1. Volume 131, Issue 2, Pages 525-537 (August 2006)
CD25+/Foxp3+ T Cells Regulate Gastric Inflammation and Helicobacter pylori Colonization In Vivo 
Roland Rad, Lena Brenner, Stefan Bauer, Susanne Schwendy, Laura Layland, Clarissa Prazeres da Costa, Wolfgang Reindl, Anar Dossumbekova, Mathias Friedrich, Dieter Saur, Hermann Wagner, Roland M. Schmid, Christian Prinz 
Gastroenterology 
Volume 131, Issue 2, Pages (August 2006)
DOI: /j.gastro
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

2. Figure 1
Dynamics of gastric pro- and anti-inflammatory gene expression during chronic H pylori infection of mice. Male C57BL/6 mice were infected with H pylori SS1 and sacrificed at different time points after challenge (30–180 days). Noninfected control animals (Hp-) received Brucella broth. Gastric expression of cytokines, cytokine receptors, and transcription factors was analyzed by real-time RT-PCR and normalized to β-actin expression. Bars indicate the arithmetic mean and standard error of the mean from pooled data including 2 identical experiments with 5 mice per group. The 2-sided P value was calculated by a Student t test. Asterisks indicate significant differences compared with the H pylori–negative controls (*P < .05; **P < .01; ***P < .001).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

3. Figure 2
Anti-CD25 mAb PC61 depletes CD4+CD25+ T cells in vivo for at least 30 days. C57BL/6 mice were treated intraperitoneally with either PBS or a single, 1-mg dose of anti-CD25 mAb (PC61). Thirty days later, splenocytes were isolated and prepared for flow cytometry analysis by using the combination of CD4-APC and either CD25-PE or CD45RB-PE antibodies. Dot plots from 1 representative untreated and 1 PC61-treated animal are shown in A and B, respectively. Values represent the percentage of cells in each quadrant. Columns in C show the arithmetic mean and standard error of the mean of absolute splenic CD4+/CD25+ T-cell numbers from groups of PC61-treated and untreated mice (n = 8). Correspondent values for CD4+/CD45RBlow cells are shown in D. P values were calculated by a 2-sided Student t test. Asterisks indicate significant differences (*P < .01; **P < .001). Similar results were obtained 3 days after PC61 application.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

4. Figure 3
Severe gastritis but no autoimmune gastritis in H pylori–infected PC61-treated mice. (A and B and E and F) H&E-stained stomach sections from representative nontreated and PC61-treated animals 4 weeks after H pylori infection. Only few scattered leukocytes can be seen in the submucosa and lamina propria (arrowhead) of a nontreated mouse 4 weeks after infection. Arrows indicate lymphocytes between the glands. Severe gastric inflammation is present in the PC61-treated mouse with dense leukocytic infiltration in the submucosa and mucosa (arrowhead). Leukocytic aggregates between the glands (arrows) are spanning the entire width of the mucosa. A concomitant hyperplasia with thickening of the gastric wall as well as gastric atrophy can be observed. (G) Histological scoring of gastritis in nontreated and PC61-treated animals. (H–P) Leukocyte subsets were stained immunohistochemically in the gastric mucosa of nontreated and PC61-treated animals 4 weeks after H pylori infection. Representative sections are shown at a magnification of 200×. Mucosal T cells (H–J), macrophages (K–M), and B cells (N–P) were stained with anti-F4/80, anti-CD3, and anti-B220 antibodies, respectively. The number of stained cells per mm2 are presented in J, M, and P. Each triangle represents one animal. Shown is 1 experiment including 10 mice per group. Similar results were obtained in 2 analogous experiments. The 2-sided P value was calculated by a Mann–Whitney U test. (C) Immunohistochemical staining of H+/K+-ATPase on paraffin-embedded mouse stomach sections by using the monoclonal Ab 2B6. (D) Representative immunohistochemical staining of H+/K+-ATPase by using serum from a PC61-treated animal at a dilution of 1:10. None of the sera from PC61-treated mice showed reactivity with parietal cells. Photographs are taken at 100× (A–D), 200× magnification (E–O), or 400× (cut-out in C).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

5. Figure 4
Expression of cytokines and T-cell markers in the stomach of H pylori–infected PC61-treated and nontreated mice. (A–I) messenger RNA expression of cytokines and cytokine receptors in the gastric mucosa of PC61-treated and nontreated mice 4 weeks after H pylori infection. Messenger RNA amounts were determined by real-time TaqMan PCR and are presented as copies per 106 β-actin copies. (J and K) Relative ratio of β-actin–normalized Foxp3 expression to β-actin–normalized CD4 and CD8 expression, respectively. (L and M) Relative ratio of β-actin–normalized CD25 expression to β-actin-normalized CD4 and CD8 expression, respectively. Bars indicate the arithmetic mean and standard error of the mean from 1 experiment including 10 mice per group. Similar results were obtained in 2 analogous experiments. The 2-sided P value was calculated by a Student t test or a Mann–Whitney rank sum test. Asterisks indicate significant differences (*P = .05; **P < .01; ***P < .001).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

6. Figure 5
H pylori–specific IgG2c and IgG1 levels in PC61-treated and nontreated mice. H pylori–specific immunoglobulin titers were determined in 1:1000 diluted sera of H pylori–infected mice 4 weeks after infection. Bars indicate the arithmetic mean and standard error of the mean from 1 experiment including 10 mice per group. Similar results were obtained in 2 analogous experiments. The 2-sided P value was calculated by a Mann–Whitney U test. Asterisks indicate significant differences (**P = .005; ***P< .001).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

7. Figure 6
Decreased bacterial colonization in PC61-treated mice. Nontreated or PC61-treated mice were sacrificed 4 weeks after H pylori infection, and bacterial colonization density was determined by quantitative culture (A) or quantification of bacterial ureB DNA (B). In A, values are expressed as colony-forming units/g stomach tissue. Each triangle represents 1 animal. Quantification of bacterial ureB DNA in B was performed by real-time PCR and normalized to β-actin DNA amounts. P values were calculated by a Student t test. One of 3 experiments yielding similar results is shown.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

8. Figure 7
FOXP3+ T cells in the H pylori–infected human gastric mucosa. (A and B) Immunohistochemical staining of the transcription factor FOXP3. Staining is clearly localized to the nucleus (insert in B). FOXP3+ cells are present in the H pylori–infected human gastric mucosa (A and B) but not in the uninfected stomach (C). Glandular epithelial cells are FOXP3 negative. (E) FOXP3 expression in the gastric mucosa of H pylori–infected (n = 43) and noninfected patients (n = 65). FOXP3 expression was analyzed by real-time quantitative RT-PCR and normalized to GAPDH expression. Bars within the box plots represent median values (50th percentile). The ends of the bars indicate the 25th and 75th percentiles, the 10th and 90th percentiles are represented by error bars, and the 5th and 95th percentiles are shown by filled circles. The P value was calculated by a Mann–Whitney U test. Photographs were taken at 100× (A and D), 200× (B), and 1000× (cut-out in B).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions