4 research outputs found
Reduction in secondary pathological features.
<p>(A) Data demonstrates the reduction in overall skeletal muscle
inflammation and fibrosis from <i>mdx</i> treated with SMT
C1100 compared to vehicle only. SMT C1100 (50 mg/kg) or vehicle was
delivered daily by oral gavage to groups of six <i>mdx</i>
mice aged around 17 d for a total of 28 days. The TA, EDL, soleus, and
diaphragm were removed and five sections from each muscle were stained
and analysed blind by a board-certified veterinary pathologist for
evidence of inflammation and fibrosis using a standard pathology scoring
method described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019189#s2" target="_blank">methods</a>
section. Scoring (0–3) was made for each section from each muscle
then averaged for all muscles to give an overall assessment of
improvement in the pathological effects of dystrophin deficiency; (B)
Qualitative assessment of EDL muscle from SMT C1100-dosed
<i>mdx</i> scored as 1 = mild -
occasional mononuclear inflammatory cells in the inter-bundle connective
tissue with focal aggregations of mononuclear inflammatory cells. The
arrows mark foci of inflammation. Qualitative assessment of EDL dosed
with vehicle and scored as 2 = moderate - multiple
foci of mononuclear inflammatory cell infiltration in the inter-bundle
connective tissue; occasional mononuclear inflammatory cells between
individual muscle fibres.</p
<i>Ex vivo</i> analysis of SMT C1100 activity in the <i>mdx</i> mouse.
<p>(A) <i>mdx</i> mice were treated with SMT C1100 (50 mg/kg/day)
or vehicle only (0.1% Tween-20/5% DMSO in PBS) via daily
i.p. injection from two weeks of age for four weeks. Whilst contracting
tetanically, EDL muscles were stretched at 15% of their fibre
length. The difference in force produced between the first and fifth
stretch is represented as an indicator of the resistance of the muscle
to stretch-induced damage. *p<0.05;
**p<1.0×10<sup>−5</sup>; (B) levels of serum
creatine kinase following oral gavage of <i>mdx</i> mice with
50 mg/kg SMT C1100 or vehicle from three weeks of age for four weeks (C)
Muscles from the dosing described in (B) were processed to assess the
percentage of centrally nucleated fibres *p<0.01;
(C)***p = 0.0001;
**p = 0.005;
*p = 0.003.</p
<i>In vitro</i> activity of SMT C1100.
<p>(A) SMT C1100 dose response in murine <i>H2k-mdx</i> utrnA-luc
cells expressing the human utrophin promoter linked to a luciferase
reporter gene. Cells were treated with compound for 48 h in standard
growth medium containing 0.3% DMSO. The chart shows relative
luminescence (RLU) in relation to five different doses of SMT C1100. A
Four Parameter Logistic Model was used to generate an EC<sub>50</sub>.
Points represent a mean ±S.E. of three experiments and are
typical of the results for all batches of SMTC1100. The structure for
SMTC1100 is shown; (B) SMT C1100 significantly increased mRNA copy
number of the utrophin transcript in SkMC cells. In this assay Gene
Expression Assay 4326315 was used for β-actin detection and Gene
Expression Assay Hu01125984_m1 was used for utrophin transcript
detection (both Applied Biosystems). Cells were exposed to SMT C1100 in
standard media with 1% DMSO (vehicle) for 72 hours with six
biological replicates. *p = 0.01 relative to
vehicle only; (C) Utrophin protein levels in human DMD cell line treated
with SMT C1100 (1 µM) or vehicle (0.1% DMSO). Blots were
stained with anti-utrophin (MANCHO3; 1∶100) and ECL HRP-conjugated
anti-mouse antibody (GE Healthcare). Bands were quantified using Image J
and arbitrary units represent utrophin levels corrected for equal
loading by α-actinin immunostaining. Results represent a mean
± S.E based on n = 3.
†p = 0.00683; §p<0.001; #p<0.005
relative to vehicle-treated cells.</p
Effect of SMT C1100 on <i>in vivo</i> parameters of exercised <i>mdx</i> mice.
<p>(A) Maximal fore limb strength after 4 weeks of either exercise and/or
drug treatment. The values are mean ± SEM from the number of
animals shown in each bar (B) Normalized force increment, i.e.
difference between the mean values of normalized fore limb strength at
time 4 and at time 0. Normalized force values have been calculated for
each mouse as the ratio of maximal fore limb strength to mouse body
weight. The values are mean ± SE. The SE of ΔNF has been
calculated as detailed elsewhere <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019189#pone.0019189-DeLuca1" target="_blank">[14]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019189#pone.0019189-DeLuca2" target="_blank">[21]</a>.
For (A) and (B) statistical significance between groups was evaluated by
ANOVA test for multiple comparison (F-values) and Bonferroni t-test
<i>post hoc</i> correction. Significantly different versus
<b>*</b>sedentary mdx and <sup>§</sup>Exer
<i>mdx</i>; p<0.05; (C) Resistance to treadmill
running, calculated as the maximal distance the mouse can run when
undergoing a single bout of treadmill exercise. The values are mean
± SEM from 3–7 mice and show the maximal distance run (in
meters) at T0 (start of forced exercise and dosing) and after four (T4)
and five weeks (T5). Statistical significance between groups was
evaluated by ANOVA test for multiple comparison (F-values) and
Bonferroni t-test <i>post hoc</i> correction. Highly
significant differences were observed between groups and within groups
at the different ages (F>10; p<0.005). The symbols show
statistical differences *versus sedentary <i>mdx</i> at T4
and #versus vehicle-treated exercised <i>mdx</i> at either T4
or T5 (p<0.05 and less).</p