen or taken for paraffin processing. Frozen sections were
analyzed for EBD-positive myofibers. Hematoxylin and eosin
(H&E) -stained paraffin sections were analyzed histologically
for inflammatory cell infiltration, myofiber necrosis, and
myotube formation and, in 12 wk samples, for myofibers with
central nuclei.
Mice and treatment groups
Inbred mdx and mdx/TNF(/) mice were used in this
investigation. Mdx mice for TNF neutralization were housed
and treated according to the Western Australian Prevention
of Cruelties to Animals Act, the National Health and Medical
Research Council, and the University of Western Australia
Animal Ethics Committee. Remicade-treated mdx mice were
sampled 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, and 28 days after
birth with numbers of mice being 4, 2, 4, 8, 5, 4, 3, 9, 7, 7, and
4, respectively, for each time point. Control MSA-injected
mdx mice were sampled 19, 20, 21, 22, 23, 24, 25, 26, and 28
days after birth with numbers of mice being 3, 4, 4, 5, 2, 5, 2,
4, and 4, respectively, for each sample point. Four additional
mice from both treatment groups were sampled at 12 wk.
Mdx/TNF(/) and control mdx mice for TNF gene
knockout analysis were housed and sampled at the University
of California, Los Angeles, using guidelines for animal use in
the United States. Duplicate mdx/TNF(/) and control
mdx mice were sampled 19, 20, 21, 22, 24, 26, and 28 days
after birth.
677

Histological assay
Inflammatory cell infiltration, muscle necrosis, and myotube
formation were analyzed using H&E stained 5 m frozen
and/or paraffin sections using light microscopy. TA muscle
for each parameter was examined in transverse section using
the following scale: 1 indicating 15% of total tissue area
affected, 2 representing 6 15%, 3 for 16 30%, 4 for 31 60%,
and 5 for 61100%. Inflammatory cell infiltration was identified by cells with basophilic nuclear staining and little cytoplasm located within the endomysium and/or necrotic tissue.
Muscle necrosis was identified by breakdown of sarcolemma
and fragmented sarcoplasm, and myotubes (indicating regeneration) were identified as plump cells with central nuclei. In
older (12 wk) mice, myofibers with persisting central nuclei
are a cumulative index of previous cycles of necrosis/regeneration (39).

RESULTS
Overall, there was no significant difference in weight
gain between Remicade injected and control MSA
injected mdx mice during the study period. The mean
gram body weight (and standard error) for the Remicade and control mdx mice, respectively, was 7.25
(/0.07) and 8.04 (/1.83) on day 7, 8.25
(/0.22) and 9.26 (/2.27) on day 14, 10.27
(/0.30) and 6.62 (/1.79) on day 21, 14.44
(/0.83) and 10.9 (/2.05) on day 28, and 32.03
(/0.41) and 30.83 (/0.6) at 12 wk. Only day 21

showed significant difference between the two groups,

with slightly higher values for the Remicade-treated
mice (t test P0.05). Therefore, neither the growth
rate of the young mdx mice nor the adult body weights
were reduced by Remicade treatment.
The histological appearance of muscles on days 21
and 24 is shown in Fig. 1; the overall patterns of
inflammatory cell infiltration, necrosis and myotube
formation from days 18 to 28, for control (MSA) mdx,
Remicade-treated mdx and mdx/TNF(/) muscles are summarized in Fig. 2. Detailed quantitation of
the tissue area occupied by muscle necrosis and myotubes at 21 and 28 days of age for control (MSA) and
Remicade-treated mdx mice (n4 mice for each sample) is presented in Fig. 3: these data strongly support
the semiquantitative analysis in Fig. 2. Inflammatory
cells were conspicuous in control (MSA) mdx muscles
at 20 days (Fig. 2a) and pronounced at 21 days throughout 50% of the mdx tissue section (Fig. 1a, Fig. 2a).
The marked increase in inflammatory cells at 20 days
preceded by 1 day the acute phase of necrosis that
occupied up to 30% of the tissue section on day 21 (Fig.
1a, Fig. 2a). In marked contrast, in Remicade-treated
mdx mice, there was little inflammation and minimal
necrosis (0 5%) at these times (Fig. 1c, Fig. 2b). The
maximum extent of necrosis in control mdx mice (seen
on day 21) was much greater than in Remicade-treated
mdx mice (seen on day 25) (Figs. 1 and 2) and the
extent of inflammatory cell infiltration were consis-

Figure 1. Histological appearance of TA muscles from

mice aged 21 and 24 days after birth. a, b) Control
(MSA injected) mdx mice, c, d) Remicade-treated
mdx mice and e, f) mdx/TNF(/) mice. In control mdx mice on a) day 21 necrosis was predominant
and on b) day 24 myotubes were prominent (b, inset:
high magnification shows myotubes with central nuclei). In contrast, necrosis is not marked in Remicade-treated mice on c) day 21 but is apparent at d)
day 24. The mdx/TNF(/) mice at e) days 21 and
f) 24 were similar to control (MSA injected) mdx
mice. Asterisks indicate sites of necrosis. Arrow indicates myotubes. Scale bar, 100 m.

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GROUNDS AND TORRISI

Figure 2. Comparison of graphs summarizing the time course

and extent of phagocyte infiltration, necrosis, and myotube
formation in a) control (MSA injected) mdx mice, b) mdx
mice treated with Remicade, and c) mdx/TNF(/) mice.
No difference in the relative time of onset of myotube
formation in response to necrosis was seen between these
groups. Each time point shows the average grading for all TA
muscles (muscle from both legs was analyzed, n2, with total
n ranging from 4 to 16; see details in Materials and Methods).

tently lower in Remicade-treated mice (Fig. 2b) than in

control mdx mice (Fig. 2a). After the initial acute peak
of necrosis, there appeared to be a lull in muscle
damage; such a cyclic pattern of necrosis/regeneration
was also noted in earlier studies in mdx mice (36). The
pattern of muscle breakdown in Remicade-treated
mice was markedly different from that seen in mdx/

TNF(/) mice, where the overall extent and pattern of damage resembled the control mdx mice (although the peak of inflammation and necrosis was
delayed by several days) (Fig. 2, compare panels b, c,
and a). The striking delay in onset of muscle breakdown in the Remicade-treated mdx mice was further
supported by analysis of EBD staining in TA muscles
from 22-day-old mice, where many EBD-positive myofibers (indicating membrane damage) were conspicuous
in control mdx mice; such myofibers but were rare in
the Remicade-treated mice (Fig. 4). Statistical analysis
of the quantitative data related to the area occupied by
necrotic tissue (Fig. 3) shows highly significant
(P0.05) reduced necrosis in Remicade-treated dystrophic muscles at 21 days compared with control
(MSA) mdx muscles.
The onset of necrosis was followed 3 days later by
the appearance of myotubes, as expected, since myotubes are not formed until 23 days after regeneration
starts (42). In control mdx mice (Fig. 2a), myotubes
were dramatically increased on day 24, 3 days after the
peak of necrosis (at 21 days). In Remicade-treated
mdx mice, myotube numbers increased gradually after
day 24 and the rise in numbers seen at 27 days with a
further increase at 28 days is 3 4 days after (the much
smaller) peaks of necrosis evident at 23 and 25 days.
The timing of myotube formation (with respect to
myofiber necrosis) appears normal and there is no hint
of impaired myogenesis in the Remicade-treated mdx
mice. This is further endorsed by the many regenerated
dystrophic myofibers (identified by central myonuclei)
in 12-wk-old Remicade-treated mdx TA muscle
In 12-wk-old mice, the Remicade-treated muscles
generally had features similar to control mdx muscles
with many central nuclei and small areas of necrosis
sometimes associated with small myotubes. The extent
of necrosis was low and similar in muscles of both
groups, being only 0.185% (/0.19) and 0.245%
(/0.294), respectively, for control mdx and Remicade-treated mice. There was no difference in EBD
staining between control and Remicade-treated mdx
mice with only a few individual or small groups of
EBD-positive myofibers in TA muscle sections; these

Figure 3. Quantitative analysis of the extent of necrosis and of myotube formation in TA muscles (n4 mice) from
control and Remicade-treated mice are
shown for samples at 21 and 28 days.
These data strongly endorse the semiquantitative data in Fig. 2. *The lack of
necrosis at 21 days in Remicade-treated
compared with control mdx mice is
highly significant (P0.05).

ANTI-TNF (REMICADE) THERAPY

679

affected muscle in the Remicade-treated (53.31%

/14.847) than control (67.11% /17.03) mdx
mice. The trend (only four mice were analyzed for each
sample) supports the proposal that Remicade treatment protects dystrophic muscle from necrosis and
reduces the dystropathology; however, this apparent
difference was not statistically significant.

DISCUSSION

Figure 4. Evans blue dye (EBD) staining as a marker of muscle

damage is shown in TA muscles of day 22 a) control (MSA
injected) and b) Remicade-treated mdx mice. On day 22
after birth, the TA muscle of a) control mdx mice show
massive necrosis and are strongly positive for EBD. This is in
contrast to b) Remicade-treated mice where the onset of
necrosis is delayed and only a few myofibers show weak
staining. Scale bar, 50 m.

stained myofibers generally corresponded to necrotic

foci visualized by H&E staining. To test the possibility
that the size of necrotic lesions might be more limited
in Remicade-treated mice, the area of all necrotic foci
was measured at 28 days and 12 wk of age in control
mdx and Remicade-treated mdx mice. Visually there
was no striking difference in necrotic foci size between
the two groups of mice. Morphometric analysis (ImagePro Plus 4.0 software, Microsoft) showed that at 28
days the mean size of the necrotic foci (expressed as
percentage of total section area) was small being only
0.08% (/0.008%) (n5) and 0.19% (/0.12%)
(n4) for Remicade-treated and control mdx mice,
respectively; the relatively high variation in the 28 day
controls was accounted for by one of the four mice
having significantly larger necrotic foci (maximum
value was 1.04%) than the other three mice (maximum
of 0.32%). At 12 wk, the mean size of the necrotic foci
was 0.02% (/0.001) (n4) and 0.04 (/0.014)
(n5) for Remicade-treated and control mdx mice,
respectively. Whereas these appeared to be fourfold
smaller than at 28 days, there was no significant differences overall between the two times of examination
(P0.05). The trend for larger necrotic foci in control
mdx vs. Remicade-treated mice at both time points was
not statistically significant (t test analysis, P0.05).
Further quantitative analysis of the area occupied by
myofibers with central myonuclei (a cumulative measure of muscle damage and repair) indicated less
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The reduced dystropathology in Remicade-treated mdx

mice in the acute phase from 21 to 28 days supports the
hypothesis that TNF contributes to the early breakdown
of dystrophic muscle. It is concluded that although a lack
of dystrophin results in sarcolemmal weakness leading to
initial breakdown of dystrophic muscle (2), the extent of
necrosis is exacerbated by TNF, which likely triggers the
associated inflammatory cascade (23, 25).
The reduced inflammatory response did not appear to
have any adverse effects on regeneration since myotube
formation was not impaired in Remicade-treated mdx
mice in response to necrosis during the acute phase. That
new muscle can be readily formed in Remicade-treated
mice was further confirmed by the presence of many
regenerated myofibers (identified by the presence of
central nuclei) in TA muscles from 12 wk mdx mice. The
marked inhibition of inflammation (with an associated
delay in regeneration) seen in our whole muscle grafts at
5 days in Remicade-treated C57Bl/10Sn mice (data not
shown) did not impair muscle formation at later times.
This graft model is a useful biological assay to demonstrate that this regime of Remicade does indeed reduce
inflammation in mice in vivo. However, the large mass of
necrotic avascular tissue in the graft is an extreme situation compared with the relatively small foci of damage
near a good blood supply that occurs in dystrophic
muscle, where probably few inflammatory cells are required to enable regeneration to proceed. The effect of
Remicade on whole muscle grafts contrasts with another
study, where TNF neutralizing antibody appeared to
have no effect on the early histological events of regeneration in response to freeze injury in C57Bl/6 mice; this
pattern corresponded to results when TNF function was
blocked in TNF double receptor knockout mice (43),
although some changes in gene expression and muscle
strength were reported. Clearly, the precise tool used to
block TNF function is of critical importance.
A striking effect was seen with Remicade during the
early acute phase of muscle necrosis (3 4 wk) in
dystrophic mice. Although the data at 12 wk suggested
that sustained Remicade treatment reduced the dystropathology of TA muscles over a longer period of
time, this effect was not statistically significant. Although it is widely considered that a cycle of necrosis
stabilizes and protects a dystrophic myofiber from
subsequent necrosis, this is clearly not the case (44).
Therefore, whereas the presence of central myonuclei
within a myofiber indicates that regeneration has occurred, it does not indicate how many times this

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GROUNDS AND TORRISI

individual myofiber has been subjected to repeated

necrosis/regeneration, and this should be taken into
account. Furthermore, although the presence of EBD
within myofibers certainly indicates sarcolemmal damage and leakiness, there is not always a close correlation
with histological myofiber necrosis (Shavlakadze et al.,
unpublished results) (40, 41). There is also great
variation between the pathology of different muscles
and individual mdx mice (40, 44, 45), and therefore
a detailed study of many muscles up to 20 wk of age
is in progress to thoroughly assess the impact of
Remicade. On the basis of this available information
in the mdx mouse (where the pathology is mild), it is
difficult to predict the potential overall benefit of
Remicade on the severe pathology seen in the dog
models of DMD (46, 47) or in clinical dystrophies
and dysferlinopathies (48).
The reduced breakdown of dystrophic muscle with
Remicade treatment suggests that this highly specific
anti-inflammatory drug may be a useful clinical intervention to treat DMD and other myopathies with a
pronounced inflammatory component such as dysferlinopathies (15, 48). The high specificity of Remicade is
advantageous compared with the existing use of antiinflammatory corticosteroids such as Prednisolone and
Deflazacort to treat DMD as these steroids are associated with severe adverse side effects such as weight gain
and osteoporosis (49, 50), and steroids should be
avoided for dysferlinopathies due to unrecoverable loss
of strength (48). The precise mode of action of these
steroidsOwhether they act by preventing muscle breakdown, suppressing inflammation, increasing the size
and strength of myofibers, enhancing regeneration, or
reducing fibrosisOremains unclear (50). In contrast,
the precise mode of action of Remicade is known: it
does not cause generalized suppression of the immune
system and has been used clinically to treat inflammatory disorders (34). Although it can have mild adverse
effects such as rash and infusion-related reactions,
there are few instances of severe reactions such as sepsis
or pneumonia (35, 51). Remicade has been used in
pediatric patients to treat disorders such as juvenile
idiopathic arthritis (52, 53), and Crohn's disease (54,
55) in children as young as 3 years (53), and children as
young as 18 months are now being treated.
The present study shows that treatment of mdx
mice with Remicade reduces the onset and intensity
of necrosis and the dystropathology. This presents a
new approach for using highly specific anti-cytokine
therapies to treat DMD. Further studies are required
to evaluate long-term benefits of Remicade treatment and to compare them with other clinically
relevant anti-cytokine drugs (34) such as the use of
soluble receptors that bind TNF (Enbrel) or interleukin-1 (56, 57). This striking protective effect combined with high specificity and few serious adverse
side effects for Remicade makes this simple strategy
a serious contender for the clinical treatment of
dystropathologies.
ANTI-TNF (REMICADE) THERAPY

We thank Dr. Melissa Spencer and Alexandra Potts of the

Department of Pediatrics and Duchenne Muscular Dystrophy
Research Centre, University of California, Los Angeles, USA, for
providing all of the muscle samples from the mdx/TNF(/)
mice for this analysis and for critical comments on the manuscript. The technical assistance and helpful comments of Marilyn Davies, Jason White, Monique Berendse, and Stuart
Hodgetts (UWA) are gratefully acknowledged.

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The FASEB Journal

Received for publication September 24, 2003.

Accepted for publication December 10, 2003.

GROUNDS AND TORRISI