1. Volume 117, Issue 5, Pages 1069-1077 (November 1999)
Enhancing lamina propria Th1 cell responses with interleukin 12 produces severe tissue injury 
Giovanni Monteleone, Thomas T. MacDonald, Neville C. Wathen, Francesco Pallone, Sylvia L.F. Pender 
Gastroenterology 
Volume 117, Issue 5, Pages (November 1999)
DOI: /S (99)
Copyright © 1999 American Gastroenterological Association Terms and Conditions

2. Fig. 1
Agarose gel stained with ethidium bromide showing RT-PCR products for IL-12 p40, IL-12Rβ2, IL-1Rrp, and GAPDH in freshly obtained fetal gut explants (day 0). +ve, positive control, mucosal tissue from a patient with CD.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

3. Fig. 2
Induction of IFN-γ and TNF-α transcripts after 4 days of culture. Transcripts were measured by competitive RT-PCR. (A) Transcripts for both IFN-γ (■) and TNF-α (□) were low in unstimulated explants. Activation of T cells in fetal gut explants with anti-CD3 alone was followed by a small increase in the number of transcripts for both IFN-γ and TNF-α. Addition of anti-CD3 and IL-12 (5 ng/mL) resulted in a marked increase in the amount of transcripts for both IFN-γ and TNF-α. In contrast, costimulation of explants with anti-CD3 and IL-18 was associated with an increase in the number of transcripts for IFN-γ only. When fetal gut explants were stimulated with anti-CD3 plus IL-12 and IL-18, a significant increase in the number of transcripts for IFN-γ, but not for TNF-α, was observed compared with that obtained in explants costimulated with anti-CD3 and IL-12 alone. (B) Effect of graded doses of IL-12 and anti-CD3 on IFN-γ and TNF-α mRNA expression. A significant increase in the number of transcript for both cytokines was observed in explants costimulated with anti-CD3 and IL-12, dependent on the amount of IL-12 added. The number of transcripts for IFN-γ obtained after costimulation with 0.5, 5, and 10 ng/mL IL-12 plus anti-CD3 was 3, 18, and 45 times, respectively, that observed after stimulation with anti-CD3 alone. One of 4 separate experiments.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

4. Fig. 3
Concentration of (A) IFN-γ and (B) TNF-α in human fetal gut explant cultures. Culture supernatants were collected after 4 days of culture with medium alone or in the presence of the indicated stimuli. Bars show the mean ± SEM of 3 separate experiments using tissue from different fetuses.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

5. Fig. 4
Addition of IL-12 at 5 ng/mL and soluble CD3 antibody (5% final dilution) to the explant culture of fetal gut caused mucosal destruction within 4 days. The epithelium is highlighted by staining with anticytokeratin, (A) showing that there are tall villi in unstimulated explants, (D) whereas there are a few short villi in the explants costimulated with anti-CD3 and IL-12. No significant change in mucosal morphology is present in (B) explants stimulated with anti-CD3 alone as well as in (C) explants costimulated with anti-CD3 and IL-18 (100 ng/mL). Results are representative of 5 experiments. (Immunoperoxidase staining; original magnification 200×.)
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

6. Fig. 5
(A) Induction of transcripts for interstitial collagenase (□) and stromelysin-1 (■) in explants costimulated with anti-CD3 and IL-12. Transcripts were measured by competitive RT-PCR. Costimulation of explants with anti-CD3 and IL-12 resulted in a strong increase in the number of transcripts for both interstitial collagenase and stromelysin-1. No further increase was observed in the explants stimulated with anti-CD3 plus IL-12 and IL-18. One of 3 separate experiments. (B) Induction of transcripts for TIMP-1 (▩) in fetal gut explants. Transcripts were measured by competitive RT-PCR. Activation of T cells in fetal gut explants with anti-CD3 resulted in a strong increase in the number of transcripts for TIMP-1 compared with that observed in unstimulated explants. No further increase was observed when explants were stimulated with anti-CD3 and IL-12 and/or IL-18. One of 3 separate experiments.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

7. Fig. 6
Identification of inactive and active forms of interstitial collagenase, stromelysin-1, and TIMP-1 in culture supernatants of explants stimulated with anti-CD3 and IL-12. Culture supernatants were collected after 4 days and run on 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis under reducing conditions. Arrows indicate molecular weight of interstitial collagenase, stromelysin-1, and TIMP-1 detected by specific polyclonal antibodies. The example is representative of 4 separate experiments.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

8. Fig. 7
Both a synthetic stromelysin-1 inhibitor, CT1399, and TNFR-IgG prevent mucosal injury of explants costimulated with anti-CD3 and IL-12. Sections were stained with H&E. (A) Morphology of an explant costimulated with anti-CD3 and IL-12 for 4 days; only the stumps of the villi remain. (B and C) Morphology of explants in which CT1399 or TNFR-IgG was added at the onset of culture with anti-CD3 plus IL-12, respectively; villus morphology is retained. (Original magnification 200×.)
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions