1. Volume 116, Issue 3, Pages 593-601 (March 1999)
Tumor necrosis factor inhibitor ameliorates murine intestinal graft-versus-host disease 
Geri R. Brown, Guy Lindberg, Jon Meddings, Maria Silva, Bruce Beutler, Dwain Thiele 
Gastroenterology 
Volume 116, Issue 3, Pages (March 1999)
DOI: /S (99)
Copyright © 1999 American Gastroenterological Association Terms and Conditions

2. Fig. 1
TNF inhibitor is present in the serum of DBA/2 → B6D2F1 recipients of TNF inhibitor–encoding adenovirus. Serum samples (0.1 μL) from 5 DBA/2 → B6D2F1 recipients of TNF inhibitor–encoding adenovirus (labeled TNFi 1–5) and 1 recipient of DBA/2 → B6D2F1 β-galactosidase–encoding adenovirus were denatured with SDS under reducing conditions and loaded into lanes 2–7, respectively, of a 10% polyacrylamide gel. Recombinant human soluble TNF receptor 1 (the 162–amino acid [18-kilodalton] extracellular domain of the 55-kilodalton TNF receptor) (50 ng) was loaded into lane 1 to serve as a positive control. The bands present in lanes loaded with the serum from the mice injected with the TNF inhibitor reflect the 55-kilodalton total molecular weight of the inhibitor monomers comprised of the extracellular domain of the human 55-kilodalton TNF receptor fused to the hinge and Fc region of IgG1.10 After electrophoresis, proteins were transferred to nitrocellulose membranes and probed with an antibody to the extracellular domain of the TNF receptor 1. Bound protein was visualized with goat anti-mouse IgG–alkaline phosphatase conjugate and bromochloroindol phosphate, nitroblue tetrazolium substrates.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

3. Fig. 2
PCR analysis of colonic DNA 25 days after adenoviral transduction of allogeneic bone marrow transplant recipients with the TNF inhibitor– or the β-galactosidase–encoding adenovirus. PCR products obtained from right, transverse, and left colonic tissue of 5 recipients of TNF inhibitor–encoding adenoviral vector and 1 recipient of the β-galactosidase–encoding adenoviral vector were electrophoresed in a 3% agarose gel and products visualized by ethidium bromide staining. The final lane represents the amplification product from a vector encoding the TNF inhibitor construct. The anticipated size of the amplification product is 150 base pairs.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

4. Fig. 3
Histological appearance of colons of allogeneic bone marrow transplant recipients transduced with the β-galactosidase–expressing adenovirus. Representative colon sections taken 25 days after allogeneic bone marrow transplantation and transduction with a virus encoding β-galactosidase were assessed by histochemical techniques for β-galactosidase expression as detailed in Materials and Methods. The staining of the colonic epithelial cell is indicated by the arrow. Probably because of absence of fixation, the colonic epithelial cells have pulled away from the basement membranes, as is often seen in postmortem biopsy sections.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

5. Fig. 4
Histological appearance of colons from DBA/2 → B6D2F1 mice that had received either the TNF inhibitor–encoding adenovirus or the β-galactosidase–encoding adenovirus. (A) Histological features of cell degeneration and vacuolization of colonic tissue observed 14 days after transplantation in recipients receiving the β-galactosidase–encoding control adenovirus. Increased numbers of intraepithelial lymphocytes were noted with some lymphocytes (open arrow) located adjacent to apoptotic colonic epithelial cells (solid arrow). (B and C) Colonic tissue obtained 25 days after transplantation from recipients receiving the β-galactosidase–encoding adenovirus had mucosal and submucosal mononuclear infiltrate invading (B) the colonic tissue and (C) mesenteric mononuclear infiltrates. (D) Colonic tissue obtained 25 days after transplantation from recipients receiving the TNF inhibitor–encoding adenovirus displayed normal histological features.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

6. Fig. 4
Histological appearance of colons from DBA/2 → B6D2F1 mice that had received either the TNF inhibitor–encoding adenovirus or the β-galactosidase–encoding adenovirus. (A) Histological features of cell degeneration and vacuolization of colonic tissue observed 14 days after transplantation in recipients receiving the β-galactosidase–encoding control adenovirus. Increased numbers of intraepithelial lymphocytes were noted with some lymphocytes (open arrow) located adjacent to apoptotic colonic epithelial cells (solid arrow). (B and C) Colonic tissue obtained 25 days after transplantation from recipients receiving the β-galactosidase–encoding adenovirus had mucosal and submucosal mononuclear infiltrate invading (B) the colonic tissue and (C) mesenteric mononuclear infiltrates. (D) Colonic tissue obtained 25 days after transplantation from recipients receiving the TNF inhibitor–encoding adenovirus displayed normal histological features.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

7. Fig. 4
Histological appearance of colons from DBA/2 → B6D2F1 mice that had received either the TNF inhibitor–encoding adenovirus or the β-galactosidase–encoding adenovirus. (A) Histological features of cell degeneration and vacuolization of colonic tissue observed 14 days after transplantation in recipients receiving the β-galactosidase–encoding control adenovirus. Increased numbers of intraepithelial lymphocytes were noted with some lymphocytes (open arrow) located adjacent to apoptotic colonic epithelial cells (solid arrow). (B and C) Colonic tissue obtained 25 days after transplantation from recipients receiving the β-galactosidase–encoding adenovirus had mucosal and submucosal mononuclear infiltrate invading (B) the colonic tissue and (C) mesenteric mononuclear infiltrates. (D) Colonic tissue obtained 25 days after transplantation from recipients receiving the TNF inhibitor–encoding adenovirus displayed normal histological features.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

8. Fig. 4
Histological appearance of colons from DBA/2 → B6D2F1 mice that had received either the TNF inhibitor–encoding adenovirus or the β-galactosidase–encoding adenovirus. (A) Histological features of cell degeneration and vacuolization of colonic tissue observed 14 days after transplantation in recipients receiving the β-galactosidase–encoding control adenovirus. Increased numbers of intraepithelial lymphocytes were noted with some lymphocytes (open arrow) located adjacent to apoptotic colonic epithelial cells (solid arrow). (B and C) Colonic tissue obtained 25 days after transplantation from recipients receiving the β-galactosidase–encoding adenovirus had mucosal and submucosal mononuclear infiltrate invading (B) the colonic tissue and (C) mesenteric mononuclear infiltrates. (D) Colonic tissue obtained 25 days after transplantation from recipients receiving the TNF inhibitor–encoding adenovirus displayed normal histological features.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

9. Fig. 5
Severity of intestinal GVHD induced by DBA/2 donor T cells in B6D2F1 recipients of either TNF inhibitor–encoding adenovirus or a β-galactosidase–encoding adenovirus. Irradiated B6D2F1 mice were infused with 5 × 106 DBA/2 bone marrow cells and 10 × 106 spleen cells and the indicated adenoviral vector. Mice were killed 25 days after transplantation, and histological sections of the colon were prepared and assessed as detailed in Materials and Methods. Each bar represents the mean ± SE for histological scores in proximal, transverse, and distal colonic sections from 6 mice in each group.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

10. Fig. 6
Total enteropathy score is significantly reduced in the TNF inhibitor–encoding adenovirus recipients. Each square represents the mean histological score from the proximal, transverse, and distal colonic sections of 6 mice in either the TNF inhibitor–encoding or the β-galactosidase–encoding adenovirus recipients. The middle bar represents the median of each group, and the box represents the 25th and 75th percentiles. P < 0.05.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

11. Fig. 7
Fractional excretion of sucralose in DBA/2 → B6D2F1 mice that had received either the TNF inhibitor–encoding adenovirus or the β-galactosidase–encoding adenovirus. Transplant recipients were gavaged with a sugar probe containing 30 mg/mL sucralose 14 days after transplantation. Urine was collected for 24 hours from 12 DBA/2 → B6D2F1 mice receiving the β-galactosidase–encoding control adenoviral vector, 8 DBA/2 → B6D2F1 mice receiving the adenoviral vector encoding the TNF inhibitor, and 5 control B6D2F1 mice after they had been housed in metabolic cages for 24 hours. The boxes present the median and 25th and 75th percentile of the values in each experimental group. The black horizontal bar represents the mean levels of fractional sucralose excretion in each experimental group.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions