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, PI_max and PE_max (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.