1. Volume 9, Issue 3, Pages 318-327 (March 2004)
Persistent Transgene Expression Following Intravenous Administration of a Liposomal Complex: Role of Interleukin-10-Mediated Immune Suppression 
Isao Ito, Tomoyuki Saeki, Imran Mohuiddin, Yuji Saito, Cynthia D Branch, Ara Vaporciyan, Jack A Roth, Rajagopal Ramesh 
Molecular Therapy 
Volume 9, Issue 3, Pages (March 2004)
DOI: /j.ymthe
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

2. Fig. 1
Cytokine profile following systemic injection of DOTAP:Chol-luc DNA complex. Serum was collected from lung TB and NTB mice at 0, 2, 6, 12, and 24 h after injection of DOTAP:Chol-luc DNA complex (50 μg) and was assayed for cytokines (TNF-α, IL-1α, IFN-γ, IL-10) using ELISA. Untreated TB and NTB animals served as controls from each group. (A) Cytokine profile in UV2337m TB and NTB mice. (B) Cytokine profile in A549 lung TB and NTB mice. Data represent the average cytokine levels in four animals per group per time point.
Molecular Therapy 2004 9, DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

3. Fig. 2
Persistent transgene expression occurs in TB animals. (A) UV2237M TB and NTB C3H mice and (B) A549 TB and NTB nude mice were injected with DOTAP:Chol-luc DNA complex via the tail vein. Their lungs were resected on days 1, 2, 3, and 7 after treatment and analyzed for luc expression. Luc activity is expressed as relative light units (RLU) per milligram of total protein. Each time point represents the average luc activity in four animals. Bars represent standard deviation.
Molecular Therapy 2004 9, DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

4. Fig. 3
Multiple treatments resulted in increased transgene expression. TB and NTB C3H or nude mice injected either once or three times with DOTAP:Chol-luc DNA complex (50 μg DNA/dose) via a tail vein were assayed for luc activity. (A) A twofold increase in luc activity was observed in TB C3H mice receiving three treatments compared with those receiving one treatment. In contrast, luc activity in NTB C3H mice receiving three treatments was significantly lower that in those receiving one treatment. (B) Luc activity in TB nude mice receiving three treatments showed two- to threefold increase in luc expression compared with those receiving one treatment. In contrast, luc activity in NTB nude mice receiving three treatments was significantly lower than in those receiving one treatment. Luc activity is expressed as RLU per milligram of total protein. Bars represent standard deviation.
Molecular Therapy 2004 9, DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

5. Fig. 4
UV2237M tumor cells produce IL-10. UV2237m TB and NTB lung tissue sections were immunohistochemically stained for mouse IL-10. IL-10 was detected in lung tissue sections as indicated by the intense brown cytoplasmic staining. Staining for IL-10 in NTB lung tissue sections was weak. Tissue sections stained only with secondary antibody served as negative controls. Arrows indicate cells staining positive for IL-10.
Molecular Therapy 2004 9, DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

6. Fig. 5
Alveolar macrophages (AM) from TB mice are less responsive to PMA stimulation. (A) Alveolar macrophages from TB and TNB C3H mice were plated in 96-well plates at various cell densities and incubated overnight at 37°C. Cells were exposed to PMA (1 μg/ml) for 1 h. After 1 h, 2′,7′ dichlorofluorescein diacetate was added and incubated for 30 min. Macrophage stimulation was detected by measuring the fluorescence intensity at 530 nm in a spectrofluorometer. AM from TNB animals responded to PMA stimulation significantly compared to those from TB animals. AM not exposed to PMA served as controls. Values shown are the means of quadruplicate wells. Bars represent standard error. (B) Alveolar macrophages from TB and TNB C3H mice were treated with LPS (1 μg/ml) for 24 h and the culture medium was assayed for murine TNF-α. A significant amount of TNF-α protein was detectable in the medium from AM harvested from NTB mice compared to that from TB mice. AM not exposed to LPS served as negative control (NT). Positive control included was provided in the kit. Data are represented as the means of quadruplicate wells. Bars represent standard error.
Molecular Therapy 2004 9, DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

7. Fig. 6
Neutralization of IL-10 in TB mice results in decreased transgene expression. Lung tumors were established in C3H mice by injecting UV2237m cells (1 × 106cells/well) via the tail vein. Three weeks later, animals were divided into three groups and treated as follows: group 1 received no treatment, group 2 received an intraperitoneal (ip) injection of isotypic control IgG antibody (20 μg), and group 3 received an ip injection of neutralizing anti-IL-10 antibody (20 μg). Twenty-four hours later, animals from all three groups were treated with DOTAP:Chol-luc DNA complex via the tail vein. Animals that did not receive any treatment served as negative controls. Animals were euthanized 48 h after injection, and their lungs were removed and analyzed for luc activity. Luc expression was significantly less in animals from group 3 than in those from groups 1 and 2. A significant reduction in luc activity was also observed in group 2 compared with group 1, indicating nonspecific inhibition. Luc activity is expressed as RLU per milligram of total protein. Bars represent standard error.
Molecular Therapy 2004 9, DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

8. Fig. 7
Schematic representation of IL-10 production by tumor cells and its effects on immune cells and inflammatory response. Tumor cells produce IL-10, which acts in an autocrine manner to promote growth and production of more IL-10 and in a paracrine fashion to suppress the functions of immune cells (macrophages, T cells) present in the tumor milieu by decreasing phagocytic activity and proinflammatory cytokine production (IL-1, TNF-α, IL-6) and rearranging receptors. Intravenous injection of liposome–DNA complex in TB animals thus results in a diminished inflammatory response, resulting in persistent and enhanced transgene expression after repeated treatments and in a therapeutic effect.
Molecular Therapy 2004 9, DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions