1. Volume 3, Issue 5, Pages 697-707 (May 2001)
Acute Cytokine Response to Systemic Adenoviral Vectors in Mice Is Mediated by Dendritic Cells and Macrophages 
Yi Zhang, Narendra Chirmule, Guang-ping Gao, Ruth Qian, Maria Croyle, Bindu Joshi, John Tazelaar, James M. Wilson 
Molecular Therapy 
Volume 3, Issue 5, Pages (May 2001)
DOI: /mthe
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

2. FIG. 1
Intravenous injection of mice with Ad-lacZ significantly induces elevation of serum inflammatory cytokines. A, B, and C show the serum concentration of cytokines IL-6, IL-12, and TNF-α in mice at 6 h after receiving an intravenous injection of the indicated doses of Ad-lacZ (A), various doses of LPS as indicated (B), and 3 × 1011 Ad-lacZ or UV-inactivated Ad-lacZ (C). Data are averages of four or five mice in each group. Error bars represent means ± 1 SD. Representative data of three experiments are shown.
Molecular Therapy 2001 3, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

3. FIG. 2
Biodistribution of intravenous Ad-lacZ. (A) Mice intravenously injected with Ad-lacZ (2 × 1011 genomes/mouse) were sacrificed at 4 h and 3 days, respectively, postvector. Total DNA were prepared from liver, spleen, lung, kidney, and heart as described under Materials and Methods for real-time PCR analysis to determine the copy numbers of lacZ gene/diploid genomic DNA. Data are averages of five mice in each group. Error bars represent means ± 1 SD. (B) Mice intravenously injected with Cy3-labeled Ad-lacZ were sacrificed at 1 h postvector. Liver and spleen sections were prepared as described under Materials and Methods. After fluorescent microphotography of the Cy3 signal, the slides were stained with hematoxylin and eosin and rephotographed using bright-field microscopy. The Cy3 label appears as a mixture of red and white dots. Magnification: upper row, 100×; lower row, 400×.
Molecular Therapy 2001 3, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

4. FIG. 3
Ad-lacZ efficiently transduces cells in the marginal zone of spleen upon intravenous injection. (A) Cryosections of spleen were taken from mice injected with PBS or Ad-lacZ at 6 h. X-gal histochemistry (X-gal) and immunohistochemistry staining by separately using anti-CD4, -B220, -CD11c, and -CD11b antibodies were performed to demonstrate the interaction of Ad-lacZ with DCs and macrophages in spleen. Magnification, × 100. (B) Splenocytes were separated from mice injected intravenously with Ad-lacZ at 6 h and stained with the antibodies as indicated. β-Gal+ cells among CD11b+, MOMA-1+, and CD11c+ cell populations were separately calculated as a percentage. (C) Purified splenic DCs were double-stained with anti-CD11c coupled with anti-Ia or anti-CD86 as described under Materials and Methods. Upon gating for CD11c+ cell population, the expression of Ia and CD86 antigens was analyzed by flow cytometry. Isotype-matched Ig was used as control. One representative experiment of at least three is shown.
Molecular Therapy 2001 3, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

5. FIG. 4
Migration of β-gal+ cells into the T lymphocyte areas of spleen. Cryosections of spleen taken from mice injected with Ad-lacZ (2 × 1011 genomes/mouse) and harvested at 24 h. X-gal histochemistry (X-gal) staining was combined with immunohistochemistry staining with anti-B220 and anti-CD11c or anti-CD11b and B220 as indicated. Magnification, ×200.
Molecular Therapy 2001 3, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

6. FIG. 5
Intravenous injection of Ad-lacZ induces the spontaneous production of high levels of inflammatory cytokines by DCs and macrophages upon ex vivo culture. Splenic DCs (A) and macrophages (B) were purified from the spleen of mice after intravenous injection of Ad-lacZ (2 × 1011 genomes/mouse) or PBS at the indicated time points as described under Materials and Methods. Cells were cultured ex vivo in the presence or absence of Ad-lacZ for an additional 72 h. The presence of IL-6 and IL-12 in supernatants was examined by ELISA. Data are means ± SD of three mice and one representative experiment of at least three is shown. The serum cytokine concentration was determined in these mice in parallel at the indicated time points for IL-12 (C) and IL-6 (D). Data are means ± SD of four mice and one representative experiment of three is shown.
Molecular Therapy 2001 3, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

7. FIG. 6
Elimination of tissue macrophages and splenic DCs by liposome–Cl2MBP significantly reduces the elevation of serum inflammatory cytokines. Mice were separately pretreated with liposome–Cl2MBP or empty liposomes, followed by 2 × 1011 particles of Ad. The other group only received PBS (no vector). (B) At day 5, the mice were injected intravenously with Ad-lacZ and serum cytokine concentrations were determined by ELISA 6 h postvector. (A) The distribution of cells expressing β-gal, B220, CD11c, and CD11b antigens in the spleens from these mice was determined by X-gal histochemistry combined with immunohistochemistry staining (magnification ×200). (C) Serum cytokine concentrations from splenectomized mice injected with Ad-lacZ were examined by ELISA at 6 h postvector. Data in B and C are averages of four mice in each group. Error bars represent one SD from the mean. Statistical comparisons were performed by Student's t test. One representative experiment of two is shown.
Molecular Therapy 2001 3, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

8. FIG. 7
Treatment of mice with liposome–Cl2MBP significantly inhibits CTL response to eliminate Ad-lacZ-transduced gene expression in liver. Mice were separately treated with liposome–Cl2MBP and empty liposomes at day 1 and day 3 as described under Materials and Methods. At day 5, these mice were injected intravenously with Ad-lacZ followed by weekly injection of liposome–Cl2MBP and sacrificed at day 28 postvector. (A) Representative macrographs of X-gal histochemical stains of the liver. Magnification ×40. (B) Lymphocytes were isolated from spleen of mice at day 28 after intravenous Ad-lacZ and restimulated with feeder cells as described under Materials and Methods. The specific lysis of CTLs was analyzed using Ad-lacZ-infected MC57 or lacZ-transduced pLj syngeneic target cells. One representative experiment of at least two is shown.
Molecular Therapy 2001 3, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions