Selected articles from ATS - 2004 - American Thoracic Society, May 21-26, Orlando
1)
Full-Length Dystrophin Gene Transfer to Dystrophic (MDX) Mouse Diaphragm
with a Gutted Adenoviral Vector
S. Matecki, M. Divangahi, R. Dudley, G. Danialou, J. Nalbantoglu, G.
Karpati, B.J. Petrof Respiratory Division, McGill University Health Center and
Meakins-Christie Laboratories, Montreal, QC, Canada; Neuromuscular Research
Group, Montreal Neurological Institute, McGill University, Montreal, QC, Canada
RATIONALE: Duchenne muscular dystrophy (DMD) is caused by defects in the
dystrophin gene, and patients most frequently die of ventilatory failure. Early
attempts at dystrophin gene transfer with adenoviral vectors have been hampered
by host immune responses against the vector.
METHODS: In this study, we used a helper-dependent adenoviral vector
encoding two full-length murine dystrophin cDNAs and regulated by a powerful
cytomegalovirus enhancer/b-actin promoter (referred to as HDCBDys), to achieve
dystrophin gene replacement in the diaphragm of immunocompetent adult (3 months
of age) mdx mice (an animal model of DMD).
RESULTS: At 1 week post-injection of HDCBDys, 165
+27
fibers (20.6 +4
% of total fibers in a diaphragm bundle) expressed dystrophin at the sarcolemma.
This level of expression remained stable over the study duration (30 days
post-injection), with 188 +37
fibers (23.8
+5
% of total fibers) expressing dystrophin. Treated diaphragms also showed
significantly improved resistance to damage induced by high-stress muscle
contractions. These benefits were achieved despite the presence of mildly
increased inflammation, characterized by CD4+ and to a lesser extent CD8+
lymphocytes, within the HDCBDys-treated diaphragms.
CONCLUSIONS: On the basis of these findings, we conclude that HDCBDys is
a promising vector for treating DMD, particularly since the mdx mouse
diaphragm exhibits severe morphological and functional changes which are similar
to those found in DMD patients.
2)
Functional Regeneration of Dystrophic (MDX) Mouse Diaphragm after
Experimentally-Induced Injury
S. Matecki, G.H. Guibinga, M. Divangahi, R. Dudley, G. Danialou, B.J.
Petrof Respiratory Division, McGill University Health Center and
Meakins-Christie Laboratories, Montreal, QC, Canada
RATIONALE: Duchenne muscular dystrophy (DMD), which is caused by a lack
of dystrophin protein, is characterized by abnormal muscle fiber fragility and
recurrent episodes of muscle necrosis. Early in the disease, muscle fiber loss
is counterbalanced by effective myofiber regeneration. However, in the later
stages of disease, a lack of regeneration leads to the progressive replacement
of muscle tissue by fibrosis. The eventual failure of compensatory regeneration
has been attributed to a loss of intrinsic regenerative capacity. Therefore, we
assessed the regenerative capacity of the dystrophin-deficient mdx mouse
diaphragm, by testing the ability of the muscle to achieve functional recovery
after experimentally-induced injury.
METHODS: The myonecrotic agent notexin, which destroys mature myofibers
while leaving myogenic precursor (satellite) cells intact, was applied directly
to the hemidiaphragmatic surface of mdx and wild-type C57BL10 control
mice (6 months old; n=18 per group). The contralateral hemidiaphragm served as a
within-animal control. At 4 hours and 60 days after notexin application, in
vitro contractile properties as well as histopathological parameters were
examined.
RESULTS: Mdx diaphragms exposed to notexin achieved vigorous
muscle fiber regeneration as well as complete functional recovery to pre-injury
values of force generation. Interestingly, normal wild-type diaphragms did not
demonstrate complete recovery to pre-injury values of force production under the
same conditions.
CONCLUSIONS: Our data suggest that despite established features of
dystrophic pathology, there is significant residual regenerative capacity within
the dystrophic diaphragm, which may even exceed that in normal diaphragm muscle.
Therefore, further efforts should be directed at exploiting this latent
regenerative potential for therapeutic purposes in DMD.
3)
Localization and Early Time Course of TGF-β1
mRNA Expression in Dystrophic (mdx) Diaphragm Muscle
L.E. Gosselin, J. Williams, M. Deering, D. Brazeau, S. Koury University
at Buffalo, Buffalo, NY
Diaphragm muscle fibrosis is a key feature in the pathological progression of
muscular dystrophy. Although we previously reported that the level of TGF-
β1
protein is significantly elevated in diaphragm from mdx mice at 12 weeks
of age, little is known about the onset of TGF-
β1
mRNA expression in mdx diaphragm, or of which cells produce the mRNA. The
purpose of this study was to examine the time course of TGF-
β1
mRNA expression in diaphragm muscle from mdx mice aged six to twelve
weeks, and to localize the cells responsible for the production of TGF-
β1
mRNA. Diaphragm muscles from control (C57BL/10ScSn) and mdx mice aged 6-,
9-, and 12-wks were removed and processed for analysis. TGF-
β1
and type I collagen mRNA was analyzed using real time quantitative PCR whereas
in situ hybridization was used to localize TGF-
β1
mRNA. Hydroxyproline concentration, assessed by HPLC, was significantly higher
in mdx diaphragm at each age group. There was a significant age- and
group effect for both TGF-
β1
and type I collagen mRNA. TGF-
β1
and type I collagen mRNA levels were significantly higher in mdx
diaphragm compared with controls, and their levels significantly decreased with
age. In situ hybridization revealed TGF-
β1
mRNA was localized to areas of cellular infiltration and not over healthy muscle
cells. Results from this study reveal the disease process in mdx mice
begins at a young age and that inflammatory cells play a role in the
pathogenesis of muscle fibrosis.
4)
Cytokine and Chemokine Expression Levels Are Higher in Diaphragm Than in Limb
Muscles of Dystrophic (mdx) Mice
A. Demoule, M. Divangahi, S. Matecki, W. Bao, B.J. Petrof Respiratory Div.
&, Meakins-Christie Laboratories, McGill University, Montreal, QC, Canada
RATIONALE: Duchenne muscular dystrophy (DMD) is caused by absence of the
dystrophin protein. In dystrophin-deficient mdx mice, the diaphragm
exhibits a histopathological and functional picture which is phenotypically
similar to human DMD, whereas the limb muscles are much less affected. Therefore,
comparisons between diaphragm and limb muscles in this model may offer insights
into disease pathogenesis.
METHODS: Ribonuclease Protection Assays were performed on total RNA
obtained from diaphragm and tibialis anterior (TA) muscles, in order to compare
expression levels of the following: TNFa, IFNg, IL1a, IL1b, IL6, IL18, MIP1a,
MIP2, and RANTES. To evaluate the effects of disease progression on cytokine/chemokine
expression in these muscles, comparisons were also made between mdx and
wild-type control BL10 mice at early (5 weeks old) and late (6 months old)
stages of the disease.
RESULTS: Levels of cytokine/chemokine expression in both diaphragm and
limb muscle were significantly higher in mdx mice than in age-matched
BL10 controls. In addition, cytokine/chemokine expression was greatly increased
(e.g., 12-fold for TNFa and IL1a) in the mdx diaphragm as compared to TA.
Expression levels for certain cytokines/chemokines within the mdx
diaphragm also showed large differences at early and late phases of the disease.
Hence the levels of expression of TNFa, MIP1a and RANTES were substantially
higher in the late (fibrogenic) phase, whereas IFNg, IL1a, and IL6 were greater
in the early (necrotic) phase.
CONCLUSIONS: Expression of multiple cytokines/chemokines is higher in the
mdx diaphragm than in limb muscle. Moreover, the kinetics and pattern of
cytokine/chemokine expression in the diaphragm differs at different stages of
the disease process. Although the precise role of these inflammatory mediators
remains to be determined, cytokine/chemokine modulation may have therapeutic
potential in DMD.
5)
The National Airway Clearance Registry (NACR) for Patients with Respiratory,
Neurological and Other Conditions Associated with Airway Clearance Complications
R.L. Morton, D.R. Hess, A.S. Gelfand, S.M. Julius, M.W. Konstan, F.J.
Accurso Kosair Children's Hospital, Louisville, KY; Massachusett's General
Hospital, Boston, MA; Pediatric Pulmonary Associates, Dallas, TX; Georgia
Pediatric Pulmonology Associates, Atlanta, GA; Rainbow Babies and Children's
Hospital, Cleveland, OH; Denver Children's Hospital, Denver, CO
Purpose: To document the use of airway clearance therapies (ACTs) and
related clinical and healthcare utilization outcomes in various patient
populations.
Methods: Consenting patients that are
>2
yo, clinically stable, and naive to high-frequency chest wall oscillation (HFCWO)
home therapy, but have one or more indications for an ACT, are enrolled into the
NACR at IRB-approved facilities. Patient data on treatments, clinical measures
and healthcare utilization are collected and entered into a Web-based data
collection system. Long-term assessment is done on all NACR patients whether or
not they continue with HFCWO therapy after a short-term trial.
Results: More than 75 facilities in the U.S. are currently enrolled in
the NACR. To date, patients with primary diagnoses of cystic fibrosis (CF),
asthma, bronchiectasis, primary ciliary dyskinesia, recurrent pneumonia,
cerebral palsy, muscular dystrophy, brain and spinal cord injury as well as
others have been enrolled (N = 222). Currently, 53% of the NACR patients have a
primary respiratory disease or condition whereas 40% have a primary neurological/neuromuscular
(neuro) disorder. At the time of NACR enrollment, the majority of neuro patients
(73%) reported current use of ACT; whereas, 43% of the respiratory patients were
naive to ACT. Healthcare utilization due to pulmonary complications for these
patients, regardless of disease category, had no significant differences in any
of the following: clinic, ER, hospital and ICU visits and antibiotic use.
Conclusion: Despite the differences in etiology, patients with primary
diagnoses in the respiratory or neuro disease category have highly comparable
healthcare utilization for pulmonary complications. The NACR may help to better
discern the ACT needs and benefits within different patient groups.
6)
Barthel Index (BI) and EK Scale (EK) Similarly Reflect Respiratory Impairment in
Duchenne Muscular Dystrophy (DMD)
J.A.B. Martinez, M. Brunherotti, J. Terra Filho, M.R. Assis, E. Vianna,
C. Sobreira Pulmonary Division, Medical School of Ribeirao Preto - University of
Sao Paulo, Ribeirao Preto, SP, Brazil; Rehabilitation Division, Medical School
of Ribeirao Preto - University of Sao Paulo, Ribeirao Preto, SP, Brazil;
Neurology Department, Medical School of Ribeirao Preto - University of Sao
Paulo, Ribeirao Preto, SP, Brazil
Background: Peripheral and respiratory muscular weakness progressively install
in DMD. Measurements of functional status and activities of daily living (ADL)
reflect peripheral muscular impairment, and may correlate to respiratory muscle
function, as well. Objective: To investigate how two commonly employed scales of
functional status, (BI and EK), correlate with respiratory parameters in DMD
patients. Material and Methods: We have studied 20 patients with DMD (age:12.6 +
3.8 years) not submitted to
any ventilatory support. They were asked to perform at a same occasion BI and EK
scales, spirometry, assessment of respiratory strength, and arterial blood
gases. Results: The group showed mean
SD
values of EK and BI of 8.5
+7,6 and 51.5
+25.3. Respiratory
function showed: CPT=84.6
+23.9 %; CVF=61.2
+23.6%; VEF1=60.7
+23.1%; MIP=73.6
+26.3%; MEP=62.5
+42.7%; pH=7.45
+0,02; PaO2=90.8
+4.1 mmHg; PaCO2=36.6
+2.7 mmHg. Both EK
and BI showed significant correlations with MEP, MIP, CPT, CVF and VEF1. Poor
scores of ADL were associated to reductions in pulmonary volumes and respiratory
muscle weakness. The values of the correlations obtained between the scores and
pulmonary function were similar for both scales. Conclusions: Measurements of
functional status reflect respiratory muscle function in patients with DMD. Both
BI and EK scales are equally useful in this setting.
7)
Controlled Trial of Intrapulmonary Percussion in Adults and Children with Stable
Severe Neuromuscular Disease
M. Chatwin, D.M. O'Driscoll, D. Corfield, M.J. Morrell, A.K. Simonds
Department of Sleep and Ventilation, Royal Brompton Hospital, England; Clinical
Academiec Unit of Sleep and Breathin, National Heart and Lung Insitute, Imperial
College, England; MacKay Institute of Communication and Neuroscience, School of
Life Sciences,Keele University, England
Intrapulmonary percussive ventilation (IPV) is a novel method of airway
clearance in which high frequency oscillation is applied to the airway. Our aim
was to investigate the effect of non-invasive IPV on ventilation and gas
exchange in adults/children with neuromuscular disease (NMD). Method 15
stable patients, median age 18, range 10-56 yrs, Duchenne MD 7, SMA 5,
congenital MD 3 were included. All had a weak cough and recurrent chest
infections. Each received
'sham treatment'
(S) or treatment (T) with the IMP II (Percussionaire, Breas, Sweden) in random
order; frequency and pressure titrated to comfort (pressure 0.7-1.0bar,
frequency 45-236 Hz).Respiratory muscle strength (RMS) was measured pre and post
intervention. SaO2 and transcutaneous CO2 (TCO2),
Ttot and RR were assessed throughout; comfort and breathlessness were measured
on VAS. Results: Baseline values (mean+SD) FEV1 0.64
+0.53L, FVC 0.77
+0.62L, PImax
and PEmax (21
+16 and 20+12)cmH2O
and unassisted peak cough flow (PCF) 128
+76 L.min-1.
TCO2 and SaO2 baseline measurements were 6.4+1.3kPa
and 98
+1.4%. For the S or T
session there was no significant difference in TCO2 , SaO2 ,
RMS or Ttot (S=2.3
1.9
and T=2.7+2.2)s or RR (S=28
+11.4 and T=25
+11.4)min-1.
Between S and T comfort on VAS (3.9
+2.6and 2.6
+2.5)cm and Borg
scale were (1.6
+1.5 and 1.6+1.8)
there was no difference. Three subjects withdrew due to poor tolerance of the
device and two could only tolerate the device at a low frequency. Conclusion:
IPV was well tolerated by the majority of steady state severe NMD patients
in this study. There was no change in RR, Ttot, RMS, SaO2 and TCO2.
Three individuals who could not tolerate IPV had features of bulbar
insufficiency and therefore may have upper airway incoordination with IPV. The
next step is to assess the effectiveness of IPV during an acute chest infection.