1. Volume 129, Issue 3, Pages 786-796 (September 2005)
Limited Efficiency of Prolyl-Endopeptidase in the Detoxification of Gliadin Peptides in Celiac Disease 
Tamara Matysiak–Budnik, Celine Candalh, Christophe Cellier, Christophe Dugave, Abdelkader Namane, Teresita Vidal–Martinez, Nadine Cerf–Bensussan, Martine Heyman 
Gastroenterology 
Volume 129, Issue 3, Pages (September 2005)
DOI: /j.gastro
Copyright © 2005 American Gastroenterological Association Terms and Conditions

2. Figure 1
In vitro degradation of (A) 3H-peptide 31–49 and (B) 56–88 (33-mer) by PEP, expressed as the percentage of intact peptides remaining after incubation, as a function of time and PEP concentration. A dose- and time-dependent disappearance of intact peptides was observed that was more rapid for peptide 31–49 than for peptide 56–88. ♦, 40 U/mL; •, 20 mU/mL; ▴, 100 mU/mL; ■, 500 mU/mL; ○, 1 U/mL.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

3. Figure 2
RP-HPLC analysis of 3H-peptide 31–49 (left panels) and 3H-33-mer (peptide 56–88) (right panels) after a 3-hour incubation in vitro in the presence of different PEP concentrations. Incomplete degradation of both peptides, with release of several intermediate metabolites, was observed. With increasing PEP concentration, there was a shift of the radioactive peaks toward the left, indicating the disappearance of large hydrophobic fragments and the appearance of smaller 3H-fragments or free 3H-proline. Mass spectrometry analysis of fragments released after digestion of peptides 31–49 and 56–88 by 20 mU/mL of PEP showed the presence of the toxic peptide 31–42 and of several known T-cell epitopes (56–69, 57–68, 62–75), respectively.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

4. Figure 3
Effect of PEP on mucosal to serosal 3H-equivalent peptides 31–49 and 56–88 fluxes across duodenal biopsy specimens of patients with active CD, measured in Ussing chambers. Fluxes of both peptides are increased in the presence of 500 mU/mL of PEP. *Significantly different from peptide 31–49 alone (P < .05). #Significantly different from peptide 56–88 alone (P < .004) and from peptide and 20 mU/mL of PEP (P < .02). □, Peptide alone (n = 7); ▩, PEP 20 mU/mL (n = 6); ■, PEP 500 mU/mL (n = 4).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

5. Figure 4
Effect of PEP on the transport and processing of 3H-peptide 31–49 by duodenal biopsy specimens from active CD patients, measured in Ussing chambers. Typical HPLC profiles of the mucosal and serosal compartments in 1 patient representative of the 7 patients studied are presented. (A) Absence of degradation of peptide 31–49 after 3 hours in the mucosal compartment of the Ussing chamber and the passage of intact peptide into the serosal compartment, as already reported.12 (B) The addition of PEP 20 mU/mL prevented the passage of intact peptide across the intestinal mucosa but an important (∼30%) fraction was recovered under the form of the toxic peptide 31–42. (C) At the highest PEP concentration studied (500 mU/mL), the peptide was found entirely degraded (>90% proline) in the serosal compartment.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

6. Figure 5
Effect of PEP on mucosal to serosal transport and processing of 3H-peptide 31–49 by duodenal biopsy specimens of patients with active CD. PEP was placed in the mucosal compartment together with 3H-peptide 31–49. After 3 hours, the percentages of peptide metabolites in (A) mucosal and (B) serosal compartments were analyzed by RP-HPLC. PEP prevented the passage of intact peptide 31–49 across the intestinal mucosa, but at 20 mU/mL, an important fraction (20%) was recovered in the serosal compartment in the form of large, potentially active fragments. ■, Intact peptide; proline (□), released free proline indicative of the percentage of fully degraded peptide; small fragments (▩), retention time of 10–15 minutes; large fragments (▨), retention time of 16–23 minutes; n = number of patients. *Significantly different from intact peptide in PEP 20 mU/mL and PEP 500 mU/mL conditions (P < .001). **Significantly different from large fragments in peptide alone and PEP 500 mU/mL conditions (P < .01). §Significantly different from small fragments in peptide alone and PEP 20 mU/mL conditions (P < .009). #Significantly different from small fragments in peptide alone (P < .04).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

7. Figure 6
Effect of PEP on the transport and processing of 3H–33-mer by duodenal biopsy specimens of active CD patients, measured in Ussing chambers. This figure presents typical HPLC profiles in 1 of the 7 patients studied. (A) Confirmation that the 33-mer is not degraded in the mucosal compartment after 3 hours and that both intact peptide and metabolites with a size compatible with immunostimulatory properties are found in the serosal compartment.12 (B) Addition of 20 mU/mL PEP to the mucosal compartment prevented the passage of intact 33-mer across the biopsy specimens but an important fraction of 33-mer (∼50%) recovered in the serosal compartment comprised metabolites with an elution time (23–28 min) corresponding to that of gliadin T-cell epitopes. (C) At the highest PEP concentration (.5 or 1 U/mL), only small nonimmunogenic metabolites (Rt < 20 min) could cross the intestinal mucosa.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

8. Figure 7
Effect of PEP on mucosal to serosal transport and processing of 3H-peptide 56–88 by duodenal biopsy specimens of patients with active CD. PEP was placed in the mucosal compartment together with 3H-peptide 56–88. After 3 hours, the percentages of peptide metabolites in (A) mucosal and (B) serosal compartments were analyzed by RP-HPLC. PEP prevented the passage of intact 33-mer across the intestinal mucosa, but at 20 mU/mL, an important fraction (35%) of the peptide was recovered in the serosal compartment in the form of large, potentially immunogenic fragments. ■, Intact peptide; proline (□), released free proline indicative of the percentage of fully degraded peptide; small fragments (▩), retention time of 10–19 minutes; large fragments (▨), retention time of 20–29 minutes; n = number of patients. *Significantly different from intact peptide in PEP 20 mU/mL and PEP 500 mU/mL conditions (P < .01). **Significantly different from large fragments in peptide alone and PEP 500 mU/mL conditions (P < .0004). §Significantly different from small fragments in peptide alone and PEP 20 mU/mL conditions (P < .008). §§Significantly different from proline in peptide alone and PEP 20 mU/mL conditions (P < .02). #Significantly different from large fragments in peptide alone and PEP 20 mU/mL conditions (P < .004).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions