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Daniel J Weiner

Publications and source records attributed to Daniel J Weiner.

9 recordsLinked to original sources
PubMed2003

Airway epithelia regulate expression of human beta-defensin 2 through Toll-like receptor 2.

The goal of this study is to investigate whether TLR2 mediates hBD2 induction through NF-kappaB in response to bacterial components in the human airway epithelia. We showed that hTLR2 is expressed in the airway epithelial cells by immunohistochemical staining and RT-PCR. The biology of hTLR2 in this context was studied initially in 293 cells transfected with a plasmid expressing hTLR2 together with an hBD2 promoter-driven luciferase reporter (hBD2-promoter-LUC). Upon incubation with lipoteichoic acid (LTA), the major cell wall component of gram-positive bacteria, luciferase activity was greatly increased compared with mock stimulation. Analysis of mutation constructs of the hBD2 promoter revealed that NF-kappaB sites are important for hTLR2-mediated hBD2 up-regulation upon LTA stimulation. When hBD2-promoter-LUC was transfected into primary human airway epithelia cells (EC), the luciferase activity was greatly increased upon LTA stimulation compared with mock stimulation. The hBD2 promoter mutation constructs were also tested in EC, which confirmed the studies in 293 cells. When a plasmid expressing a dominant-negative mutant of hTLR2 was co-transfected with hBD2-promoter-LUC into EC, LTA could not stimulate hBD2 expression. These data provide convincing evidence that up-regulation of hBD2 can be induced through hTLR2-mediated NFkappaB/IkappaB pathway in the human airway epithelial cells.

Cell Line↗
PubMed2003

Human fetal trachea-SCID mouse xenografts: efficacy of vesicular stomatitis virus-G pseudotyped lentiviral-mediated gene transfer.

BACKGROUND/PURPOSE: Postnatal gene transfer in respiratory epithelium has been inefficient, particularly in submucosal gland cells, the target cells for cystic fibrosis transmembrane regulator (CFTR) gene transfer in cystic fibrosis. The authors hypothesized that fetal tracheobronchial epithelium may be more receptive to gene transfer in that precursor cells of the respiratory epithelium may be more accessible. METHODS: Vesicular stomatitis virus-G (VSV-G) pseudotyped lentiviral vector was first tested in human fetal tracheal organ culture then validated in a human fetal tracheal xenograft model in severe combined immunodeficiency (SCID) mice. Between 7 x 10(7) and 1.6 x 10(8) transducing units of lentiviral vector encoding the transgene LacZ under the control of the human CMV promoter were administered onto the lumenal surface of the xenografts (n = 6). Biopsy specimens were taken from the xenografts at one month (n = 1), 2 months (n = 2), 3 months (n = 1), 6 months (n = 4), and 9 months (n = 1) after vector administration. Analysis of transgene expression was performed on X-gal stained sections. RESULTS: Transgene expression was observed in 20.2% to 99% of the surface epithelial cells (mean, 70.8 +/- 32.3% SD) and in 28.4% to 99% of the submucosal gland cells (mean, 68.5 +/- 26.2% SD) out to 9 months after vector administration in the tracheal xenografts, equivalent to 63 weeks postconceptual age. No staining was seen in the controls. CONCLUSIONS: Excellent gene transfer in human fetal tracheal xenografts after VSV-G pseudotyped lentiviral vector administration, which may result from more accessible target precursor cells during development, suggests the feasibility of fetal gene therapy for the treatment of congenital airway disease such as cystic fibrosis.

Animals↗
PubMed2003

Infant pulmonary function testing.

Pulmonary function tests in infants have been used for many years, but they have been largely relegated to specialized centers and research purposes. More recently, development of commercial equipment and newer techniques has allowed for more broad use and application of these tests. This review describes different techniques that have been used to study infants and toddlers, and their clinical applications.

Child, Preschool↗
PubMed2003

Noninvasive measurement of the tension-time index in children with neuromuscular disease.

Respiratory muscle weakness is common in children with neuromuscular disease (NMD). We hypothesized that weakness puts them at risk for respiratory muscle fatigue, a harbinger of chronic respiratory failure. We therefore measured a noninvasive index of respiratory muscle fatigue, the tension-time index of the respiratory muscles (TT(mus)), in 11 children with NMD and 13 control subjects. Spirometric flow rates and maximal inspiratory pressure were significantly lower in the NMD group than in controls (43 +/- 23 vs. 99 +/- 21 cmH2O, P < 0.001). The mean TT(mus) was significantly higher in the NMD group than in controls (0.205 +/- 0.117 vs. 0.054 +/- 0.021, P < 0.001). The increase in TT(mus) was primarily due to an increase in the ratio of average mean inspiratory pressure to maximal inspiratory pressure, indicating decreased respiratory muscle strength reserve. We found a significant correlation between TT(mus) and the residual volume-to-total lung capacity ratio (r = 0.504, P = 0.03) and a negative correlation between TT(mus) and forced expiratory volume in 1 s (r = -0.704, P < 0.001). In conclusion, children with NMD are prone to respiratory muscle fatigue. TT(mus) may be useful in assessing tolerance during weaning from mechanical ventilation, identifying impending respiratory failure, and aiding in the decision to institute therapies.

Adolescent↗
PubMed2002

The antimicrobial activity of the cathelicidin LL37 is inhibited by F-actin bundles and restored by gelsolin.

Antimicrobial peptides are part of the innate host defense system, and inactivation of these peptides is implicated in airway infections in cystic fibrosis (CF). The sputum of patients with CF contains anionic polyelectrolytes, including F-actin and DNA not found in normal airway fluid. These anionic filaments aggregate to contribute to the altered viscoelastic properties of CF sputum. We hypothesized that the airway components stabilizing bundles of F-actin and DNA are in part cationic antimicrobial agents, and that appropriate modification of diseased airway fluid of patients with CF might dissociate these bundles and restore antimicrobial activity. We demonstrate that the human cathelicidin peptide LL37 forms bundles with F-actin and DNA, which are dissolved by gelsolin and DNase, respectively. Coincident with bundle formation, antimicrobial activity of LL37 is inhibited by F-actin and DNA. Pseudomonas bacteria were killed by low concentrations of LL37, but killing was significantly reduced in the presence of F-actin. The actin filament-fragmenting protein gelsolin restored bactericidal activity nearly completely. In a growth inhibition assay, the effects of F-actin were confirmed, and DNA was also shown to inhibit the activity of LL37. When added to CF sputum, gelsolin significantly reduced the growth of bacteria, suggesting activation of endogenous antimicrobial factors. These findings may have therapeutic implications for treatments previously thought to alter only the viscoelastic properties of airway secretions and amplify the possible advantage of gelsolin in CF treatment.

Actin Cytoskeleton↗
PubMed2002

Toll-like receptor 4 mediates innate immune responses to Haemophilus influenzae infection in mouse lung.

Toll-like receptors (TLRs) have been implicated in the regulation of host responses to microbial Ags. This study characterizes the role of TLR4 in the innate immune response to intrapulmonary administration of Haemophilus influenzae in the mouse. Two different strains of mice efficiently cleared aerosolized H. influenzae concurrent with a brisk elaboration of IL-1beta, IL-6, TNF-alpha, macrophage-inflammatory protein (MIP)-1alpha, and MIP-2 in bronchoalveolar lavage and a corresponding mobilization of intrapulmonary neutrophils. Congenic strains of mice deficient in TLR4 demonstrated a substantial delay in clearance of H. influenzae with diminished IL-1beta, IL-6, TNF-alpha, MIP-1alpha, and MIP-2 in bronchoalveolar lavage and a notable absence of intrapulmonary neutrophils. In TLR4-expressing animals, but not TLR4-deficient animals, TNF-alpha and MIP-1alpha expression was up-regulated in epithelial cells of the conducting airway in response to H. influenzae which was preceded by an apparent activation of the NF-kappaB pathway in these cells based on the findings of decreased overall IkappaB and an increase in its phosphorylated form. This study demonstrates a critical role of TLR4 in mediating an effective innate immune response to H. influenzae in the lung. This suggests that the airway epithelia might contribute to sensing of H. influenzae infection and signaling the innate immune response.

Administration, Inhalation↗
PubMed2002

Development of novel antibacterial peptides that kill resistant isolates.

The rapid emergence of bacterial strains that are resistant to current antibiotics requires the development of novel types of antimicrobial compounds. Proline-rich cationic antibacterial peptides such as pyrrhocoricin kill responsive bacteria by binding to the 70 kDa heat shock protein DnaK and inhibiting protein folding. We designed and synthesized multiply protected dimeric analogs of pyrrhocoricin and optimized the in vitro antibacterial efficacy assays for peptide antibiotics. Pyrrhocoricin and the designed dimers killed beta-lactam, tetracycline- or aminoglycoside-resistant strains of Escherichia coli, Salmonella typhimurium, Klebsiella pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis in the submicromolar or low micromolar concentration range. One of the peptides also killed Pseudomonas aeruginosa. The designed dimers showed improved stability in mammalian sera compared to the native analog. In a murine H. influenzae lung infection model, a single dose of a dimeric pyrrhocoricin analog reduced the bacteria in the bronchoalveolar lavage when delivered intranasally. The solid-phase synthesis was optimized for large-scale laboratory preparations.

Anti-Bacterial Agents↗
PubMed2002

beta-Defensin 1 contributes to pulmonary innate immunity in mice.

Innate immunity serves as a first line defense in vertebrate organisms by providing an initial barrier to microorganisms and triggering antigen-specific responses. Antimicrobial peptides are thought to be effectors of innate immunity through their antibiotic activity and direct killing of microorganisms. Evidence to support this hypothesis in vertebrates is indirect, based on expression profiles and in vitro assays using purified peptides. Here we investigated the function of antimicrobial peptides in vivo using mice deficient in an antimicrobial peptide, mouse beta-defensin-1 (mBD-1). We find that loss of mBD-1 results in delayed clearance of Haemophilus influenzae from lung. These data demonstrate directly that antimicrobial peptides of vertebrates provide an initial block to bacteria at epithelial surfaces.

Animals↗