Volume 132, Issue 4, Pages 1245-1253 (April 2007) Humoral Immune Response to Tissue Transglutaminase Is Related to Epithelial Cell Proliferation in Celiac Disease  Maria V. Barone, Ivana Caputo, Maria T. Ribecco, Maria Maglio, Roberto Marzari, Daniele Sblattero, Riccardo Troncone, Salvatore Auricchio, Carla Esposito  Gastroenterology  Volume 132, Issue 4, Pages 1245-1253 (April 2007) DOI: 10.1053/j.gastro.2007.01.030 Copyright © 2007 AGA Institute Terms and Conditions

Figure 1 Representative images of several similar experiments (>3). CUB 7402 dose-dependent induction of actin rearrangement in (A) Caco-2, (B) MCF7, and (C) NIH 3T3 cells. Arrows indicate membrane ruffles. (Original magnification 40×.) Gastroenterology 2007 132, 1245-1253DOI: (10.1053/j.gastro.2007.01.030) Copyright © 2007 AGA Institute Terms and Conditions

Figure 2 Representative images of several similar experiments (>3). (A) Effect on actin rearrangement in Caco-2 cells of single-chain antibodies (clones 4.2 and 3.7) from patients with celiac disease and of a single-chain anti-tTG antibody from a healthy donor (clone D51). The effect of the anti-gliadin single-chain antibody (G2 clone) is also shown. (B) Effect of the recombinant anti-tTG mini-antibody clone 2.8 on actin rearrangement in Caco-2, MCF7, and NIH 3T3 cells. Arrows indicate membrane ruffles. (Original magnification 40×.) Gastroenterology 2007 132, 1245-1253DOI: (10.1053/j.gastro.2007.01.030) Copyright © 2007 AGA Institute Terms and Conditions

Figure 3 (A) CUB 7402 dose-dependently induced G0 → S transition in synchronized NIH 3T3 fibroblasts. Nonspecific rabbit IgG1 served as control. Values are normalized to BrdU incorporation of cells treated with 10% serum. Data are reported as means ± SDs from 3 separate experiments. *P < .05 vs untreated. (B) Induction of G0 → S transition in synchronized NIH 3T3 fibroblasts by commercial anti-tTG antibodies (CUB 7402, 2.5 μg/mL; TG100, 2.5 μg/mL; and rabbit polyclonal antibody, 1:500) and by single-chain anti-tTG antibodies (10 μg/mL). Values are normalized to BrdU incorporation of cells treated with 10% serum. Data are reported as means ± SDs from 3 separate experiments. *P < .05 vs untreated. (C) TUNEL assay of NIH 3T3 fibroblasts treated with 2.5 μg/mL IgG1, CUB 7402, or clone 2.8 (green). A positive control obtained by treatment with deoxyribonuclease I before the TUNEL assay is shown. Hoechst staining of nuclei is also shown (blue). Gastroenterology 2007 132, 1245-1253DOI: (10.1053/j.gastro.2007.01.030) Copyright © 2007 AGA Institute Terms and Conditions

Figure 4 (A) Surface tTG staining of unpermeabilized living cells (Caco-2, MCF7, and NIH 3T3). (Original magnification 40×.) (B) Effect of PD9859 plus CUB 7402 (2.5 μg/mL) on the G0 → S transition of synchronized NIH 3T3 fibroblasts. Values are normalized to BrdU incorporation of cells treated with 10% serum. Data are reported as means ± SDs from 2 separate experiments. *P < .05 vs untreated. Gastroenterology 2007 132, 1245-1253DOI: (10.1053/j.gastro.2007.01.030) Copyright © 2007 AGA Institute Terms and Conditions

Figure 5 Surface tTG activity of whole NIH 3T3 cells in the presence of different amounts of CUB 7402 and 20 μg/mL of 2 single-chain anti-tTG antibodies. Data are reported as means ± SDs from 3 separate experiments performed in triplicate. Gastroenterology 2007 132, 1245-1253DOI: (10.1053/j.gastro.2007.01.030) Copyright © 2007 AGA Institute Terms and Conditions

Figure 6 (A) Confocal images of cytokeratin staining (green) and BrdU incorporation (red) in celiac disease epithelial mucosal cells of representative samples treated with medium alone or with 2.0 μg/mL antibodies (IgG1, CUB 7402, and clone 2.8 alone or in the presence of PD98059) (original magnification 20×). (B) Mean values ± SDs of BrdU incorporation in enterocytes of several patients with celiac disease. The number of biopsy specimens used for each experiment is indicated. *P < .05 vs cultures with medium. (C) Mean values ± SDs of BrdU incorporation in enterocytes of several nonceliac control patients. The number of biopsy specimens used for each experiment is indicated. (D) TUNEL assay of sections from celiac disease biopsy specimens cultured with medium alone or with 2.5 μg/mL antibodies (IgG1, CUB 7402, or clone 2.8) (green). A positive control obtained by treatment with deoxyribonuclease I before TUNEL assay is shown. Hoechst staining of nuclei is also shown (blue). Gastroenterology 2007 132, 1245-1253DOI: (10.1053/j.gastro.2007.01.030) Copyright © 2007 AGA Institute Terms and Conditions