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Effect of anti-gliadin IgY antibody on epithelial intestinal integrity and inflammatory response induced by gliadin.

Gujral N, Suh JW, Sunwoo HH - BMC Immunol. (2015)

Bottom Line: Caco-2 monolayers were then evaluated for effects of gliadin and/or anti-wheat gliadin IgY after 24 h exposure.Among other conditions, anti-wheat gliadin IgY at a ratio of 1:3,000 (anti-gliadin IgY: PT-gliadin) significantly prevented gliadin toxicity on Caco-2 by maintaining intestinal integrity, inhibiting phenol red permeation, and inhibiting gliadin absorption and production of proinflammatory cytokines (TNF-α and IL-1β) as compared to PT-gliadin stimulated cultures (P < 0.05).Anti-gliadin IgY, therefore has potential to be used as an oral passive antibody therapy to treat CD.

View Article: PubMed Central - PubMed

Affiliation: 3142G Katz Group Centre for Pharmacy & Health Research, Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, 11361 - 87 Ave, Edmonton, AB, T6G 2E1, Canada. gujral@ualberta.ca.

ABSTRACT

Background: Pepsin-trypsin resistant gliadin (PT-gliadin) promotes intestinal tissue inflammation and increases paracellular permeability of immunogenic gliadin peptides into the lamina propria. This leads to the complications seen in the pathogenesis of celiac disease (CD). In this study, specific anti-gliadin IgY antibody was produced and evaluated for its efficacy on gliadin induced intestinal integrity impairment and proinflammatory effects on intestinal epithelial (Caco-2) cell culture model for CD.

Methods: Caco-2 (passages 20-24) monolayers were subjected to 7 experimental conditions (n=3 each): phosphate buffered saline (PBS; control), pancreatic digested-casein (PD-casein; negative control), PT-gliadin (positive control), non-specific IgY with PT-gliadin, and anti-wheat gliadin IgY with PT-gliadin at a ratio of 1:6,000, 1:3,000 and 1:1,500. Caco-2 monolayers were then evaluated for effects of gliadin and/or anti-wheat gliadin IgY after 24 h exposure. Enzyme-linked immunosorbent assay (ELISA) was used to quantify anti-inflammatory markers (TNF-α and IL-1β) 5 days after cells were exposed to PT-gliadin and/or anti-wheat gliadin IgY.

Results: Among other conditions, anti-wheat gliadin IgY at a ratio of 1:3,000 (anti-gliadin IgY: PT-gliadin) significantly prevented gliadin toxicity on Caco-2 by maintaining intestinal integrity, inhibiting phenol red permeation, and inhibiting gliadin absorption and production of proinflammatory cytokines (TNF-α and IL-1β) as compared to PT-gliadin stimulated cultures (P < 0.05).

Conclusion: The anti-wheat gliadin IgY antibody produced in this study has proved to inhibit absorption of gliadin and gliadin-induced inflammatory response in Caco2 cell culture model of CD. Anti-gliadin IgY, therefore has potential to be used as an oral passive antibody therapy to treat CD.

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Related in: MedlinePlus

Relative TEER and phenol red permeation measurements to determine effects of anti- gliadin IgY in Caco-2 cells. Pancreatic digested casein (PD-casein) (60 µg); PT-gliadin (60 µg); Non-specific IgY (40 ng); Anti-gliadin IgY (10 ng) and PT-gliadin (60 µg) [1:6,000]. † indicates statistically significant decrease in TEER value (P < 0.05). ‡ indicates statistically significant permeation of phenol red (P < 0.05). Values are shown as mean ± SD. Analysis of each group was done in triplicates per plate. Five plates were repeated (n = 15)
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Fig4: Relative TEER and phenol red permeation measurements to determine effects of anti- gliadin IgY in Caco-2 cells. Pancreatic digested casein (PD-casein) (60 µg); PT-gliadin (60 µg); Non-specific IgY (40 ng); Anti-gliadin IgY (10 ng) and PT-gliadin (60 µg) [1:6,000]. † indicates statistically significant decrease in TEER value (P < 0.05). ‡ indicates statistically significant permeation of phenol red (P < 0.05). Values are shown as mean ± SD. Analysis of each group was done in triplicates per plate. Five plates were repeated (n = 15)

Mentions: To evaluate the neutralizing effect of anti-gliadin IgY on gliadin-induced intestinal integrity deterioration, Caco-2 cells were exposed to different ratios of anti-gliadin IgY and PT-gliadin. At 21 days, after seeding cells on the transwell inserts, the TEER values were in the range of 305–310 Ω cm2 in well-formed Caco-2 monolayers. Caco-2 monolayers have non-significant changes in TEER values after 4 h in PBS, PD-casein and non-specific IgY (control conditions) (P > 0.05). Upon 4 h exposure to PT-gliadin there was a significant decrease in TEER value of 52 %, as compared to exposures to negative control conditions in TEER value of 85 %. Basal TEER value of Caco2 monolayers at time zero is considered to have TEER value of 100 %. When the Caco2 monolayers were exposed to anti-gliadin IgY and PT-gliadin at a ratio of 1: 6,000 [anti-gliadin IgY (10 ng) and PT-gliadin (60 μg)] for 4 h, (P > 0.05) (Fig. 4), there was no significant TEER value change. This indicates that anti-gliadin IgY neutralized the toxic gliadin and prevented gliadin-induced impairment of intestinal integrity. Anti-gliadin IgY at higher ratio (1:3,000 and 1:1,500) showed similar effects to Caco2 monolayers exposed to anti-gliadin IgY and PT-gliadin at a ratio of 1: 6,000 (data not shown).Fig. 4


Effect of anti-gliadin IgY antibody on epithelial intestinal integrity and inflammatory response induced by gliadin.

