1. Volume 127, Issue 2, Pages 502-513 (August 2004)
Active delivery of trefoil factors by genetically modified Lactococcus lactis prevents and heals acute colitis in mice 
Klaas Vandenbroucke, Wolfgang Hans, Jacques Van Huysse, Sabine Neirynck, Pieter Demetter, Erik Remaut, Pieter Rottiers, Lothar Steidler 
Gastroenterology 
Volume 127, Issue 2, Pages (August 2004)
DOI: /j.gastro

2. Figure 1
Time course of heterologous mTFF1, mTFF2, and mTFF3 production by genetically modified L. lactis (LL-mTFF1, LL-mTFF2, LL-mTFF3). LL-pTREX1, vector control; LL-mIL10, L. lactis strain secreting murine IL-10; LL-mTFF1, +UV, UV-killed LL-mTFF1. (A) Western blot analysis of proteins secreted from the various strains revealed by anti-Myc antibody. Each lane on the blot represents 0.5 mL of L. lactis culture supernatant obtained after different periods of growth (2 × 107 CFU at time zero). Purified Myc-mTFF1 (+) was used as positive control. (B) Concentrations of secreted heterologous Myc-tagged proteins in culture supernatants of LL-pTREX1 (open diamonds), LL-mTFF1 (closed squares), LL-mTFF2 (closed triangles), LL-mTFF3 (closed diamonds), LL-mIL10 (open circles), and UV-killed LL-mTFF1 (open triangles), as determined by enzyme-linked immunosorbent assay.
Gastroenterology  , DOI: ( /j.gastro )

3. Figure 2
TFF produced by L. lactis enhances CMT-93 cell monolayer reconstitution. CMT-93 cell monolayers were wounded as described. The negative control was left untreated (white bar). The gray bars represent wells where purified mTFF1 was added at 2 different concentrations (100 ng/mL and 10 ng/mL). The black bars represent wells where 100 μL of filtered (0.22 μm) lactococcal supernatant was added. The effective amounts of TFF in the aliquots of lactococcal supernatants added were 35 ng for LL-mTFF1, 55 ng for LL-mTFF2, and 31 ng for LL-mTFF3. The total volume in each well after adding the lactococcal supernatant was 1 mL. ∗P < 0.005, statistically significant difference compared with the negative control and the LL-pTREX1 supernatant.
Gastroenterology  , DOI: ( /j.gastro )

4. Figure 3
Analysis of mortality and morbidity in acute DSS-induced colitis. (A) Mortality in the different treatment groups: control (n = 33, closed diamonds), mock-treated (n = 42, closed squares), LL-pTREX1 (n = 32, closed triangles), LL-mTFF1 (n = 31, open squares), LL-mTFF2 (n = 20, open triangles), LL-mTFF3 (n = 20, open diamonds), LL-mIL10 (n = 12, closed circles), and UV-killed LL-mTFF1 (n = 11, asterisks). (B) Evolution of relative body weight during the induction of DSS colitis in the different groups: control (n = 23, closed diamonds), mock-treated (n = 16, closed squares), LL-pTREX1 (n = 17, closed triangles), LL-mTFF1 (n = 21, open squares), LL-mTFF2 (n = 10, open triangles), LL-mTFF3 (n = 10, open diamonds), LL-mIL10 (n = 6, closed circles), and UV-killed LL-mTFF1 (n = 7, asterisks). All groups treated with LL-mTFF1, LL-mTFF2, and LL-mTFF3 have significantly higher relative body weight than mock-treated and vector control groups starting at day 4 (∗P < 0.05) and increasing to ∗∗P < from day 5 onward. (C-F) Statistical evaluation of the (C and E) MPO levels per square millimeter of colon tissue and (D and F) histologic score of the middle colon. C and D represent the prophylactic LL-mTFF experiment, and E and F represent the therapeutic LL-mTFF experiment. Bars represent the mean ± SEM. White bars represent the healthy control group. Mice with DSS-induced colitis were either mock treated (hatched bars), received different L. lactis cultures (black bars), or received purified mTFF1 (gray bars). Statistical significant differences compared with the mock-treated and the vector control groups: ∗∗∗P < 0.001, ∗∗P < 0.01, and ∗P < The UV-killed LL-mTFF1 strain is abbreviated as LL-mTFF1, + UV.
Gastroenterology  , DOI: ( /j.gastro )

5. Figure 4
Representative histology of the middle colon in the prophylactic LL-mTFF experiment on day 8 from (A) healthy control mice and mice with acute DSS-induced colitis either (B) mock treated or treated with (C) LL-pTREX1, (D) LL-mTFF1, (E) LL-mTFF2, (F) LL-mTFF3, (G) LL-mIL10, (H) UV-killed LL-mTFF1, purified mTFF1 administered orally at (I) 5 μg/day or (J) 50 μg/day, and purified mTFF1 administered rectally at (K) 5 μg/day or (L) 50 μg/day (H&E staining).
Gastroenterology  , DOI: ( /j.gastro )

6. Figure 5
Induction of Ptgs2 expression by LL-TFF mediates protection against acute colitis. All treatments were given before installation of acute DSS colitis. The evaluation was performed on day 8. (A) Relative expression levels of Ptgs2 mRNA in colon samples isolated from mice treated with LL-mTFF1, LL-mTFF2, LL-mTFF3, or purified mTFF1 (50 μg/day rectally). The fold change in Ptgs2 mRNA expression levels relative to LL-pTREX1 is expressed as the ratio of the TBP-normalized value for each treated sample to the TBP-normalized value for LL-pTREX1. Bars represent the mean ± SD. (B) Immunohistochemical score of Ptgs2 expression, ranging from 0 to 4 depending on the strength of the Ptgs2 signal. Bars represent the mean ± SEM. Statistically significant differences compared with the mock-treated group: ∗∗∗P < and ∗P < Statistical significant difference compared with the vector control group LL-pTREX1 of †P < (C–H) Representative immunohistochemical images for Ptgs2 expression in the distal colon of (C) healthy control mice and mice with DSS-induced colitis either (D) mock treated or treated with the empty expression vector (E) LL-pTREX1, (F) LL-mTFF1, (G) LL-mTFF2, or (H) LL-mTFF3. (I and J) Effect of Ptgs2 inhibition on the prevention of acute DSS-induced colitis by LL-TFF2. One mock and one LL-TFF2-treated group received the Ptgs2 inhibitor meloxicam (3 mg/kg daily intraperitoneally). Statistical evaluation of (I) the MPO levels per square millimeter of colon tissue and (J) histologic score of the middle colon. Bars represent the mean ± SEM. White bars represent the healthy control group. Mice with DSS-induced colitis were either mock treated (hatched bars) or received LL-mTFF2 (black bars). Statistically significant differences compared with the mock-treated groups: ∗∗∗P < 0.001, ∗∗P < 0.01, and ∗P < 0.05.
Gastroenterology  , DOI: ( /j.gastro )

7. Figure 6
Statistical evaluation of the (A) MPO levels per square millimeter of colon tissue and (B) histologic score of the distal colon in 129Sv/Ev IL-10−/− mice. Each group received daily for 7 or 14 days 2 × 109 CFU LL-pTREX1 (vector control) or LL-mTFF2, except the untreated group. Bars represent the mean ± SEM. The black bars represent the 129Sv/Ev IL-10−/− mice that were untreated, the white bars represent the 129Sv/Ev IL-10−/− mice that received the vector control LL-pTREX1, and the gray bars represent the 129Sv/Ev IL-10−/− mice that were treated with LL-mTFF2. Statistically significant difference compared with the vector control group: ∗P < 0.05 and ∗∗P < 0.01.
Gastroenterology  , DOI: ( /j.gastro )