1. Volume 133, Issue 3, Pages 862-874 (September 2007)
Prevention and Treatment of Colitis With Lactococcus lactis Secreting the Immunomodulatory Yersinia LcrV Protein 
Benoit Foligne, Rodrigue Dessein, Michael Marceau, Sabine Poiret, Mathias Chamaillard, Bruno Pot, Michel Simonet, Catherine Daniel 
Gastroenterology 
Volume 133, Issue 3, Pages (September 2007)
DOI: /j.gastro
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2. Figure 1
In vitro production of LcrV by recombinant L lactis. One hundred microliters of supernatants from stationary-phase lactococcal cells (OD600 = 2) were separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and proteins were electrotransferred on a nitrocellulose membrane. LcrV was detected by immunoblotting using a specific mouse monoclonal antibody. Y pseudotuberculosis was used as a positive control for LcrV production, as described elsewhere.54 LcrV migrated as a mixture of dimeric and multimeric species.55
Gastroenterology  , DOI: ( /j.gastro )
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3. Figure 2
Cytokine production by LPS-stimulated macrophages pretreated with L lactis strains. BALB/c peritoneal macrophages (2 × 106 cells/well) were incubated for 3 hours with phosphate-buffered saline containing stationary-phase L lactis (106 colony-forming units/well, Ll), L lactis producing LcrV (106 CFU/well, Ll-LcrV), or no bacteria at all (non-Ll) before a 24-hour contact period with 2.5 μg/mL LPS. IL-6 and IL-10 levels in the cell culture supernatants were measured by ELISA. Bars represent mean values ± SEM of 3 independent assays. Baseline levels of cytokine production by untreated cells in the absence of any stimulation are indicated by the dashed lines. *.01< P < .05; **.001< P < .01; ***P < .001; ns, not significant (P > .05).
Gastroenterology  , DOI: ( /j.gastro )
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4. Figure 3
Colonic IL-10 and IL-6 levels in BALB/c mice fed with L lactis strains. Three groups of mice were fed for 5 days with either buffer or 2 × 108 live Ll, Ll-LcrV. IL-10 and IL-6 levels in the entire homogenized colon of each individual mouse were measured by ELISA. Three independent assays were performed, and a representative experiment is depicted. Bars represent mean values from 5 animals ± SEM. *.01< P < .05; ns, not significant (P > .05).
Gastroenterology  , DOI: ( /j.gastro )
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5. Figure 4
Preventive effects of recombinant L lactis strains against TNBS-induced acute colitis in BALB/c mice. Loss of body weight, colonic inflammation (macroscopically and microscopically scored according to the Wallace and Ameho criteria, respectively), and infiltration by polymorphonuclear neutrophils (estimated by tissue myeloperoxidase [MPO] activity) on day 3 after intrarectal administration of TNBS are shown. Mice were pretreated (or not, as the case may be) with recombinant lactococcal strains. Three independent assays were performed, and a representative experiment is depicted. Bars represent mean values of 10 animals ± SEM. *.01< P < .05; **.001< P < .01; ***P < .001; ns, not significant (P > .05). Myeloperoxidase from human polymorphonuclear neutrophils was used for calibration; one enzyme unit degrades 1 μmol hydrogen peroxide·min−1·mL−1 at 25°C.
Gastroenterology  , DOI: ( /j.gastro )
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6. Figure 5
Histologic features of LcrV-mediated protection. The figure shows representative May–Grünwald–Giemsa—stained colon sections on day 3 after instillation of TNBS in BALB/c mice pretreated with lactococcal strains (A) Ll or (B) Ll-LcrV as compared with (C) healthy mice not given TNBS (original magnification 40×). A corresponds to an Ameho score of 5, showing transmural inflammation and important thickening of the colon wall, characterized by prominent inflammatory infiltrates of mononuclear cells (mainly neutrophils) in the lamina propria, extensive ulceration, and focal epithelial necrosis extending into the muscle layers, in addition to massive goblet cell depletion. B yields a score of 2, with only slight thickening of the submucosa, mild, localized submucosal inflammatory infiltrates, moderate superficial erosions, and the presence of a few crypts with minor hyperplasia, resulting in a well-conserved mucosal architecture more similar to the normal appearance of healthy mice colons (C).
Gastroenterology  , DOI: ( /j.gastro )
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7. Figure 6
Blood and colon markers of inflammation in TNBS-inoculated BALB/c mice pretreated with recombinant lactococci. Samples were collected on day 3 after instillation of TNBS after pretreatment with Ll, Ll-LcrV, and Ll-IL-10. (A) Quantification (using real-time PCR) of tnf-α, cox-2, il-1β, and il-10 transcripts in colon extracts. (B) Blood IL-6 and SAA levels, measured by ELISA. Two independent assays were performed, and a representative experiment is shown. Values are expressed as the relative increase in specific mRNA levels compared with healthy colons. Bars represent mean values of 10 animals ± SEM. *.01< P < .05; **.001< P < .01; ns, not significant (P > .05); #P = .075.
Gastroenterology  , DOI: ( /j.gastro )
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8. Figure 7
Preventive effects of LcrV-secreting L lactis against TNBS-induced colitis in C57BL/6 wild-type and IL-10−/− mice. Data for body weight loss, colonic inflammation (scored macroscopically according to the Wallace criteria), and infiltration by polymorphonuclear neutrophils (estimated by tissue myeloperoxidase [MPO] activity) on day 3 after intrarectal administration of TNBS are shown. Mice were pretreated with recombinant lactococcal strains. Bars represent mean values of 10 animals ± SEM. *.01< P < .05; not significant (P > .05). MPO from human polymorphonuclear neutrophils was used for calibration; 1 enzyme unit degrades 1 μmol hydrogen peroxide·min−1·mL−1 at 25°C.
Gastroenterology  , DOI: ( /j.gastro )
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9. Figure 8
The preventive potential of orally administered recombinant L lactis strains in DSS-induced colitis in BALB/c mice. A total of 2 × 108 live lactococci were administered orally 2 days before and then during the 9-day 6% DSS regimen. (A) Mice were weighed daily throughout the experiment. Each time point represents the mean percentage change from day 0 for 10 mice: open squares, healthy mice not given DSS; closed circles, non–Ll-treated mice with DSS; gray circles, Ll-treated mice receiving DSS; open circles, Ll-LcrV–treated mice receiving DSS. (B) Colon length and histologic scores were measured at necropsy on day 8; bars represent mean values of 10 animals ± SEM. (C) Histologic features on day 8. The panels show representative May–Grünwald–Giemsa—stained middle-colon sections of (a) non–DSS-challenged mice, (b) DSS-challenged mice treated with buffer, and (c) lactococcal strains Ll and (d) Ll-LcrV, respectively (original magnification 10×). (a) corresponds to a histologic score of 0. (b) and (c) correspond to scores of 4 and 3, respectively, showing marked numbers of inflammatory cells, including neutrophils in the lamina propria extending into the submucosa, together with mucosal erosion/ulceration and disruption of crypt architecture. (d) has a score of 1, exhibiting a few inflammatory cells in the lamina propria and discrete focal epithelial lesions. *.01< P < .05; **.001< P < .01; ***P < .001; ns, not significant (P > .05).
Gastroenterology  , DOI: ( /j.gastro )
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10. Figure 9
Preventive properties and accelerated epithelium-healing abilities of recombinant L lactis in DSS-induced colitis. Mice were given 5% DSS for 8 days and then normal drinking water for the next 5 days. The animals were fed daily with 2 × 108 live lactococci throughout the experimental procedure. (A) Mice were weighed daily throughout the experiment. Each time point represents the mean percentage change from day 0 for 10 mice: open squares, healthy mice not given DSS; closed circles, non–Ll-treated mice receiving DSS; gray circles, Ll-treated mice receiving DSS; open circles, Ll-LcrV–treated mice receiving DSS. (B) Colon length was measured at necropsy on day 12; bars represent mean values of 10 animals ± SEM. (C) May–Grünwald–Giemsa—stained middle-colon section of DSS-challenged mice treated with Ll-LcrV (original magnification 20×). The photograph shows re-epithelialization and cryptal regeneration of colonic mucosa. *P < .001; ns, not significant (P > .05).
Gastroenterology  , DOI: ( /j.gastro )
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11. Figure 10
Therapeutic potential of L lactis strains in DSS-induced colitis. Mice were given a 5% DSS solution for a 9-day period and fed daily with 2 × 108 lactococci from day 4 to day 8. (A) Mice were weighed daily throughout the experiment. Each time point represents the mean percentage change from day 0 for 10 mice: open squares, healthy mice not given DSS; closed circles, non–Ll-treated mice receiving DSS; gray circles, Ll-treated mice receiving DSS; open circles, Ll-LcrV–treated mice receiving DSS. (B) Colon length and histologic scores were measured at necropsy on day 8; bars represent mean values of 10 animals ± SEM. *.01< P < .05; **.001< P < .01; ***P < .001; ns, not significant (P > .05); #P = .07.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions