A third-generation adenoviral vector containing recombinant human cystic fibrosis transmembrane conductance regulator (CFTR) gene was delivered by bronchoscope in escalating doses to the conducting airway of 11 volunteers with cystic fibrosis. Assessments of dose-limiting toxicity (DLT), efficiency of gene transfer, and cell-mediated and humoral immune responses to vector administration were performed. DLT, manifest by flulike symptoms and transient radiographic infiltrates, was seen at 2.1 x 10(11) total viral particles. A highly specific assay for gene transfer was developed using in situ hybridization with an oligoprobe against unique vector sequence. Detectable gene transfer was observed in harvested bronchial epithelial cells (<1%) 4 days after vector instillation, which diminished to undetectable levels by day 43. Adenovirus-specific cell-mediated T cells were induced in most subjects, although only mild increases in systemic humoral immune response were observed. These results demonstrate that gene transfer to epithelium of the lower respiratory tract can be achieved in humans with adenoviral vectors but that efficiency is low and of short duration in the native CF airway.
Inhaled dehydroepiandrosterone-3-sulfate (DHEAS), but not dehydroepiandrosterone (DHEA), possesses anti-inflammatory activity in in vitro assays and in models of allergen and lipopolysaccharide challenges. We postulated whether an inhaled suspension of DHEAS delivered via nebulizer would improve asthma control in moderate-to-severe asthma patients. We also characterized the safety profile of an inhaled suspension of DHEAS. Patients receiving at least 500 μg of fluticasone equivalent plus long-acting beta-agonists (LABA) entered a 5-week run-in where the dose of inhaled corticosteroids was reduced to 200 μg of fluticasone plus LABA per day. Patients were randomized to 70 mg of DHEAS or placebo if their Asthma Control Questionnaire (ACQ) score was ≥2.0 and their FEV(1) ≥ 50%. When compared with control, a statistically significant improvement in ACQ in 6 weeks of treatment with 70 mg of DHEAS was observed. The median improvement in ACQ was -0.72 and -0.43 for the active and placebo groups, respectively (p = 0.0389); the percentage of patients with at least minimally clinically important difference of -0.50 from baseline was significantly greater in the DHEAS group versus the placebo, (59.4% versus 45.7%; p = 0.0236). Asthma symptom scores, the proportion of symptom-free days and symptom nights, although not statistically significant, had positive trends supporting the improvement in ACQ. Fewer patients were withdrawn from the study for respiratory events on DHEAS compared with placebo. There were few adverse events and no changes in sex hormones despite increases in circulating levels of DHEAS. An inhaled suspension of DHEAS delivered via nebulizer improved asthma control scores in subjects with poorly controlled moderate-to-severe asthma. AUSTRALIAN NEW ZEALAND CLINICAL TRIALS REGISTRY ANZCTR.ORG.AU IDENTIFIER: 012607000192482.
Advances in our understanding of asthma pathogenesis and delineation of the human genome project are yielding novel candidate targets for therapeutic intervention. In parallel with target identification, the past decade has produced novel approaches to normalizing expression genes that are upregulated in disease processes. Single-stranded antisense oligonucleotides and double-stranded short-interfering RNA molecules, which specifically mark target transcripts for degradation, are being investigated for their ability to modulate disease processes. In both cases, the targets are RNA transcripts, and not protein; therefore, all types of molecular gene products can be inhibited, including historically undrugable species such as transcription factors and phosphatases. Various RNA interference strategies have been successfully tested in vitro and in animal models of disease, and data is beginning to accumulate from human clinical trials.EPI-2010, a 21-mer phosphorothioate against the adenosine A1 receptor promoter region,has completed preclinical pharmacology testing and its initial clinical trials. The rationale for EPI-2010 is that overactivity of the adenosine signaling pathway in asthmatic lungs contributes to airway inflammation and hyperresponsiveness. Phase I/IIa clinical trials have shown EPI-2010 to be safe and well-tolerated, with modest indications of efficacy in patients with mild asthma.
We previously demonstrated that human tracheobronchial epithelial (TBE) cells synthesize mucin and form mucous granules in culture when they are maintained on a collagen gel (CG) substratum, but not on a plastic tissue culture surface or a thin collagen-coated surface (Wu et al., Am. J. Respir. Cell Mol. Biol., 3:467-478; 1990). This observation led us to examine the effects of CG thickness on cell growth and differentiation in primary human/monkey TBE cell cultures. Using the same CG preparation, culture dishes with different thicknesses of CG substratum were prepared. In general, equivalent degrees of cell attachment and proliferation were observed in all cultures maintained on a collagen gel, independent of the thicknesses of CG substratum. However, a greater degree of mucin synthesis and secretion by the cells was observed as the thickness of the CG substratum was increased. Cultures maintained on a thick collagen gel (1 mm) exhibited greater apical membrane complexity, more pseudostratification, and more mucous granules than did cultures maintained on a thin CG substratum. The optimal culture surface for airway mucous cell differentiation contains more than 1-mm thickness of collagen gel substratum.
Previous studies using the CA 19-9 antibody have demonstrated that serum mucin levels in patients with cystic fibrosis (CF) are elevated and that the degree of elevation relates to the age of the patient and possibly to his or her clinical status. However, CA 19-9 only recognizes the mucin-associated blood group sialyl Le(a+) antigen, so mucin levels cannot be measured in patients without Lewis antigens. The present study used the 17B1 monoclonal antibody to measure serum mucin levels in normal subjects, and in patients with CF, patients with chronic obstructive pulmonary disease (COPD), and patients with lung transplants. Serum mucin levels were 25 ng/ml (+/- 1 SEM, n = 8) in normal subjects, 13,853 ng/ml (+/- 1,281, n = 25) in patients with CF, and 25.5 ng/ml (+/- 1.9, n = 17) in patients with COPD. Patients with CF who were sialyl Le(a-b-) also had elevated serum mucin levels (715 +/- 152, n = 2). Serum mucin levels of six lung transplant recipients with CF were elevated compared with those in normal subjects (4,621 +/- 765 ng/ml), but they were not different from serum mucin levels in six lung transplant recipients without CF (5,307 +/- 1.677 ng/ml). Preliminary characterization of the serum mucin antigen showed that: (1) in CF sera, the antigen is polydisperse and smaller than the antigen in normal sera; (2) the mucin antigen is distinct from ABO blood group antigens. Serum mucin levels may be a useful marker to follow a specific patient's response to therapy.
Although reversible airway obstruction in part defines asthma, lung function as measured by spirometry alone inadequately predicts the value of new therapeutic agents in the treatment of severe asthma. Our objectives are 1) to review whether pulmonary function and bronchodilator reversibility are endpoints for drug discovery and 2) to identify parameters that predict efficacy in drug development in severe asthma. An English language literature search using MedLine and PubMed was conducted from 1997 to present concerning pathophysiology, diagnosis and therapy of severe asthma using the terms “severe asthma,” “irreversible asthma,” “difficult asthma,” “airway remodeling,” “fixed airway obstruction,” “reversibility” and “bronchodilator reversibility” as index terms. Eight studies were characterized that encompass 1,424 subjects with asthma. Our review identified the limitations of using bronchodilator reversibility as a predictor in drug development for severe asthma. Neither improvement in lung function nor bronchodilator reversibility characterized the benefit of new drugs in the treatment of severe asthma. Newly approved drugs in the treatment of severe asthma show decreased asthma exacerbations and improved quality of life associated with steroid-sparing benefits without altering bronchodilator responsiveness or improving lung function. Although changes in lung function predict asthma control in mild/moderate asthma, lung function alone is inadequate to assess improvement in asthma control in severe asthma manifested by fixed airway obstruction. Endpoints that focus on asthma control, as defined by the Expert Panel Report 3 and GINA guidelines, may predict the value of new therapeutics in the management of severe asthma.
Acid hydrolases are synthesized as precursors that undergo several posttranslational modifications including proteolytic processing to a smaller mature enzyme. The amount of proteolytic processing varies for different acid hydrolases, and many details of the intracellular pathways are not known. The processing of alpha-L-fucosidase was distinguished from that of other acid hydrolases reported when studied in systematic pulse-chase labeling experiments. Only one form of alpha-L-fucosidase, Mr 56,000-57,000, was demonstrated intra- and extracellularly. Under the same conditions, N-acetyl-beta-D-glucosaminidase was shown to be processed with several forms, as previously reported by Hasilik and Neufeld (1980a). To obtain these results, human skin fibroblasts were labeled metabolically with L-[3H]leucine for periods of 20 min to 8 hr with varying periods of chase from 1 to 96 hr with nonradioactive L-leucine. alpha-L-Fucosidase was immunoprecipitated by a polyclonal antibody from material extracted from cells and ammonium sulfate precipitated medium and was examined by polyacrylamide gel electrophoresis under denaturing conditions. N-Acetyl-beta-D-glucosaminidase was examined with similar procedures and served as a control for the methods. Tunicamycin treatment of the cells was used to show that glycosylation did not obscure proteolytic processing because, again, only one form of the intra- and extracellular enzyme was observed, although of smaller size, Mr 52,000-53,000. In addition, separation of the cells into prelysosomal and lysosomal fractions showed only one form of the enzyme. It is concluded that alpha-L-fucosidase does not undergo proteolytic processing in human skin fibroblasts in the usual manner described for other acid hydrolases.
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