1. Unexpected Role of Surface Transglutaminase Type II in Celiac Disease
Luigi Maiuri, Carolina Ciacci, Ida Ricciardelli, Loredana Vacca, Valeria Raia, Antonio Rispo, Martin Griffin, Thomas Issekutz, Sonia Quaratino, Marco Londei 
Gastroenterology 
Volume 129, Issue 5, Pages (November 2005)
DOI: /j.gastro
Copyright © 2005 American Gastroenterological Association Terms and Conditions

2. Figure 1
p31-43 induces up-regulation of TG2 in TCD biopsy specimens. Expression of TG2 protein as detected by CUB 7402 mAb (green) and TG activity (red) in TCD biopsy specimens (A) before challenge, (B) after 20 minutes of challenge with medium alone, and after (C and D) 20 minutes and (E and F) 3 hours of challenge with (C and E) p31-43 or (D and F) pα-9. (A) Before challenge, TG2 protein and activity are detected within the subepithelial extracellular matrix, as revealed by a yellow color. (B) After incubation with medium alone, the pattern of expression is similar to that found before challenge. (C and D) TG activity is induced within the epithelial nuclei (red) and within the subepithelial extracellular matrix where it colocalizes with TG2 protein (yellow) after 20 minutes of challenge with (C) p31-43 but not with (D) pα-9. (E and F) Intense up-regulation of TG2 protein and activity within the extracellular matrix (yellow) and presence of TG enzymatic activity in enterocytes (red) are observed after 3 hours of challenge with (E) p31-43 but not with (F) pα-9. The pattern observed after challenge with pα-2, deamidated pα-2, or pα-9 is similar to that observed after incubation with pα-9 or with medium alone. (Two-color immunofluorescence, TG2 protein, as revealed by CUB 7402 mAb [green] and TG activity [red]. Overlap of labels results in a yellow color. Original magnification 200×.)
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

3. Figure 2
Expression and distribution of TG2 protein as revealed by CUB 7402 mAb or 6B9 mAb in celiac disease biopsy specimens and T84 cell lines. (A–E) Expression and distribution of TG enzymatic activity (red) and TG2 protein (green), as detected by (A, C, and E) 6B9 mAb or (B and D) CUB 7402 mAb in (A and B) treated and (C and D) untreated celiac mucosa. TG2 protein localization, as detected by (A and C) 6B9 mAb, is evident at the surface of enterocytes. Many lamina propria cells are labeled. No overlap with TG enzymatic activity is found. At the higher magnification (E), note the absence of overlap between TG protein (green) and TG enzymatic activity (red), which is mainly located at the level of subepithelial extracellular matrix. TG2 protein localization, as revealed by (B and D) CUB 7402 mAb, is evident at the level of the extracellular matrix, mainly in the subepithelial region of the lamina propria. TG protein expression colocalizes with TG enzymatic activity (yellow). No epithelial staining is found. (F) Surface TG protein expression in T84 cells detected by 6B9 (solid line), CUB 7402 (dotted line), and isotype control (thin line). Dark shaded area. ([A–E] Two-color immunofluorescence. Original magnification: A–D, 200×; E, 400×.)
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

4. Figure 3
p31-43 and pα-9 are both substrates of TG2 in patients with celiac disease and in controls. Detection of TG enzymatic activity (green) in (A–C) control and (D–H) untreated celiac disease biopsy specimens by using (A, D, and F–H) biotinylated p31-43 (A and D) without and with pretreatment with (F) R283, (G) CUB 7402 mAb, or (H) 6B9 mAb, (C and E) biotinylated p-α9, and (B) the biotinylated deamidated form of pα-9 (100 μg/mL) as TG substrates instead of biotinylated MDC. In (A) control and (D) celiac duodenum, TG enzymatic activity is detected within the extracellular matrix, mainly of the subepithelial compartment, with detectable label within the epithelium when biotinylated p31-43 is used as TG substrate. (C and E) A similar pattern is observed in (C) control and (E) celiac disease biopsy specimens when biotinylated pα-9 is used as substrate. (B) In control biopsy specimens, no label is detected when the deamidated form of pα-9 is used; the same pattern is observed in celiac disease biopsy specimens. (F) No label is detected when tissue sections are pretreated with R283 inhibitor. (G) Pretreatment of sections with CUB 7402 mAb is only partially effective in preventing p31-43 tissue binding. (H) No inhibitory effect is provided by 6B9 mAb pretreatment, and the pattern is similar to that observed after incubation with p31-43 without pretreatment. A similar pattern of inhibition by R283, CUB 7402, or 6B9 mAb is found for pα-9 or pα-2. pα-2 shows the same pattern as pα-9. TCD biopsy specimens show the same behavior as control tissues. (Indirect immunofluorescence; original magnification 200×.)
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

5. Figure 4
Effect of the inhibition of TG2 activity and protein on gliadin peptide-induced modifications of celiac disease biopsy specimens after 24 hours of in vitro challenge. (A and B) Effect on T-cell activation, as revealed by CD25 expression by CD3+ lamina propria cells, induced by sequential incubation with (A) p31-43 (3 hours) and pα-9 (21 hours) or (B) deamidated pα-9 (21 hours) (number of CD25+CD3+ cells/mm2 of lamina propria; Wilcoxon signed rank test; *P = .027 vs cultures with medium; **P = .027 vs p31-43 added with TG inhibitor R283 or 6B9 mAb; P = .08 and P = .5 vs p31-43 added with CUB 7402 mAb or isotype control mAb, respectively). (C and D) Effect on T-cell activation, as revealed by CD69 expression by lamina propria cells, induced by sequential incubation with (C) p31-43 (3 hours) and pα-9 (21 hours) or (D) deamidated pα-9 (21 hours) (number of CD69+ cells/mm2 of lamina propria; Wilcoxon signed rank test; *P = .02 vs cultures with medium; **P = .02 vs p31-43 added with TG inhibitor R283 or 6B9 mAb; P = .08 and P = .5 vs p31-43 added with CUB 7402 mAb or isotype control mAb, respectively). (E) Real-time polymerase chain reaction assay of interferon gamma messenger RNA expression in 3 patients after sequential incubation with p31-43 (3 hours) and pα-9 (21 hours) with and without R283, CUB7402, or 6B9 mAb. Bars represent individual values of 3 tested patients. The data are expressed as fold of increase compared with biopsy specimens cultured in medium alone. The standard curve was constructed of triplicates, and all results were analyzed using 2-ΔΔCT method. The level of interferon gamma was calculated as the ratio to the expression of glyceraldehyde-3-phosphate dehydrogenase in each sample. (F, H, and I) Effect on intraepithelial migration/redistribution of CD8+ lymphocytes induced by 24 hours of challenge with p (F) Number of CD8+ cells/mm of epithelium; Wilcoxon signed rank test; *P = .028 vs cultures with medium; **P = .028 vs 6B9 mAb; P = .2, P = .9, and P = .1 vs R283, CUB 7402 mAb, and isotype control mAb, respectively. (H and I) Impressive intraepithelial infiltration of CD8+ cells after (E) 24 hours of challenge of celiac disease biopsy specimens with p31-43 and dramatic inhibition after (F) challenge with p31-43 added with 6B9 mAb. The latter pattern is similar to that observed after incubation with medium alone. (G) Effect on enterocyte apoptosis induced by 24 hours of challenge with p31-43 (number of terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling—positive enterocytes/100 enterocytes; Wilcoxon signed rank test; *P = .028 vs medium; **P = .027 vs 6B9 mAb; P = .3, P = .1, and P = .1 vs R283 [250 μmol/L], CUB 7402 mAb, and isotype control mAb, respectively). a, medium alone; b, p31-43 (3 hours) + pα-9 (21 hours); c, p31-43 (3 hours) + deamidated pα-9 (21 hours); d, plus R283; e, plus CUB 7402; f, plus 6B9; g, plus isotype control (IC). (H and I, indirect immunofluorescence, original magnification 200×.)
Gastroenterology  , DOI: ( /j.gastro )
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6. Figure 5
p31-43 induces early epithelial modifications in TCD biopsy specimens and T84 cell lines. (A–F) p31-43 but not pα-9 (20 μg/mL) induces epithelial phosphorylation in treated celiac disease biopsy specimens after (A–C) 20 minutes and (D–F) 3 hours of challenge. The challenge with p31-43 induces PY-99 expression at the apical membrane of the villus enterocytes, after 20 minutes of incubation (A and B, high magnification of A), and at the apical and basolateral membrane of the villus enterocytes after 3 hours of challenge (D and E, high magnification of D). No staining is evident at the enterocyte level after (C) 20 minutes or (F) 3 hours of challenge with pα-9. The latter pattern is similar to that observed after incubation with medium alone. pα-2 and the deamidated forms of pα-2 or pα-9 provide comparable results to that observed with pα-9. (G and H) p31-43 but not pα-9 induces actin reorganization after 20 minutes of challenge in treated celiac disease biopsy specimens. (G) Strong staining is evident at the apical membrane and at the basolateral membranes of villus enterocytes after challenge with p31-43, whereas (H) fainter staining is evident only at the apical membrane of the enterocytes after challenge with pα-9. The latter pattern is similar to that observed after incubation with medium alone. pα-2 and the deamidated forms of pα-2 or pα-9 show a similar behavior as pα-9. (I and J) p31-43 but not pα-9 induces actin reorganization after 30 minutes of challenge in T84 cell lines. Actin reorganization and induction of filopodia on T84 cells are induced by stimulation for 30 minutes with (I) p31-43 but not with (J) pα-9. pα-2 and the deamidated forms of pα-2 or pα-9 show a similar behavior as pα-9. ([A–F]) Indirect or [G–J] direct immunofluorescence [green]; counterstain with [I–J] propidium iodide [red]. Original magnification: A, C, D, and F, 200×; B, E, G, and H, 1000×.)
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

7. Figure 6
6B9 mAb controls p31-43–induced early epithelial modifications in celiac disease biopsy specimens and T84 cell lines. (A and B) 6B9 controls early epithelial phosphorylation induced by p31-43 in TCD biopsy specimens. PY-99 expression induced by 20 minutes of challenge with (A) p31-43 is dramatically inhibited by (B) 6B9 mAb. (C and D) 6B9 mAb controls early actin reorganization induced by p31-43 in TCD biopsy specimens. Actin reorganization induced by 20 minutes of challenge with (C) p31-43 is dramatically inhibited by (D) 6B9 mAb. (E and F) 6B9 controls early epithelial protein phosphorylation, as revealed by PY-99 expression, induced by p31-43 in T84 cell lines. Induction of PY-99 expression by (E) epithelial cells (green) is neutralized by (F) 6B9 mAb. (G and H) 6B9 controls early actin reorganization induced by p31-43 in T84 cell lines. Induction of actin reorganization and stress fiber induction upon (G) p31-43 challenge is neutralized by (H) 6B9 mAb. ([A, B, E, and F] Indirect or [C, D, G, and H] direct immunofluorescence [green], [E–H] counterstain with propidium iodide [red], original magnification 1000×.)
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

8. Figure 7
p31-43 induces apoptosis of T84 cells via Fas engagement. Apoptosis of confluent T84 cells was analyzed by propidium iodide and annexin V expression as described in Patients and Methods. (A) T84 cells cultured in medium alone; 2.1% of cells are propidium iodide and annexin V positive, and 2.9% are only annexin V positive. (B) T84 cells cultured in the presence of p31-43; 13.9% double-positive cells and 6.8% single-positive cells for annexin V. (C) T84 cells cultured in the presence of p31-43 and neutralizing anti-Fas antibody M38; 5.6% cells are double positive and 4.5% are annexin V single positive.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

9. Figure 8
p31-43–induced apoptosis is controlled by mAb 6B9, and pα-9 fails to induce apoptosis of T84 cells. Apoptosis of T84 confluent cells. (A) Baseline apoptosis of T84 cells cultured in medium alone; 4.9% cells are double positive for propidium iodide and annexin V, and 3.5% are annexin V positive (B) Apoptosis induced by p31-43 challenge; 15.5% cells are both propidium iodide and annexin V positive, and 6.7% are only annexin V positive. (C) Apoptosis of T84 cells stimulated with p31-43 in the presence of 6B9; 4.8% of the cells are propidium iodide and annexin V positive, and 5.3% are only annexin V positive. (D) Apoptosis of T84 cells stimulated with p31-43 in the presence of CUB 7402 mAb; 11.5% of the cells are propidium iodide and annexin V positive, and 8.5% are only annexin V positive. (E) Apoptosis of cells cultured with medium alone and baseline for the next series of experiments; 6.4% of cells are both propidium iodide and annexin V positive, and 4.7% of cells are only annexin V positive. (F) A total of 15.7% of cells are positive for both propidium iodide and annexin V when cultured with p31-43, and 7.2% are only positive for annexin V. (G) A total of 6.2% of cells are both positive for propidium iodide and annexin V when cultured with pα-9, with 4.4% positive only for annexin V. (H) A total of 4.8% of cells are positive when cultured with p31-43 in the presence of 6B9, and 5.7% are only annexin V positive. (I) A total of 16.3% of cells are positive when cultured with p31-43 in the presence of isotype control for both propidium iodide and annexin V, and 7.8% are only annexin V positive. All the data reported in this figure were reproduced in at least 4 separate experiments.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions