[show abstract][hide abstract] ABSTRACT: Use of perfluorochemical liquids during intratracheal vector administration enhances recombinant adenovirus and adeno-associated virus (AAV)-mediated lung epithelial gene expression. We hypothesized that inhalation of nebulized perfluorochemical vapor would also enhance epithelial gene expression after subsequent intratracheal vector administration. Freely breathing adult C57BL/6 mice were exposed for selected times to nebulized perflubron or sterile saline in a sealed Plexiglas chamber. Recombinant adenoviral vector was administered by transtracheal puncture at selected times afterward and mice were killed 3 days after vector administration to assess transgene expression. Mice tolerated the nebulized perflubron without obvious ill effects. Vector administration 6 hr after nebulized perflubron exposure resulted in an average 540% increase in gene expression in airway and alveolar epithelium, compared with that with vector alone or saline plus vector control (p<0.05). However, vector administration 1 hr, 1 day, or 3 days after perflubron exposure was not different from either nebulized saline with vector or vector alone and a 60-min exposure to nebulized perflubron is required. In parallel pilot studies in macaques, inhalation of nebulized perflubron enhanced recombinant AAV2/5 vector expression throughout the lung. Serial chest radiographs, bronchoalveolar lavages, and results of complete blood counts and serum biochemistries demonstrated no obvious adverse effects of nebulized perflubron. Further, one macaque receiving nebulized perflubron only was monitored for 1 year with no obvious adverse effects of exposure. These results demonstrate that inhalation of nebulized perflubron, a simple, clinically more feasible technique than intratracheal administration of liquid perflubron, safely enhances lung gene expression.
Human gene therapy methods. 05/2012; 23(2):98-110.
[show abstract][hide abstract] ABSTRACT: Recent literature suggests that adult bone marrow-derived cells can localize to lung and acquire immunophenotypic characteristics of lung epithelial cells. We speculated this might be a potential therapeutic approach for correcting defective lung epithelium in cystic fibrosis.
To determine whether adult bone marrow-derived cells containing normal cystic fibrosis transmembrane conductance regulator protein (CFTR) could repopulate lung epithelium in transgenic mice deficient in that protein.
Stromal marrow cells or total marrow obtained from adult male wild-type mice were transplanted into adult female Cftr knockout mice. To increase marrow cell recruitment naphthalene was used to induce airway epithelial injury in recipient mice.
At 1 wk, 1 mo, and 3 mo after transplantation, Cftr mRNA was detected in lung homogenates of recipient mice by reverse transcription-polymerase chain reaction. Cftr mRNA was not found in either donor marrow cells or mature circulating leukocytes. In situ examination of recipient mouse lungs demonstrated rare (0.025%) chimeric airway epithelial cells, some of which (0.01%) expressed CFTR protein. Naphthalene-induced airway remodeling nonsignificantly increased the number of chimeric airway epithelial cells expressing Cftr.
These results demonstrate that adult marrow cells can be recruited to airway epithelium and induced to express Cftr in mice otherwise lacking this protein. However, the number of observed chimeric epithelial cells is small and new strategies for enhancing airway epithelial remodeling by adult bone marrow-derived cells will be necessary for correction of defective CFTR-dependent chloride transport.
American Journal of Respiratory and Critical Care Medicine 02/2006; 173(2):171-9. · 11.04 Impact Factor
[show abstract][hide abstract] ABSTRACT: Adult marrow-derived stem cells can localize to lung and acquire immunophenotypic characteristics of lung epithelial cells. Lung injury increases recruitment of the marrow-derived cells. We speculated that comparing patterns of lung engraftment following different lung injuries would provide insight into potential mechanisms by which marrow-derived cells were recruited to lung. To evaluate this, adult female C57Bl/6 mice irradiated and engrafted with marrow from adult male transgenic GFP mice were exposed to either intranasal inhalation of endotoxin (25 microg/mouse) or 3 days of 25 ppm NO(2) and then compared 1 or 3 months later to transplanted but otherwise uninjured mice. In all cases, the majority of marrow-derived cells recruited to lung were CD45(+) leukocytes. In lungs of transplanted but otherwise uninjured mice, small numbers of CD45(-) donor-derived cells in alveolar septae stained positively for pro-surfactant protein C. Rare donor-derived cells located in the airway epithelium stained positively with cytokeratin. Subsequent exposure of engrafted mice to NO(2) or endotoxin did not significantly increase the number or pattern of donor-derived CD45(-) cells found in recipient lungs. These results suggest that NO(2) or endotoxin lung injury does not result in significant engraftment of marrow-derived cells in lung.
[show abstract][hide abstract] ABSTRACT: Several recent studies have demonstrated localization of donor bone marrow-derived cells in recipient lungs following transplantation from male to female mice or patients. Donor cells are identified by detection of the Y chromosome by fluorescence in situ hybridization (FISH). However, protein digestion pretreatments usually required for tissue FISH significantly limit the ability to detect cell type-specific markers by immunohistochemistry. We have used an alternative protein digest approach that entails heating the slides in 10 mM sodium citrate rather than utilizing a protease digestion. This approach preserves cell proteins following FISH, and allows lung tissue to remain intact for subsequent detection of cell-specific markers by immunohistochemistry. We have examined this technique in mouse lungs using a Y chromosome paint and three cell-specific markers, a pan-cytokeratin for epithelial cells, PECAM-1 for endothelial cells, and CD45 for leukocytes. Excellent visualization of both the Y chromosome and cell-specific surface protein markers was obtained on a single slide. This approach will significantly enhance the ability to detect and identify donor bone marrow cells in recipient mouse lungs following male to female transplantation.
[show abstract][hide abstract] ABSTRACT: Intratracheal instillation of perfluorochemical (PFC) liquids enhances lung epithelial transgene expression by improved vector propulsion throughout lung airways. We now demonstrate that PFC liquids also facilitate gene transfer by increasing transepithelial permeability. Apical application of PFC liquid to well-differentiated human airway epithelial cells resulted in a transient decrease in transepithelial resistance peaking approximately 2 h after PFC liquid administration and returning to normal approximately 24 h later. The permeability change was sufficient to enhance access of apically applied 100-nm latex beads and adenoviral vectors to the basolateral side of the culture. Adenovirus-mediated gene expression was concurrently enhanced. Following intratracheal instillation of PFC liquid into mouse lungs, tight junction permeability, as assessed by electron microscopic evaluation of lanthanum deposition, was increased with peak effect between 6 h and 1 day after instillation. Importantly, alveolar-capillary permeability remained unchanged with the treatment. Administration of PFC liquid 6 h or 1 day, but not 3 days, prior to instillation of a recombinant adenovirus vector enhanced gene expression comparable to that observed with concurrent administration of PFC liquid and vector. We conclude that transient increase in epithelial permeability after PFC liquid administration contributes to the enhancement of adenovirus vector-mediated gene expression in lung epithelium.
[show abstract][hide abstract] ABSTRACT: A novel approach for treatment of cystic fibrosis and other lung diseases is suggested by recent studies demonstrating that airway and alveolar epithelium in adult mice can be repopulated with cells derived from bone marrow of adult donor mice. The mechanisms by which marrow-derived cells transform into epithelial cells are unknown. However pre-existing lung injury increases localization of bone marrow cells to lung epithelium suggesting that active repair or remodeling of lung epithelium may create a more conducive environment for bone marrow cells to lodge and transform into epithelial cells. We have previously demonstrated that donor-marrow-derived cells could localize to lungs of CFTR KO mice and that different experimentally-induced injury patterns could influence the distribution of the recruited marrow cells in the recipient lungs. We now demonstrate that wildtype CFTR mRNA is expressed in CFTR-KO lungs following administration of donor marrow cells from animals containing wtCFTR. Adult female CFTR-KO mice aged 6-12 weeks received intraperitoneal administration of naphthalene (275 mg/kg body weight of a stock solution of 10 ml/kg in sterile soybean oil) to induce airway lung epithelial injury. 3-4 days later, at the time of peak lung injury, treated and naïve control CFTR KO mice received tail vein injection of 1 × 106 stromal marrow cells originally obtained from transgenic constitutive GFP-expressing donors (C57Bl/6 background) and cultured for 10 days prior to use. Mice were euthanized 1 week and 3 months after donor marrow cell administration and the lungs flash frozen in liquid N2, homogenized, and RNA extracted with Trizol. DNAse-treated purified RNA was reverse transcribed followed by PCR amplification using specific primers for murine CFTR (mCF11/mCF12). wtCFTR was observed in naphthalene-injured CFTR-KO mouse lungs collected 3 months but not 1 week after donor marrow administration. No wtCFTR expression was detected in un-injured transplanted CFTR-KO mice at either time point. Importantly, wtCFTR mRNA expression was not detected in donor marrow cells or in mature circulating leukocytes. These results suggest that donor marrow cells that traffic to lung subsequently express wtCFTR as they acquire an epithelial cell phenotype. Lung injury appears to increase this process. Importantly, these results suggest that marrow cells can directly traffic to lung and express a specific lung epithelial protein. Engraftment of the recipient marrow may not be necessary.
[show abstract][hide abstract] ABSTRACT: In female rats, ovariectomy (OVX) is associated with increased body fat and insulin resistance, and estradiol replacement prevents these alterations. These metabolic changes related to the estrogen-deficient state might be due, in part, to alterations in skeletal muscle substrate metabolism. We tested the hypothesis that estradiol affects the regulation of enzymes involved in substrate oxidation and storage within skeletal muscle. Specifically, we examined enzymes involved in the regulation of glycogen synthesis (glycogen synthase [GS]), glycolysis (phosphofructokinase [PFK]), tricarboxylic acid cycle activity (citrate synthase [CS]), and beta-oxidation (beta-hydroxyacyl-CoA dehydrogenase [beta-HADH]). Twenty-two, female Sprague-Dawley rats (7 to 8 weeks old) were separated into 3 groups: OVX + placebo (P; n = 8), OVX + estradiol (E(2); n = 8), and sham-operated (S; n = 6). Rats from E(2) and P groups were pair-fed to the S group to control for OVX-induced changes in food intake. After 16 days, activities of GS, PFK, CS, and beta-HADH were measured in vastus medialis muscle. GS fractional velocity was significantly lower (P <.05) in P (mean +/- SE; 39.7% +/- 6.2%) compared with both S (61.9% +/- 8.8%) and E(2) (65.8% +/- 8.4%) rats. In addition, E(2) rats (41.4 +/- 2.0) had significantly higher (P <.05) CS activity than P (34.9 +/- 2.0) and S (33.9 +/- 1.4 micromol/min/g) groups. There was no effect of OVX or estradiol replacement on beta-HADH or PFK. Our results suggest that, independent of alterations in food intake, estradiol availability affects the regulation of enzymes involved in nonoxidative glucose disposal (GS) and oxidative metabolism (CS) in skeletal muscle.
[show abstract][hide abstract] ABSTRACT: There exists considerable controversy regarding the impact of different modes of exercise training on total daily energy expenditure (TEE). To examine this question, young, nonobese women were randomly assigned to a supervised 6-month program of endurance training, resistance training, or control condition. TEE was measured before and 10 d after a 6-month exercise program was completed with doubly labeled water. Body composition was determined from dual energy x-ray absorptiometry, maximum aerobic capacity from a treadmill test to exhaustion, and muscular strength from one-repetition maximum tests. Results showed that body composition did not change in endurance-trained women, but maximum aerobic capacity increased by 18%. Resistance-trained women increased muscular strength and fat-free mass (1.3 kg). TEE did not significantly change when measured subsequent to the endurance or resistance training programs. Absolute resting metabolic rate increased in resistance-trained women but not when adjusted for fat-free mass. No change in physical activity energy expenditure was found in any of the groups. These results suggest that endurance and resistance training does not chronically alter TEE in free-living young women. Thus, the energy-enhancing benefits of exercise training are primarily derived from the direct energy cost of exercise and not from a chronic elevation in daily energy expenditure in young, nonobese women.