Gujral N, Suh JW, Sunwoo HH - BMC Immunol. (2015)

Relative TEER and phenol red permeation measurements to determine effects of anti- gliadin IgY in Caco-2 cells. Pancreatic digested casein (PD-casein) (60 µg); PT-gliadin (60 µg); Non-specific IgY (40 ng); Anti-gliadin IgY (10 ng) and PT-gliadin (60 µg) [1:6,000]. † indicates statistically significant decrease in TEER value (P < 0.05). ‡ indicates statistically significant permeation of phenol red (P < 0.05). Values are shown as mean ± SD. Analysis of each group was done in triplicates per plate. Five plates were repeated (n = 15)
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4495697&req=5

Fig4: Relative TEER and phenol red permeation measurements to determine effects of anti- gliadin IgY in Caco-2 cells. Pancreatic digested casein (PD-casein) (60 µg); PT-gliadin (60 µg); Non-specific IgY (40 ng); Anti-gliadin IgY (10 ng) and PT-gliadin (60 µg) [1:6,000]. † indicates statistically significant decrease in TEER value (P < 0.05). ‡ indicates statistically significant permeation of phenol red (P < 0.05). Values are shown as mean ± SD. Analysis of each group was done in triplicates per plate. Five plates were repeated (n = 15)
Mentions: To evaluate the neutralizing effect of anti-gliadin IgY on gliadin-induced intestinal integrity deterioration, Caco-2 cells were exposed to different ratios of anti-gliadin IgY and PT-gliadin. At 21 days, after seeding cells on the transwell inserts, the TEER values were in the range of 305–310 Ω cm2 in well-formed Caco-2 monolayers. Caco-2 monolayers have non-significant changes in TEER values after 4 h in PBS, PD-casein and non-specific IgY (control conditions) (P > 0.05). Upon 4 h exposure to PT-gliadin there was a significant decrease in TEER value of 52 %, as compared to exposures to negative control conditions in TEER value of 85 %. Basal TEER value of Caco2 monolayers at time zero is considered to have TEER value of 100 %. When the Caco2 monolayers were exposed to anti-gliadin IgY and PT-gliadin at a ratio of 1: 6,000 [anti-gliadin IgY (10 ng) and PT-gliadin (60 μg)] for 4 h, (P > 0.05) (Fig. 4), there was no significant TEER value change. This indicates that anti-gliadin IgY neutralized the toxic gliadin and prevented gliadin-induced impairment of intestinal integrity. Anti-gliadin IgY at higher ratio (1:3,000 and 1:1,500) showed similar effects to Caco2 monolayers exposed to anti-gliadin IgY and PT-gliadin at a ratio of 1: 6,000 (data not shown).Fig. 4

Bottom Line: Caco-2 monolayers were then evaluated for effects of gliadin and/or anti-wheat gliadin IgY after 24 h exposure.Among other conditions, anti-wheat gliadin IgY at a ratio of 1:3,000 (anti-gliadin IgY: PT-gliadin) significantly prevented gliadin toxicity on Caco-2 by maintaining intestinal integrity, inhibiting phenol red permeation, and inhibiting gliadin absorption and production of proinflammatory cytokines (TNF-α and IL-1β) as compared to PT-gliadin stimulated cultures (P < 0.05).Anti-gliadin IgY, therefore has potential to be used as an oral passive antibody therapy to treat CD.

View Article: PubMed Central - PubMed

Affiliation: 3142G Katz Group Centre for Pharmacy & Health Research, Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, 11361 - 87 Ave, Edmonton, AB, T6G 2E1, Canada. gujral@ualberta.ca.

ABSTRACT

Background: Pepsin-trypsin resistant gliadin (PT-gliadin) promotes intestinal tissue inflammation and increases paracellular permeability of immunogenic gliadin peptides into the lamina propria. This leads to the complications seen in the pathogenesis of celiac disease (CD). In this study, specific anti-gliadin IgY antibody was produced and evaluated for its efficacy on gliadin induced intestinal integrity impairment and proinflammatory effects on intestinal epithelial (Caco-2) cell culture model for CD.

Methods: Caco-2 (passages 20-24) monolayers were subjected to 7 experimental conditions (n=3 each): phosphate buffered saline (PBS; control), pancreatic digested-casein (PD-casein; negative control), PT-gliadin (positive control), non-specific IgY with PT-gliadin, and anti-wheat gliadin IgY with PT-gliadin at a ratio of 1:6,000, 1:3,000 and 1:1,500. Caco-2 monolayers were then evaluated for effects of gliadin and/or anti-wheat gliadin IgY after 24 h exposure. Enzyme-linked immunosorbent assay (ELISA) was used to quantify anti-inflammatory markers (TNF-α and IL-1β) 5 days after cells were exposed to PT-gliadin and/or anti-wheat gliadin IgY.

Results: Among other conditions, anti-wheat gliadin IgY at a ratio of 1:3,000 (anti-gliadin IgY: PT-gliadin) significantly prevented gliadin toxicity on Caco-2 by maintaining intestinal integrity, inhibiting phenol red permeation, and inhibiting gliadin absorption and production of proinflammatory cytokines (TNF-α and IL-1β) as compared to PT-gliadin stimulated cultures (P < 0.05).

Conclusion: The anti-wheat gliadin IgY antibody produced in this study has proved to inhibit absorption of gliadin and gliadin-induced inflammatory response in Caco2 cell culture model of CD. Anti-gliadin IgY, therefore has potential to be used as an oral passive antibody therapy to treat CD.

Show MeSH
Related in: MedlinePlus