1. Use of Perfluorochemical Liquid Allows Earlier Detection of Gene Expression and Use of Less Vector in Normal Lung and Enhances Gene Expression in Acutely Injured Lung 
Daniel J. Weiss, Laura Bonneau, Denny Liggitt 
Molecular Therapy 
Volume 3, Issue 5, Pages (May 2001)
DOI: /mthe
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

2. FIG. 1
Instillation of FC-75 with Adlac-Z results in increased total amount and in earlier detection of transgene expression. Sprague-Dawley rats instilled with Adlac-Z (5 × 1010 particles/rat) ± FC-75 (10 cc/kg) were sacrificed at the indicated times and quantitative β-gal activity was determined in lung homogenates. Increased and earlier expression was observed in lungs from rats receiving Adlac-Z with FC-75. No β-gal activity was observed in naive control animals or in animals receiving FC-75 alone. Representative results from one of three similar experiments are shown (n = 3-5 for each experimental condition).
Molecular Therapy 2001 3, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

3. FIG. 2
Instillation of FC-75 with Adlac-Z results in increased in situ transgene expression. Sprague–Dawley rats instilled with Adlac-Z (5 × 1010 particles/rat) ± FC-75 (10 cc/kg) were sacrificed at the indicated times and in situ β-gal expression was determined. No in situ expression was observed at 6 h after instillation. At 1 and 3 days, rats receiving Adlac-Z with FC-75 demonstrated increased overall expression compared to that observed in lungs from rats receiving Adlac-Z alone. No β-gal expression was observed in naïve control animals or in animals receiving FC-75 alone. A transient inflammation consisting of inflammatory cell infiltration and alveolar septal thickening was occasionally observed in lungs harvested 6 h after receiving Adlac-Z with FC-75 but not in lungs receiving either Adlac-Z or FC-75 alone. Representative photomicrographs from one of three similar experiments are shown (n = 3–5 for each experimental condition). Original magnification 200X.
Molecular Therapy 2001 3, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

4. FIG. 3
Instillation of PFC liquid with Adlac-Z results in β-gal expression in a variety of upper and lower airway epithelial cells. Sprague–Dawley rats instilled with Adlac-Z (5 × 1010 particles/rat) ± FC-75 (10 cc/kg) were sacrificed at the indicated times and in situ β-gal expression was determined. Representative high-power (oil immersion) photomicrographs of lungs of mice instilled with Adlac-Z + FC-75 demonstrate β-gal activity in upper airway epithelium and in type 1 and type 2 alveolar epithelial cells. Similar photomicrographs of lungs of mice instilled with FC-75 alone demonstrate predominantly normal-appearing upper and lower airway epithelium without obvious epithelial cell damage. Original magnification 1000X.
Molecular Therapy 2001 3, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

5. FIG. 4
Use of PFC liquid allows use of less adenoviral vector to obtain in situ transgene expression comparable to use of higher amount of vector alone. Sprague–Dawley rats were instilled with Adlac-Z at the indicated concentrations (particles/rat) ± FC-75 (10 cc/kg). Animals were sacrificed 3 days later and lungs processed for in situ β-gal expression. No β-gal expression was observed in naïve control animals or in animals receiving FC-75 alone. Representative photomicrographs from one of nine similar experiments are shown (n = 3–5 for each experimental condition). Original magnification 100X.
Molecular Therapy 2001 3, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

6. FIG. 5
Use of PFC liquid allows use of less adenoviral vector to obtain total transgene expression comparable to use of higher amount of vector alone. Sprague-Dawley rats were instilled with Adlac-Z at the indicated concentrations (particles/rat) ± FC-75 (10 cc/kg). Animals were sacrificed 3 days later and lungs processed for quantitative β-gal expression. No β-gal activity was detected in naïve control animals or in animals receiving FC-75 alone. Representative results from one of nine similar experiments are shown (n = 3-5 for each experimental condition).
Molecular Therapy 2001 3, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

7. FIG. 6
Use of less adenovirus vector is associated with less TNFα in BAL fluid and use of PFC liquid inhibits BAL fluid TNFα accumulation provoked by instillation of a high titer of adenovirus vector. Sprague-Dawley rats were instilled with Adlac-Z at the indicated concentrations (particles/ml) ± FC-75 (10 cc/kg). Animals were sacrificed 3 days later and BAL fluid was assayed for TNFα content by ELISA. TNFα content increased with increasing dose of Adlac-Z instilled. Instillation of FC-75 with the highest dose of Adlac-Z inhibited accumulation of TNFα. Representative results from one of three similar experiments are shown (n = 3-5 for each experimental condition).
Molecular Therapy 2001 3, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

8. FIG. 7
Use of PFC liquid enhances in situ β-gal expression when instilled with Adlac-Z at either 1 or 3 days following induction of acute lung injury with bleomycin. One (A-D) or three (E-H) days after intratracheal instillation of bleomycin, a second intratracheal instillation of Adlac-Z ± FC-80 was performed. Animals were sacrificed 3 days after the second instillation (4 and 6 days after the initial bleomycin instillation, respectively). No in situ activity was observed in lungs from rats receiving bleomycin alone (A, E) or from rats receiving bleomycin followed by FC-80 (B, F). Rats receiving bleomycin followed by Adlac-Z alone exhibited scattered activity mostly in uninjured lung regions (C, G). In contrast, rats receiving bleomycin followed by Adlac-Z + FC-80 exhibited more diffuse activity in both normal and injured lung regions (D, H). Representative photomicrographs from one of four similar experiments are shown (n = 3–5 for each experimental condition). Arrows in A and E denote representative areas of acute lung injury characterized by alveolar septal thickening, edema, and inflammatory cell infiltrates. Original magnification 100X.
Molecular Therapy 2001 3, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

9. FIG. 8
Instillation of PFC liquid with Adlac-Z results in β-gal expression in areas of injured lung. Representative high-power photomicrographs (original magnifications 400 and 1000X) of lungs of rats receiving Adlac-Z + FC-80 3 days after bleomycin administration demonstrate β-gal activity in grossly abnormal lung regions. A variety of both identifiable and unidentifiable cell types exhibit β-gal activity. The arrow indicates a macrophage containing ingested FC-80.
Molecular Therapy 2001 3, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

10. FIG. 9
Use of PFC liquid increases total β-gal activity when instilled with Adlac-Z 1 or 3 days after bleomycin instillation. Rats received intratracheal instillation of bleomycin and 1 (A) or 3 (B) days later of Adlac-Z ± FC-80. Animals were sacrificed 3 days later and quantitative β-gal activity was determined in left lung homogenates. Representative results from one of four similar experiments are shown (n = 3-5 for each experimental condition).
Molecular Therapy 2001 3, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

11. FIG. 10
Use of PFC liquid transiently increases BAL fluid total protein and inflammatory cytokine content and augments the increase provoked by adenovirus vector from lungs of normal rats. Sprague–Dawley rats instilled with Adlac-Z (5 × 1010 particles/rat) ± FC-75 (10 cc/kg) were sacrificed at the indicated times, BAL fluid was collected, and total protein and cytokine contents were determined. Representative results from one of at least two similar experiments for each time point are shown (n = 3-5 per experimental condition).
Molecular Therapy 2001 3, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

12. FIG. 11
Use of PFC liquid had no effect on total protein and inflammatory cytokine content in BAL fluid from lungs of rats with acute bleomycin-induced lung injury. Sprague-Dawley rats received instillation of bleomycin sulfate and 1 day later were instilled with Adlac-Z (5 × 1010 particles/rat) ± FC-75 (10 cc/kg). Rats were sacrificed 3 days later, BAL fluid was collected, and total protein (A) and cytokine (B) contents were determined. Representative results from one of at least three similar experiments for each time point are shown (n = 3-5 per experimental condition).
Molecular Therapy 2001 3, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions