1. Orally administered TGF-β is biologically active in the intestinal mucosa and enhances oral tolerance 
Takashi Ando, MD, PhD, Kyosuke Hatsushika, MD, Masanori Wako, MD, PhD, Tetsuro Ohba, MD, Kensuke Koyama, MD, Yuko Ohnuma, Ryohei Katoh, MD, PhD, Hideoki Ogawa, MD, PhD, Ko Okumura, MD, PhD, Jian Luo, MD, PhD, Tony Wyss-Coray, PhD, Atsuhito Nakao, MD, PhD 
Journal of Allergy and Clinical Immunology 
Volume 120, Issue 4, Pages (October 2007)
DOI: /j.jaci
Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2. Fig 1
The oral administration of TGF-β results in the activation of the SBE-luc reporter in the intestine. A, Bioluminescence emission from live SBE-luc mice before, 3 hours after, and 5 hours after oral administration of TGF-β. Before or after the oral administration of TGF-β (5 μg per mouse), SBE-luc mice (line T9-55F) were anesthetized, luciferin was administered (150 mg/kg administered intraperitoneally), and then bioluminescence emissions from live SBE-luc mice were recorded, as described in the Methods section. Note a focal induction of luciferase activity in the intestinal region and also in the nose, lower jaw, paws, and tail. Images from representative mice are shown. B, Relative induction of reporter activity in dissected intestine expressed as fold induction of bioluminescence in mice treated orally with TGF-β (at 3 and 5 hours after the TGF-β treatment) compared with nontreated mice. Values represent the mean ± SD in 4 mice per group. C, Luciferase activity in different tissue specimens after the oral administration of TGF-β. Before and 3 hours after oral administration of TGF-β (5 μg per mouse), SBE-luc mice (line T9-55F) were killed, and luciferase activities in different organs were measured. Note the increase in the luciferase activity in the intestine 3 hours after the oral administration of TGF-β. Values represent the mean ± SD in 4 mice per group. ∗P < .05 compared with corresponding control value.
Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci )
Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3. Fig 2
Oral administration of TGF-β induces the phosphorylation of Smad2 in the mouse intestine. BALB/c mice were treated orally with 5 μg of TGF-β. Three hours after the treatment, the mouse stomach, ileum, and colon tissues were obtained, and then the tissue sections were stained with hematoxylin and eosin (HE; upper panels) or anti-phosphorylated Smad2 antibody. Representative photographs are shown. Positive staining is indicated as a brown color.
Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci )
Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4. Fig 3
The oral administration of TGF-β increases the TGF-β1 and Smad7 mRNA expression in the mouse intestine and the serum TGF-β1 levels. A, BALB/c mice were treated orally with 5 μg of TGF-β. Three hours after the treatment, the indicated tissue specimens were obtained, RNA was extracted, and then real-time RT-PCR was performed with specific primers for TGF-β1, Smad7, and GAPDH. The ratio of each gene to that of GAPDH was calculated, and the relative expression levels are shown. B, BALB/c mice were treated orally with 5 μg of TGF-β. Three hours after the treatment, TGF-β1 levels in serum were determined by means of ELISA. Values represent the mean ± SD in triplicate samples. ∗P < .05 compared with the corresponding control. Representative results of 4 independent experiments with similar results are shown.
Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci )
Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5. Fig 4
The effects of orally administered TGF-β on the induction of oral tolerance by a high-dose antigen. BALB/c mice were treated orally with 100 mg of OVA and/or 5 μg of TGF-β or control vehicle 3 times every 24 hours. One week after the final antigen feeding, the mice were immunized twice intraperitoneally with OVA for a 1-week interval. A, Seven days after the final immunization, anti-OVA IgE, IgG1, and IgG2a antibody levels in serum were determined by means of ELISA. B, Seven days after the final immunization, the splenocytes were obtained from the mice and then were cultured in the presence or absence of 100 μg/mL OVA for 3 days, and the concentrations of IL-4 and IFN-γ in the culture supernatants were determined by means of ELISA. C, Seven days after the final immunization, the immediate-type skin reaction to OVA was evaluated. Evans blue dye was injected intravenously into the mice, and then OVA was applied intradermally into the abdominal skin. After 20 minutes, mice were killed and skinned, and the diameter of the color reaction was evaluated on the inside of the abdominal skin. Please note that a striking immediate skin reaction, as indicated by the blue-colored area, occurred in the skin of the mice orally treated with control vehicle (control) or TGF-β alone (TGF-β). The striking immediate skin reaction was reduced in the mice orally treated with OVA (OVA; please note the reduction of the blue-colored area), and it was further reduced in the mice orally treated with OVA and TGF-β (the blue-colored area was almost undetectable). Values represent the mean ± SD in triplicate samples. ∗P < .05 compared with corresponding control. Representative results of 4 independent experiments with similar results are shown.
Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci )
Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions