1. Mast Cells Are Critical Mediators of Vaccine-Induced Helicobacter Clearance in the Mouse Model 
Dominique Velin, Daniel Bachmann, Hanifa Bouzourene, Pierre Michetti 
Gastroenterology 
Volume 129, Issue 1, Pages (July 2005)
DOI: /j.gastro
Copyright © 2005 American Gastroenterological Association Terms and Conditions

2. Figure 1
Kinetics of Helicobacter clearance after vaccination. Balb/c mice were treated intranasally (at days 0, 7, 14, and 28) with either 30 μg urease + 10 μg cholera toxin (CT) as adjuvant or CT alone. At day 42, the mice were challenged with H felis (5 × 107) and killed 4 or 5 days postchallenge. At sacrifice, stomachs were recovered for urease testing (A) or quantification of bacterial density by histology (B; cresyl violet staining). In another set of experiments, mice were immunized as described above and challenged at days 42 and 44 with 5 × 108 H pylori 49 (Hp49) and killed at day 49. At death, stomachs were recovered for urease testing (C) and for colony-forming-unit counts (D). *5 mice per group, P < .02; **10 mice per group, P < (2 independent experiments with 5 mice per group); ***6 mice per group, P < .003; ****6 mice per group, P < .003 (Mann-Whitney test). (−) means.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

3. Figure 2
Mast cells hyperplasia in the stomach of urease + CT-vaccinated mice during the Helicobacter clearance. Flow cytometry analysis of lymphoid cell populations recovered from the stomachs of vaccinated (CT + urease) or CT control mice (CT) at day 4 post–H felis challenge. (A) Percentages of CD4+ cells. (B) Percentages of CD3−CD117+ cells. (C) Cresyl violet staining of a stomach section of a urease + CT-vaccinated mouse at day 5 post–H felis challenge. Relative expression of the mMCP-1 (E) and 2 (D) mRNA in total mRNA extracts from stomachs of vaccinated (CT + urease) or CT-vaccinated mice (CT) at day 5 post–H felis challenge. Results are shown as ratios between mRNA copy numbers of mMCP-1 or mMCP-2 and mRNA copy numbers of the housekeeping gene GAPDH. mMCP-1 levels were determined by ELISA at day 5 post–H felis challenge in the serum of vaccinated (CT + urease) or CT control mice (CT) (F). *5 mice per group, P < .02; **5 mice per group, P < .008; ***6 mice per group, P < .005; ****4 mice per group, P < .005; *****5 mice per group, P < .008 (Mann-Whitney test). (−) means.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

4. Figure 3
Critical role of mast cells in anti-Helicobacter vaccination. Wild-type WBB6 F1 mice were vaccinated (at days 0, 7, 14, and 28) either with urease + CT or CT alone. At day 42, mice were challenged with H felis (5 × 107). At death (day 56), urease tests were performed on gastric samples (A). W/Wv mast cell-deficient mice were vaccinated, challenged, and killed. At death, urease tests were performed on gastric specimens (B). W/Wv mast cell-deficient mice were vaccinated as described above, and, at day 35, they were reconstituted with bone marrow-derived mast cells. At day 42, the mice were challenged with H felis and, at day 56, killed to perform urease tests on gastric specimens (C). Balb/c mice were vaccinated as described above and challenged with H felis at day 42. The group of CT + urease-vaccinated mice were CD4+ cell-depleted (CT + urease CD4+ depletion) or injected with control antibody (CT + urease). At death (day 47), urease tests were performed on gastric specimens (D). W/Wv mice were immunized, mast cell repopulated, and challenged at day 42 with H felis and CD4+ cells depleted or injected with control antibody as described above. At death (day 47), urease tests were performed on gastric specimens (E). *12 mice per group, P < .001; **12 mice per group, P < .001; ***12 or 10 mice per group, P < .01; ****5 mice per group; P < .008; *****5 mice per group P < .04 (Mann-Whitney test). (−) means.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

5. Figure 4
Serum antiurease IgE levels in urease + CT-vaccinated mice. Balb/c or W/Wv mice were intranasally vaccinated (at days 0, 7, 14, and 28) with CT + urease or CT administered. Serum of vaccinated mice were recovered at day 41 and tested for the presence of antiurease IgE antibodies. As positive control for presence of serum antiurease IgE antibodies, we subcustaneously immunized 20 Balb/c mice with 0.1 μg urease adjuvanted with alum. (−) means.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

6. Figure 5
Neutrophil recruitment is not the main mechanism involved in vaccination-induced Helicobacter clearance. Balb/c mice were vaccinated with CT + urease or CT administered as described previously. Twelve hours before H felis challenge, mice were either injected with antineutrophil-depleting antibody (RB6-8C5) or rat control IgG (250 μg/mouse intraperitoneal injection) (A). Antibody injections were repeated at days 1 and 4 postchallenge. At day 4 postinfection, peripheral bloods were recovered to perform blood smears. After May Grünewald Giemsa staining, the percentages of circulating neutrophils were calculated in the white blood cells by microscopic examination. In mice injected with rat IgG antibodies, the percentages were 30% ± 7%, and, in mice injected with neutrophil-depleting antibodies, the percentages were 1.8% ± 9% (P = .0022). At day 5 postchallenge, mice were killed to perform the urease test on gastric specimens. *7 mice per group, P < .008; ** 7 mice per group, P < .002 (Mann-Whitney test). TNF-α produced by mast cells is dispensable for efficacy of anti-Helicobacter vaccination (B). As described previously, W/Wv mice were immunized with CT + urease and repopulated with mast cells derived from bone marrow of B6 TNF-α −/− mice or B6 wild-type mice. Mice were challenged with H felis and, 12 days later, killed, and their stomachs evaluated by urease tests. *7 mice per group, P = .9, no significant difference (Mann-Whitney test). (−) means.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

7. Figure 6
Gastric mastocytosis does not induce Helicobacter clearance. IL-9 transgenic mice do not clear Helicobacter infection spontaneously (A). An IL-9 transgenic mouse develops a massive mast cell infiltration in the stomach. IL-9 transgenic and nontransgenic mice (8 weeks old) were infected with H felis and, 14 days later, killed and their stomachs evaluated by urease tests. *9 mice per group, P = .9, no significant difference (Mann-Whitney test). Balb/c mice developing a gastric mastocytosis do not clear Helicobacter infection spontaneously (B). Balb/c mice (8 weeks old) were injected daily with rIL-3 from day 4 to day 0, infected with H felis, and, 14 days later, killed and their stomachs evaluated by urease tests. *6 mice per group, P = .07, no significant difference (Mann-Whitney test). (−) means.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions